Гибридные системы на основе полисахаридов и магнетита, золота или меди: физико-химические свойства, коллоидная стабильность и применения тема диссертации и автореферата по ВАК РФ 00.00.00, кандидат наук Трэйси Шантал Талена

• Трэйси Шантал Талена
• кандидат наук
• 2022

Оглавление диссертации кандидат наук Трэйси Шантал Талена

Реферат

Synopsis

Introduction

Chapter 1. Literature review

1.1. Hybrid materials

1.2. Polysaccharides

1.2.1. Cellulose nanocrystals

1.2.2. Chitin nanoparticles

1.3. Metallic nanoparticles

1.4. Approaches to hybridisation

1.5. Advantages of polysaccharide nanoparticle/metallic nanoparticle hybridisation

1.6. Disperse system colloidal stability: the DLVO Theory

1.7. Conclusion

Chapter 2. Experimental section

2.1. Materials

2.2. Synthetic methods

2.2.1. Cellulose nanocrystal synthesis

2.2.2. Chitin nanocrystal synthesis

2.2.3. Gold nanoparticle synthesis

2.2.4. Magnetite nanoparticle synthesis

2.2.5. Copper nanoparticle synthesis

2.2.6. Cellulose nanocrystal/magnetite nanoparticle (CNC/Fe3O4NP) hybrid synthesis

2.2.7. Cellulose nanocrystal/gold nanoparticle (CNC/AuNP) hybrid synthesis

2.2.8. Cellulose nanocrystal/copper nanoparticle (CNC/CuNP) and chitin nanocrystal/copper nanoparticle (ChNC/CuNP) hybrid synthesis

2.3. Methods for investigating physicochemical properties

2.4. Application testing

2.4.1. Glucose sensing

2.4.2. Humidity sensing

2.4.3. Antibacterial activity testing

Chapter 3. Synthesis and characterisation of polysaccharide and metallic nanoparticles

3.1. Introduction

3.2. Metallic nanoparticles

Magnetite nanoparticles

Gold nanoparticles

Copper nanoparticles

3.3. Cellulose nanocrystals

3.4. Chitin nanocrystals

3.5. Conclusion

Chapter 4. Colloidal-chemical basis of interactions between metallic and polysaccharide nanoparticles

4.1. Introduction

4.2. Cellulose nanocrystal/magnetite nanoparticle hybrid systems

4.3. Cellulose nanocrystal/gold nanoparticle hybrid systems

4.4. Polysaccharide nanoparticle/copper nanoparticle hybrid systems

4.5. Conclusion

Chapter 5. Physicochemical properties of CNC/FesO4NP, CNC/AuNP, CNC/CuNP, and ChNC/CuNP hybrid systems

5.1. Introduction

5.2. Cellulose nanocrystal/magnetite nanoparticle hybrid systems

5.3. Cellulose nanocrystal/gold nanoparticle hybrid systems

5.4. Polysaccharide nanoparticle/copper nanoparticle hybrid systems

1.5. Conclusion

Chapter 6. Potential applications of CNC/FesO4NP, CNC/AuNP, CNC/CuNP, and ChNC/CuNP hybrid systems

6.1. Introduction

6.2. Cellulose nanocrystal/magnetite nanoparticle hybrid for glucose sensing

6.3. Hybrid cellulose nanocrystal/gold nanoparticle humidity sensor

6.4. Antibacterial and bactericidal cellulose nanocrystal/copper nanoparticle and

chitin nanocrystal/copper nanoparticle hybrids

6.5. Conclusion

Chapter 7. Conclusions

Благодарность

Acknowledgements

List of abbreviations

Glossary

Bibliography

Министерство науки и высшего образования Российской Федерации Федеральное государственное автономное образовательное учреждение высшего образования "Национальный исследовательский университет ИТМО" (Университет ИТМО)

На правах рукописи

Трэйси Шантал Талена Chantal Talena Tracey

«Гибридные системы на основе полисахаридов и магнетита, золота или меди: физико-химические свойства, коллоидная стабильность и применения»

"Magnetite-, gold-, and copper-polysaccharide hybrid systems: Physicochemical properties, colloidal stability, and applications"

«02.00.04 (1.4.4)» - «Физическая химия»

«2022»

Реферат

Общая характеристика диссертации

Введение диссертации (часть автореферата) на тему «Гибридные системы на основе полисахаридов и магнетита, золота или меди: физико-химические свойства, коллоидная стабильность и применения»

Актуальность темы работы.

Необходимость в экологичных, прочных, легких и функциональных материалов привела к разработке гибридных материалов. Гибридные системы -это композиционные материалы, состоящие как минимум из одного органического и одного неорганического компонента, представленных как на молекулярном, так и на наноуровне. В следствии наноразмерности таких материалов, они проявляют физические и химические свойства нетипичные для объемных материалов. Кроме того, органические и неорганические компоненты взаимодействуют друг с другом, улучшая уже имеющиеся свойства или приводят к появлению абсолютно новых физико-химических свойств. Уникальная синергия компонентов побудила новые исследования, а также привела к использованию гибридных материалов в нескольких областях промышленности.

Металлические или металлоксидные наночастицы реагируют на электрические и магнитные поля, а также часто проявляют оптическую и каталитическую активности, таким образом, они играют значительную роль в процессах визуализации, оптоэлектронике и в производстве сенсоров. Однако, неорганические наночастицы склонны к агрегации, что приводит к снижению их активности. Материалы, такие как оксиды кремния, титана и синтетические полимеры часто используются в качестве стабилизаторов металлических наночастиц. В настоящее время фокус исследований стабилизаторов смещен к более экологичным альтернативам, в частности, к биополимерам - полимерным материалам природного происхождения, состоящих из нуклеиновых кислот, липидов, протеинов и полисахаридов. Широко исследовались полисахаридные наночастицы благодаря их распространенности, возобновляемости и низкому влиянию на окружающую среду. Целлюлоза и хитин используются во многих гибридных материалах вследствие их высокой прочности на растяжении, большому модулю упругости, большой удельной поверхности, оптической прозрачности и химической активности. Эта уникальная комбинация свойств

способствует разработке уникальных биоразлагаемых, биосовместимых, функциональных гибридных систем.

Актуальность данной диссертации заключается в разработке функциональных гибридных систем на основе металлических или металлоксидных и полисахаридных наночастиц с широким спектром применения. В данной работе представлено исследование взаимодействий между металлическими или металлоксидными и полисахаридными наночастицами с использованием подходов теории Дерягина-Ландау-Фервея-Овербека (ДЛФО) для объяснения влияния на коллоидно-химические свойства гибридных систем таких факторов, как поверхностные функциональные группы, поверхностный заряд, концентрация неорганических наночастиц и концентрация электролита. Кроме того, было исследовано влияние гибридизации на структуру компонентов, проанализированы и охарактеризованы физико-химические свойства гибридного материала. Были предложены области применения разработанных систем.

Целью диссертационной работы является разработка физико-химических основ синтеза гибридных систем на основе наночастиц полисахаридов и магнетита, золота или меди; изучение механизмов межчастичного взаимодействия одноименно и разноименно заряженных наночастиц в дисперсных системах, установление взаимосвязи между составом, структурой и свойствами для полученных материалов.

Для достижения данной цели в рамках диссертации были поставлены и решены следующие задачи:

• Разработать синтетические методы синтеза гибридных систем, обеспечивающих взаимодействие одинаково и противоположно заряженных металлических или металлоксидных и полисахаридных наночастиц;

• Провести комплексное исследование коллоидно-химических свойств чистых и гибридных дисперсий на основе металлических или металлоксидных и полисахаридных наночастиц и их агрегативной устойчивости, изучить механизмы образования гибридных систем;

• Оценить межчастичные взаимодействия между металлическими или металлоксидными и полисахаридными наночастицами в коллоидных системах на основе классических и обобщенных теорий взаимодействия, позволяющих предсказать наличие или отсутствие гетероагрегации;

• Изучить состав, морфологию и физико-химические свойства полученных гибридных материалов и установить особенности процессов их формирования, в том числе при гидротермальной обработке;

• Установить взаимосвязь состава и структуры гибридных материалов и их оптических, каталитических и биохимических свойств.

Основные положения, выносимые на защиту:

1. Предложена физико-химическая модель, позволяющая оценить вероятность протекания процессов взаимодействия наночастиц оксидов металлов или металлов с поверхностью наноразмерных частиц полисахаридов. Установлена решающая роль дальнодействующих сил в растворе, которые основаны на возникновении расклинивающего давления и распределении наночастиц в объеме раствора.

2. Взаимодействие между противоположно заряженными наночастицами оксидов металлов или металлов и полисахаридными наночастицами сопровождается отсутствием потенциального энергетического барьера и происходит по механизму необратимой гетерокоагуляции.

3. Взаимодействие одинаково заряженных наночастиц оксидов металлов или металлов и полисахаридных наночастиц сопровождается наличием высокого барьера потенциальной энергии из-за электрических сил отталкивания двойного электрического слоя.

4. Методический подход к формированию гибридных систем из одинаково заряженных наночастиц оксидов металлов или металлов и полисахаридных наночастиц путем гидротермальной обработки для преодоления энергетического барьера и облегчения гетероагрегации.

5. Физико-химические основы получения гибридных материалов, предназначенных для мониторинга высокого уровня глюкозы в крови, уровня

влажности окружающей среды, применения в качестве антибактериального агента и/или бактерицида.

Научная новизна диссертации отражена в следующих пунктах:

Впервые установлены закономерности формирования гибридных систем на основе металлических или металлоксидных и полисахаридных наночастиц в зависимости от соотношения компонентов, поверхностного заряда и состава дисперсионной среды.

Показано, что межчастичное взаимодействие между противоположно заряженными наночастицами определяется электростатическим притяжением. Для гибридных систем на основе металлических или металлоксидных и полисахаридных наночастиц определены области агрегативной устойчивости и соотношения компонентов при образовании гибридных структур за счет электростатических взаимодействий и образования водородных связей.

Предложен механизм образования гибридных систем на основе одинаково заряженных частиц путем преодоления энергетического барьера, вызванного отталкивающими силами, посредством гидротермальной обработки, что позволяет прогнозировать свойства систем с заданными параметрами.

Предложена физико-химическая модель, предсказывающая образование гибридных структур на основе межчастичных взаимодействий между металлическими или металлоксидными и полисахаридными наночастицами. Модель основана на полуэмпирических расчетах энергий взаимодействия пар частиц в процессе коагуляции, которые позволяют оценить вероятность взаимодействия металлических или металлоксидных и полисахаридных наночастиц.

Изучены механизмы гетероагрегации между противоположно заряженными наночастицами магнетита и нанокристаллами целлюлозы. Исследована каталитическая активность полученного гибридного материала в реакции окисления глюкозы, а также влияние температуры, pH, времени и концентрации глюкозы. Благодаря высокой каталитической активности при низком pH и температуре тела, а также пределу обнаружения 5 мМ, был получен неинвазивный

биосенсор для определения уровня глюкозы в биологических жидкостях, таких как пот и слюна.

Изучены физико-химические свойства гибридных систем на основе одинаково заряженных наночастиц золота и нанокристаллов целлюлозы после обработки при повышенной температуре и давлении. Такой подход позволяет преодолеть энергетический барьер, вызванный отталкивающими силами, и увеличить площадь контактов за счет образования водородных связей. Благодаря гигроскопичности гибрида он был предложен для применения в качестве датчика влажности. Оптическая активность датчика проявляет линейную зависимость от влажности и может работать в диапазоне от 35% до 80% относительной влажности.

Взаимодействие между противоположно заряженными нанокристаллами целлюлозы и наночастицами меди, а также одинаково заряженными нанокристаллами хитина и наночастицами меди было предсказано с помощью модели, предложенной в данной диссертации. Гидротермальная обработка была использована для преодоления высокого барьера потенциальной энергии, существующего между нанокристаллами хитина и наночастицами меди. В то время как между противоположно заряженными наночастицами меди и нанокристаллами целлюлозы существовало электростатическое притяжение, наночастицы меди окислялись до оксида меди (I). Поэтому для обработки гибридной системы нанокристаллs целлюлозы/наночастицы меди также использовалась гидротермальная обработка. Результаты показали, что гидротермальная обработка стабилизирует наночастицы меди.

Были изучены физико-химические свойства гибридных систем нанокристаллы хитина/наночастицы меди и нанокристаллы целлюлозы/наночастицы меди. Гибридные системы эффективны при низких концентрациях против грамотрицательных и грамположительных бактерий (минимальная ингибирующая концентрация = 0.4 мкг/мл; минимальная бактерицидная концентрация = 1.6 мкг/мл). Гибриды могут быть использованы

для подавления видимого роста и уничтожения грамотрицательных и грамположительных бактерий.

Теоретическая значимость результатов диссертационной' работы состоит в исследовании зависимости коллоидно-химических свойств гибридных систем на основе металлических или металлоксидных наночастиц/полисахаридных наночастиц от величины поверхностных зарядов наночастиц, массовой доли неорганических наночастиц и концентрации электролита, а также манипуляция и оптимизация полученных свойств для создания функциональных гибридных систем.

Практическая значимость результатов диссертационной' работы состоит в разработке функциональных гибридных систем на основе металлических или металлоксидных наночастиц/полисахаридных наночастиц, которые могут быть использованы в медицине, фармацевтике и пищевой индустрии. Это показывает, что металлические или металлоксидные и полисахаридные наночастицы могут быть использованы для создания функциональных материалов. Может быть разработано множество новых функциональных материалов, благодаря изобилию видов металлических или металлоксидных и полисахаридных наночастиц.

Достоверность полученных результатов обусловлена тем, что в работе использованы надежные и проверяемые синтетические методы, стандартизированные методы определения характеристик материалов, теоретические и вычислительные модели, которые признаны и приняты научным сообществом. Кроме того, результаты были подтверждены в ходе рецензирования работ в процессе их публикации.

Апробация результатов работы.

Основные результаты работы докладывались и обсуждались на следующих конференциях: Конгресс Молодых Ученых, Университет ИТМО, Россия (2019); 20th International Sol-Gel Conference, Россия (2019); XIII Международный конкурс-конференция научных работ учащихся имени А.А. Яковкина, Россия (2019); Конгресс Молодых Ученых, Университет ИТМО, Россия (2020); Конгресс Молодых Ученых, Университет ИТМО, Россия (2021); Food Biotech Conference,

Россия (2021); 2022: International Conference on Advanced Plasmonics, Magnetics & Magneto-Optical Technologies, Canada (2022).

Связь темы с планируемыми исследованиями и финансовое обеспечение работы.

Работа выполнялась в соответствии с дорожной картой развития федерального государственного автономного образовательного учреждения высшего образования "Национальный исследовательский университет ИТМО". Диссертационная работа выполнялась в рамках ряда научных проектов: РФФИ, проект № 18-33-20230, и Министерства науки и высшего образования РФ, проект № 075-15-2019-1896.

Личный вклад автора.

Автор диссертации внес ключевой вклад в проекты, изложенные в работе. Автор лично участвовал в проектировании и планировании работ, получении и характеристике всех описанных в работе систем. Автор принимал участие в сборе, обработке и представлении полученных данных. Также она играла важную роль в публикации исследовательских работ.

Структура и объем диссертации.

Диссертация состоит из введения, шесты глав, заключения и списка литературы. Диссертация 225 страниц, включая библиографию 163 литературных источников. Работа содержит 79 рисунков и 6 таблиц. Список рисунков с указанием номера страницы приводится после библиографии.

Основное содержание работы.

Первая глава представляет собой обзор литературы, включающий краткое описание гибридных материалов. Здесь приведено определение систем, классификации и различные типы гибридных материалов, а также кратко описываются различные силы, действующие между составляющими частицами. В главе также освещаются характеристики и свойства полисахаридных и металлических или металлоксидных наночастиц, которые делают их подходящими для получения гибридных материалов, и описываются преимущества гибридных

систем на основе металлических или металлоксидных наночастиц/полисахаридных наночастиц. Кроме того, приведены различные подходы к получению гибридных систем. Также описаны зависимости стабильности коллоидных систем от соотношения между потенциальной энергией и расстоянием между компонентами.

Во второй главе описаны используемые реактивы и методы синтеза необходимых материалов, методы получения гибридных систем, методы определения характеристик и условия проведения тестирования материалов. В этой главе описаны синтетические методы, использованные для получения нанокристаллов целлюлозы (СКС) и хитина (СКЫС), а также наночастиц магнетита (Рез04КР), золота (АиКР) и меди (СиЫР), а также применение просвечивающей электронной микроскопии (ПЭМ), ультрафиолетовой/видимой (УФ/Вид) спектроскопии, динамического рассеяния света (ДЛС), лазерного доплеровского электрофореза (ЛДЭ), рентгеновской фотоэлектронной спектроскопии (РФЭС), инфракрасной (ИК) спектроскопии с преобразованием Фурье и рентгеновской дифракции (РФА) для определения характеристик полученных наночастиц. В этой главе описаны методы получения систем на основе нанокристаллов целлюлозы/наночастиц магнетита, нанокристаллов целлюлозы/наночастиц золота, нанокристаллов целлюлозы/наночастиц меди и нанокристаллов хитина/наночастиц меди. Для определения характеристик гибридных систем использовались УФ/Вид спектроскопия, ДЛС, ЛДЭ, ИК-Фурье спектроскопия, РФА, сканирующая электронная микроскопия (СЭМ), атомно-силовая микроскопия (АСМ) и энергодисперсионная рентгеновская спектроскопия (ЭДРС). Наконец, описаны методы тестирования гибридных систем на основе нанокристаллов целлюлозы/наночастиц магнетита, нанокристаллов целлюлозы/наночастиц золота, нанокристаллов целлюлозы/наночастиц меди и нанокристаллов хитина/наночастиц меди на содержание глюкозы, уровня влажности и антибактериальных свойств.

Третья глава посвящена синтезу и определению характеристик суспензий нанокристаллической целлюлозы и хитина, а также золей наночастиц магнетита, золота и меди. Здесь приведены данные ИК-Фурье спектроскопии, РФА, УФ/Вид спектроскопии, РФЭС, ПЭМ и ДЛС. Для определения поверхностного заряда

частиц использовался метод ЛДЭ. Также были синтезированы два типа нанокристаллов целлюлозы: нанокристаллы сульфатированной целлюлозы CNC), которые имели сульфатные группы на поверхности, и нанокристаллы ацетилированной целлюлозы (N-CNC), которые имели ацетильные группы вместо сульфатных групп и имели поверхность очень похожую на поверхность натуральной целлюлозы. РФА показал, что кристаллические структуры обоих типов нанокристаллов целлюлозы были очень похожи, что указывает на то, что различия были только в составе функциональных групп.

Рисунок Р1 - XRD-шаблоны №СЖ! (серый) и S-CNC (красный).

Четвертая глава посвящена использованию полуэмпирических расчетов энергий парного взаимодействия частиц в процессе коагуляции для определения вероятности взаимодействия металлических или металлоксидных наночастиц с полисахаридными наночастицами (Р№) на основе поверхностных зарядов органических и неорганических компонентов. Системы, состоящие из противоположно заряженных полисахаридных и металлических или металлоксидных наночастиц (т.е. гибридные системы на основе нанокристаллы целлюлозы/наночастицы магнетита и нанокристаллы целлюлозы/наночастицы меди) не имели потенциального энергетического барьера, препятствующего гетероагрегации, и частицы электростатически притягивались друг к другу; поэтому для получения гибридных систем было достаточно перемешивания.

(200)

N-CNC S-CNC

5 10 15 20 25 30 35 40 20(degrees)

300 250

■100

Рисунок Р2 - Потенциальные энергетические барьеры для необратимой агрегации между наночастицами CNC/CNC (серый), FeзO4/FeзO4 (синий) и СКС/Без04 (красный). Вставка: Ван-дер-Ваальсовое притяжение между отрицательно заряженными нанокристаллами целлюлозы и положительно заряженными наночастицами магнетита.

Рисунок Р3 - Потенциальные энергетические барьеры для необратимой агрегации между наночастицами CNC/CNC (серый), CuNP/CuNP (синий) и CNC/CuNP (красный). Вставка: Ван-дер-Ваальсовое притяжение между отрицательно заряженными нанокристаллами целлюлозы и положительно заряженными наночастицами меди.

Однако для систем с одинаково заряженными наночастицами полисахаридов и металлическими наночастицами (например, гибридные системы

на основе нанокристаллов целлюлозы/наночастиц золота и нанокристаллов хитина/наночастиц меди) существует очень высокий потенциальный энергетический барьер, который препятствует получению гибридных материалов. Поэтому для повышения энергии системы и получения гибридных систем посредством образования водородной связи была использована гидротермальная обработка. Анализ показал, что гидротермальная обработка оказывает минимальное влияние на полисахаридные наночастицы при температурах до 130 °С.

Рисунок Р4 - Потенциальные энергетические барьеры для необратимой агрегации между наночастицами CNC/CNC (серый), AuNP/AuNP (синий) и CNC/AuNP (красный). Вставка: Электрическое отталкивание между отрицательно заряженными нанокристаллами целлюлозы и отрицательно заряженными наночастицами золота.

1750 1600

■600

Рисунок Р5 - Потенциальные энергетические барьеры для необратимой агрегации между наночастицами ChNC/ChNC (серый), CuNP/CuNP (синий) и ChNC/CuNP (красный). Вставка: электрическое отталкивание между положительно заряженными нанокристаллами хитина и положительно заряженными наночастицами меди.

Были исследованы коллоидно-химические свойства гибридов, а также влияние соотношения органических и неорганических компонентов (изменение массовой доли металлических или металлоксидных наночастиц) и концентрации электролита на стабильность раствора. Результаты показали, что при увеличении массовой доли неорганических наночастиц устойчивость раствора снижается, а затем восстанавливается. Для систем, состоящих из противоположно заряженных наночастиц полисахаридов и металлических или металлоксидных наночастиц (т.е. гибридных систем на основе нанокристаллов целлюлозы/наночастиц магнетита и нанокристаллов целлюлозы/наночастиц меди), небольшой барьер потенциальной энергии, существующий между наночастицами, либо снижается, что означает, что гибридным системам требуется меньше энергии для гомоагрегации, либо смещается влево, что означает, что притягивающие силы сохраняются на больших расстояниях и вероятность гомоагрегации увеличивается.

Рисунок Р6 - Зависимость энергии взаимодействия CNC/Fe3O4NP от концентрации KCl.

0,4

Рисунок Р7 - Влияние увеличения концентрации KCl на систему CNC/CuNP.

Сопоставимые результаты наблюдались для систем, состоящих из одинаково заряженных наночастиц полисахаридов и металлических наночастиц. По мере увеличения массовой доли металлических наночастиц устойчивость раствора снижалась, а затем стабилизировалась. Однако вместо небольшого барьера потенциальной энергии между гибридными наночастицами, как это наблюдалось при взаимодействии противоположно заряженных наночастиц друг с другом, существует вторичный минимум для одинаково заряженных частиц. Независимо от этого, при увеличении концентрации электролита вторичный минимум либо смещается влево, что означает, что силы притяжения сохраняются на больших

расстояниях и гомоагрегация более вероятна, либо количество необходимой энергии снижается и коагуляция более вероятна.

Рисунок Р8 - Зависимость энергии взаимодействия CNC/AuNP от концентрации KCl.

Рисунок Р9 - Влияние увеличения концентрации KCl на гибридную систему ChNC/CuNP.

В пятой главе физико-химические свойства гибридных систем анализируются с помощью СЭМ, РФА, ИК-Фурье спектроскопии, АСМ, ЭДРС, которые использовались для изучения взаимодействия наночастиц полисахаридов и металлических или металлоксидных наночастиц друг с другом. Было обнаружено, что методы получения систем оказывает минимальное влияние на составляющие различных гибридных систем; однако было установлено, что

полисахаридные наночастицы стабилизируют металлические наночастицы, сохраняя их каталитическую и оптическую активности.

В шестой главе рассматриваются потенциальные области применения гибридных систем. Гибридная система на основе нанокристаллов целлюлозы/наночастиц магнетита была предложена в качестве потенциального сенсора мониторинга гипергликемии, поскольку наночастицы магнетита могут разлагать перекись водорода, которая является побочным продуктом распада глюкозы. Глюкозооксидаза и 2,2'-азинобис[3-этилбензотиазолин-6-сульфокислота] (ABTS) были включены в систему для создания самоиндикаторной тест-полоски, способной определять концентрацию глюкозы до 5 ммоль/л. Система демонстрирует высокую каталитическую активность при pH 2 и при температуре тела (37 °C); поэтому ее можно использовать для мониторинга уровня глюкозы в поте и слюне, которые имеют тенденцию быть слегка кислыми. Система, содержащая нанокристаллы сульфатированной целлюлозы, была более каталитически активной, что объясняется наличием сульфатных групп на поверхности. По сравнению с другими системами мониторинга глюкозы, предложенная система на основе наночастиц целлюлозы и магнетита является недорогим, быстрым, безболезненным, самоиндикаторным методом мониторинга гипергликемии.

0,8

5 wt.% Fe,0,NP N-CNC

<

0,2

i

0 10 20 30 40 50 Glucose concentration (mM)

Рисунок Р10 - Зависимость каталитической активности 5 мас.% Fe3O4NP N-CNC/Fe3Ü4NP (серый) и S-CNC/Fe3O4NP (красный) от концентрации глюкозы.

В ходе анализа методом АСМ гибридная система на основе нанокристаллов целлюлозы/золотых наночастиц показала высокую гигроскопичность; таким образом, она была предложена в качестве потенциального датчика влажности. Метод лазерной дифракции показал, что целлюлозная матрица активна и участвует в детектировании уровня влажности. Здесь молекулы воды адсорбируются на поверхности целлюлозной матрицы и десорбируются с нее по мере увеличения и уменьшения влажности. Лазерная дифракция также показала, что система является обратимой и автономной.

Рисунок Р11 - Картина лазерной дифракции 3 мас.% Аи№ СКС/АиИР гибридной пленки (а) в 0 мин, (Ь) 1 мин после обработки 90% влажности, (с) 2 мин после обработки 90% влажности, 1 мин после обработки влажности и (е) 2 мин после обработки влажности. Размер каждого квадрата составлял 17 х 17 мм.

Тушение флуоресценции использовалось как альтернативный метод детектирования изменений уровня влажности окружающей среды. Показана линейная зависимость оптического сигнала от уровня влажности, что предпочтительно для получения сенсоров из-за простоты калибровки и меньшей неопределенности при масштабировании выходного сигнала. Было установлено, что систему возможно использовать в диапазоне между 35% и 80% относительной влажности, что является типичным для датчиков влажности.

Рисунок Р12 - Снижение интенсивности флуоресценции с увеличением влажности для пленок N-CNC/AuNP (серий) и чистых пленок CNC (красный), допированных флуоресцентным красителем Родамин B.

Наконец, гибридные системы на основе нанокристаллов целлюлозы/наночастиц меди и нанокристаллов хитина/ наночастиц меди были предложены в качестве потенциальных антибактериальных агентов. Системы показали эффективность против грамотрицательных Escherichia coli и грамположительных Bacillus subtilis бактерий, с минимальной ингибирующей концентрацией 0.4 мкг/мл и минимальной бактерицидной концентрацией 1.6 мкг/мл. Гибридные системы подавляли рост E. coli в течение 18 ч, а B. subtilis - в течение 36 ч. Также показана эффективность против грамотрицательных бактерий через 24 ч после обработки; однако эффективность снизилась через 24 ч против грамположительных бактерий. Для предположения потенциального механизма действия был использован анализ методом СЭМ. Несколько исследований также показали, что наночастицы меди имеют тенденцию высвобождать ионы меди (II) в растворе. На микрофотографиях были видны питтинги в клетках бактерий и полностью лизированные клетки после обработки. Таким образом, была выдвинута гипотеза, что наночастицы проникают в клеточную стенку и мембрану бактерий, а ионы меди (II) переносятся через мембрану через канальные белки, нарушая биопроцессы, повреждая биомолекулы и приводя в конечном итоге к лизису клеток.

Список литературы диссертационного исследования кандидат наук Трэйси Шантал Талена, 2022 год

Публикации.

Ключевые результаты исследования описаны в четырех публикациях, все из которых опубликованы в международных журналах первого квартиля, индексируемых в Scopus и Web of Science:

A1.Torlopov, M.A., Martakov, I.S., Mikhaylov, V.I., Legki, P.V., Golubev, Y.A., Krivoshapkina, E.F., Tracey, C.T., Sitnikov, P.A., Udoratina, E.V. Manipulating the colloidal properties of (non-)sulfated cellulose nanocrystals via stepwise cyanoethylation/carboxylation. European Polymer Journal 115. p. 225-233. (2019) A2.Koroleva, M.S., Tracey, C.T., Sidunets, Y.A., Torlopov, M.A., Mikhaylov, V.I., Krivoshapkin, P.V., Martakov, I.S., Krivoshapkina, E.F. Environmentally friendly Au@CNC hybrid systems as prospective humidity sensors. RSC Advances 10(58). p. 35031 - 35038. (2020) A3. Tracey, C.T., Torlopov, M.A., Martakov, I.S., Vdovichecko, E.A., Zhukov, M., Krivoshapkin, P.V., Mikhaylov, V.I., Krivoshapkina, E.F. Hybrid cellulose nanocrystal/magnetite glucose biosensors. Carbohydrate Polymers 247. p. 116704. (2020)

A4.Tracey, C.T., Predeina, A.L., Krivoshapkina, E.F., Kumacheva, E. A 3D printing approach to intelligent food packaging. Trends in Food Science & Technology. (2022)

Synopsis General thesis summary

Relevance of the chosen topic.

The need for environmentally friendly, durable, lightweight, and functional materials has led to the development of hybrid materials. Hybrid systems are composite materials consisting of at least one organic and one inorganic component that occur on the nanoscale or at the molecular level. Due to the nanoscale nature of such materials, they exhibit physical and chemical properties atypical of bulk materials. In addition, organic and inorganic components interact with each other, improving already existing properties or giving rise to entirely new physical and chemical properties. The unique synergy of components has prompted new research and has also led to the use of hybrid materials in several industries.

Metal and metal oxide nanoparticles respond to electric and magnetic fields, and often exhibit optical and catalytic activities; therefore, they play a significant role in imaging processes, optoelectronics, and sensor production. However, these nanoparticles are prone to aggregation, resulting in a decrease in their activity. Materials such as silicon oxides, titanium oxides, and synthetic polymers are often used to stabilise metallic nanoparticles. The focus of stabiliser research is now shifting to more environmentally friendly alternatives, in particular biopolymers, naturally occurring polymeric materials consisting of nucleic acids, lipids, proteins, and polysaccharides. Polysaccharide nanoparticles have been extensively studied due to their prevalence, renewability, and low environmental impact. Cellulose and chitin are used in many hybrid materials due to their high tensile strengths, high elastic moduli, large specific surface areas, optical transparencies, and chemical activities. This unique combination of properties contributes to the development of unique biodegradable, biocompatible, functional hybrid systems.

The relevance of this thesis is to develop functional hybrid systems based on metal or metal-oxide and polysaccharide nanoparticles with a wide range of applications. This work presents a study of the interactions between metal or metal-oxide and polysaccharide nanoparticles using Derjaguin-Verwy-Landau-Overbeek (DLVO) theory approaches to explain the effects of factors such as surface functional groups, surface charge, inorganic nanoparticle concentration, and electrolyte concentration on the colloidal-chemical properties of hybrid systems. The effect of hybridization on the structure of the components is also investigated, and the physicochemical properties of the hybrid material are analysed and characterised. Finally, potential applications of the developed systems are suggested.

The aim of this dissertation is to develop a physicochemical basis for the synthesis of hybrid systems based on polysaccharide nanoparticles and magnetite, gold, or copper nanoparticles; to study the mechanisms of interparticle interaction of single- and multiple-charged nanoparticles in disperse systems; and to establish the relationship between the composition, structure, and properties for the obtained materials.

To achieve this goal in the framework of the thesis, the following objectives have been established:

• To develop synthetic methods for the synthesis of hybrid systems providing for the interaction of similarly and oppositely charged metal/metal oxide and polysaccharide nanoparticles;

• To comprehensively study the colloid-chemical properties of pure and hybrid dispersions based on metal/metal oxide and polysaccharide nanoparticles and their aggregative stability as well as the mechanisms of hybrid system formation;

• To evaluate interparticle interactions between metal/metal oxide and polysaccharide nanoparticles in colloidal systems based on classical and generalized interaction theories to predict the presence or absence of heteroaggregation;

• To study the composition, morphology, and physicochemical properties of the obtained hybrid materials and to establish the peculiarities of their formation processes, including hydrothermal treatment;

• To determine the relationship between the composition and structure of hybrid materials and their optical, catalytical, and biochemical properties.

Assertions that are presented for defence:

1. A physicochemical model that estimates the probability of interaction of metal/metal oxide metal nanoparticles with the surface of polysaccharide nanoparticles has been proposed. The decisive role of long-range forces in a sol is established, which is based on the initiation of disjoining pressure and nanoparticle distribution in a solution volume.

2. The interaction between oppositely charged metal/metal oxide nanoparticles and polysaccharide nanoparticles is accompanied by the absence of a potential energy barrier and occurs by the mechanism of irreversible heterocoagulation.

3. The interaction between similarly charged metal/metal oxide nanoparticles and polysaccharide nanoparticles is accompanied by the presence of a high potential energy barrier due to the electrical repulsive forces of the electrical double layer.

4. A methodical approach hybridisation between similarly charged metal/metal oxide nanoparticles and polysaccharide nanoparticles is to use hydrothermal treatment to overcome the energy barrier and facilitate heteroaggregation.

5. Physicochemical bases for the production of hybrid materials designed to monitor high blood glucose levels, ambient moisture levels, and to use as an antibacterial agent and/or bactericide.

Novelty of research

The formation of hybrid systems based on metal/metal oxide and polysaccharide nanoparticles depending on the ratio of components, surface charge, and composition of dispersion medium was established for the first time.

It is shown that interparticle interaction between oppositely charged nanoparticles is governed by electrostatic attraction. For hybrid systems based on metal/metal oxide and polysaccharide nanoparticles, the areas of aggregative stability and component ratios for the formation of hybrid structures due to electrostatic interactions and formation of hydrogen bonds are determined.

A mechanism of hybrid system formation between similarly charged particles by overcoming the energy barrier caused by repulsive forces via hydrothermal treatment is proposed, which allows the properties of the systems with the given parameters to be predicted.

A physicochemical model predicting the formation of hybrid structures based on interparticle interactions between metal/metal oxide and polysaccharide nanoparticles has been proposed. The model is based on semiempirical calculations of the interaction energies of particle pairs in the coagulation process, which allow the probability of interaction between metal/metal oxide and polysaccharide nanoparticles to be estimated.

The mechanisms of heteroaggregation between oppositely charged magnetite nanoparticles and cellulose nanocrystals were studied. The catalytic activity of the obtained hybrid material in the glucose oxidation reaction was investigated, as well as the effect of temperature, pH, time, and glucose concentration. Due to the high catalytic activity at low pH and body temperature and a detection limit of 5 mM, a non-invasive biosensor was obtained for the determination of glucose levels in biological fluids such as sweat and saliva.

The physicochemical properties of hybrid systems based on similarly charged gold nanoparticles and cellulose nanocrystals were studied after treatment at elevated temperature and pressure. This approach makes it possible to overcome the energy barrier caused by repulsive forces and to increase the contact area through the formation of hydrogen bonds. Due to the hygroscopic nature of the hybrid, it is proposed for use as a moisture sensor. The sensor exhibits a linear dependence on humidity and can operate between 35% and 80% relative humidity.

The interactions between oppositely charged cellulose nanocrystals and copper nanoparticles and similarly charged chitin nanocrystals and copper nanoparticles were predicted using the model proposed in this thesis. Hydrothermal treatment was used to overcome the high potential energy barrier that exists between chitin nanocrystals and copper nanoparticles. While an electrostatic attraction existed between the oppositely charged copper nanoparticles and the cellulose nanocrystals, the copper nanoparticles were oxidized to copper (I) oxide. Therefore, hydrothermal treatment was also used to

treat the cellulose nanocrystal/copper nanoparticles hybrid system. The results showed that hydrothermal treatment stabilizes the copper nanoparticles.

The physicochemical properties of the chitin nanocrystal/copper nanoparticle and cellulose nanocrystal/copper nanoparticle hybrid systems were studied. The hybrid systems are effective at low concentrations against gram-negative and gram-positive bacteria (minimum inhibitory concentration = 0.4 ^g/mL; minimum bactericidal concentration = 1.6 ^g/mL). The hybrids can be used to inhibit the visible growth of and kill gram-negative and gram-positive bacteria.

The theoretical significance of the results of the thesis work consists in the study of the dependence of the colloid-chemical properties of hybrid systems based on metal/metal oxide nanoparticles and polysaccharide nanoparticles on nanoparticle surface charge, the mass fraction of inorganic nanoparticles, and background electrolyte concentration as well as the manipulation and optimization of the obtained properties to create functional hybrid systems.

The practical significance of the results of the thesis work consists in the development of functional hybrid systems based on metal/metal oxide nanoparticles and polysaccharide nanoparticles, which can be used in medicine, pharmaceuticals, and the food industry. This shows that metal/metal oxide and polysaccharide nanoparticles can be used to create functional materials. Many new functional materials can be developed due to the many different types of metal/metal oxide and polysaccharide nanoparticles.

The accuracy of the results obtained is due to the fact that reliable and verifiable synthetic methods, standardized methods for determining the characteristics of materials, theoretical and computational models, which are recognized and accepted by the scientific community, have been used in the work. In addition, the results were confirmed in the course of reviewing the papers during their publication.

Approbation of research results.

Key research results were presented and discussed at the following conferences: Congress of Young Scientists, ITMO University, Russia (2019); XX International SolGel Conference, Russia (2019); XIII International Competition-Conference of Scientific Works for Students named A.A. Yakovkin, Russia (2019); Congress of Young

Scientists, ITMO University, Russia (2020); Congress of Young Scientists, ITMO University, Russia (2021); International Conference on Advanced Plasmonics, Magnetics & Magneto-Optical Technologies, Canada (2022).

Connection of the topic with planned research and financial support for the

work.

The work was carried out in accordance with the roadmap for the development of the federal state autonomous educational institution of higher education "National Research University ITMO". The dissertation work was carried out within the framework of a number of scientific projects: Russian Foundation for Basic Research Project, №18-33-20230, and the Ministry of Science and Higher Education of Russia, Project № 075-15-2019-1896.

Personal contribution of the author.

The author of the thesis made key contributions to the projects described in this dissertation. The author was personally involved in the design and planning of the work, and the synthesis and characterisation all of the systems described herein. The author was involved in the collection, processing, and presentation of the data obtained. She also played an important role in the publication of the research papers.

Thesis structure and number of pages.

This thesis consists of an introduction, six chapters, a conclusion, and a list of references. The dissertation is 225 pages, including a bibliography of 163 references. The work contains 79 figures and 6 tables. A list of figures and their page number is included after the bibliography.

Main contents of the work.

The first chapter is a literature review that includes a brief summary of hybrid materials. Here, the definition, classification system, and different types of hybrid materials are described and the different forces between the constituent particles are summarised. The chapter also highlights the characteristics and properties of polysaccharide and metal/metal oxide nanoparticles that make them suitable for hybridisation and the advantages of polysaccharide nanoparticle/metallic nanoparticle hybrid systems are described. Furthermore, different approaches to hybridisation are also

described. Finally, the dependence of sol stability on the relationship between potential energy and distance is explored.

The second chapter presents the materials and methods used to obtain the necessary materials, the hybridisation techniques, characterisation methods, and application testing. This chapter describes the synthetic methods used to obtain cellulose nanocrystals (CNC) and chitin nanocrystals (ChNC), as well as magnetite nanoparticles (Fe3O4NP), gold nanoparticles (AuNP), and copper nanoparticles (CuNP). It also describes the use of transmission electron microscopy (TEM), ultraviolet/visible (UV/Vis) spectroscopy, dynamic light scattering (DLS), laser Doppler electrophoresis (LDE), X-ray photoelectron spectroscopy (XPS), Fourier transform infrared (FTIR) spectroscopy, and X-ray diffraction (XRD) analysis to characterise the resulting nanoparticles. In this chapter, the hybridisation methods for obtaining cellulose nanocrystal/magnetite nanoparticle, cellulose nanocrystal/gold nanoparticle, cellulose nanocrystal/copper nanoparticle, and chitin nanocrystal/copper nanoparticles are recounted. UV/Vis spectroscopy, DLS, LDE, FTIR, spectroscopy, XRD analysis, scanning electron microscopy (SEM), atomic force microscopy (AFM), and energy-dispersive X-ray (EDX) spectroscopy were used to characterise the hybrid nanoparticles. Finally, methods for testing the cellulose nanocrystal/magnetite nanoparticle, cellulose nanocrystal/gold nanoparticle, and cellulose nanocrystal/copper nanoparticle and chitin nanocrystal/copper nanoparticle hybrid systems for measuring glucose content, moisture level, and antibacterial and bactericidal properties are reported.

The third chapter concerns the synthesis and characterisation of pure cellulose nanocrystal and chitin nanocrystal sols as well as colloidal magnetite, gold, and copper. Here, FTIR spectroscopy, XRD analysis, and UV/Vis spectroscopy, XPS, TEM, and DLS confirmed the synthesis of the polysaccharide and metal/metal oxide nanoparticles. LDE was used to determine the surface charges of the particles. Also, two types of cellulose nanocrystals were synthesised: sulphated cellulose nanocrystals (S-CNC), which had sulphate groups on the surface; and acetylated cellulose nanocrystals (N-CNC), which had acetyl groups instead of sulphate groups and had a surface very similar to that of native cellulose. XRD analysis showed that the crystalline structures of both

types of cellulose nanocrystals were very similar, indicating that the differences were only the surface functional groups.

Figure S1 - XRD patterns of N-CNC (grey) and S-CNC (red).

The fourth chapter is dedicated to the use of semiempirical calculations of particle pair interaction energies during coagulation to determine the probability of metal/metal oxide nanoparticle-polysaccharide nanoparticle interactions based on the surface charges of the organic and inorganic constituents. The systems composed of oppositely charged polysaccharide and metal/metal oxide nanoparticles (i.e., cellulose nanocrystal/magnetite nanoparticle and cellulose nanocrystal/copper nanoparticle hybrid systems) had no potential energy barriers preventing heteroaggregation, and the particles are electrostatically attracted to each other; therefore, stirring was sufficient to obtain hybrid systems.

(200)

N-CNC S-CNC

5 10 15 20 25 30 35 40 20(degrees)

300 r

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<-e

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Figure S2 - Potential energy barriers for irreversible aggregation between CNC/CNC (grey), Fe3O4/Fe3O4 (blue), and CNC/Fe3O4 (red) nanoparticles. Inset: van der Waals attraction between negatively charged cellulose nanocrystals and positively charged magnetite nanoparticles.

Figure S3 - Potential energy barriers for irreversible aggregation between CNC/CNC (grey), CuNP/CuNP (blue), and CNC/CuNP (red) nanoparticles. Inset: van der Waals attraction between negatively charged cellulose nanocrystals and positively charged copper nanoparticles.

However, for systems with similarly charged polysaccharide and metal/metal oxide nanoparticles (i.e., cellulose nanocrystal/gold nanoparticle and chitin nanocrystal/copper nanoparticle hybrid systems), there exists a very high potential energy barrier that hinders hybridisation. Therefore, hydrothermal treatment was used to

125

100

■50

increase the energy of the system and facilitate hybridisation via hydrogen bonding. Analysis showed that hydrothermal treatment had minimal effect on polysaccharide nanoparticles at temperatures as high as 130 °C.

Figure S4 - Potential energy barriers for irreversible aggregation between CNC/CNC (grey), AuNP/AuNP (blue), and CNC/AuNP (red) nanoparticles. Inset: electrical repulsion between negatively charged cellulose nanocrystals and negatively charged gold nanoparticles.

Figure S5 - Potential energy barriers for irreversible aggregation between ChNC/ChNC (grey), CuNP/CuNP (blue), and ChNC/CuNP (red) nanoparticles. Inset: electrical repulsion between positively charged chitin nanocrystals and positively charged copper nanoparticles.

The colloidal-chemical properties of the hybrids were investigated, and the effect of organic-to-inorganic component ratio (changing the metal/metal oxide nanoparticle mass fraction) and electrolyte concentration on sol stability examined. The results showed that, as metal/metal oxide nanoparticle mass fraction increases, sol stability decreases then restabilises. For systems composed of oppositely charged polysaccharide and metallic nanoparticles (i.e., cellulose nanocrystal/magnetite nanoparticles and cellulose nanocrystal/copper nanoparticle hybrid systems), the small potential energy barrier that exists between hybrid nanoparticles is either lowered, meaning that hybrid particles require less energy for homoaggregation, or shifts to the left, which means that attractive forces persist over longer distances and the likelihood of homoaggregation increases.

Figure S6 - Dependence of CNC-Fe3O4NP interaction energy on KCl concentration.

Figure S7 - Effect of increasing KCl concentration on CNC/CuNP hybrid system.

Comparable results were observed for systems composed of similarly charged polysaccharide and metal/metal oxide nanoparticles. As metal/metal oxide nanoparticle mass fraction increased, sol stability decreased and then restabilised. However, instead of a small potential energy between hybrid nanoparticles, as was observed when oppositely charged nanoparticles interacted with each other, there exists a secondary minimum for like-charged particles. Regardless, as electrolyte concentration increases, the secondary minimum either shifts to the left, meaning that attractive forces persist over longer distances and homoaggregation is more likely or the amount of energy required is lowered and coagulation is more likely.

0.5 -, 0.0 o -0.5

T*

-1.0 -1.5 -I

Figure S8 - Dependence of CNC-AuNP interaction energy on KCl concentration.

Figure S9 - Effect of increasing KCl concentration on ChNC/CuNP hybrid system.

50 100 150 200 250 300 350 100

h (nm)

In the fifth chapter, the physicochemical properties of the hybrid systems are analysed using SEM, XRD, FTIR, AFM, and EDX, which were used to investigate how the polysaccharide and metal/metal oxide nanoparticles interacted with each other within the hybrid. Hybridisation was found to have minimal effect on the constituents of the different hybrid systems; however, the polysaccharide nanoparticles were found to stabilise the metallic nanoparticles, preserving their catalytical and optical activities.

The sixth chapter considers potential applications of the hybrid systems. The cellulose nanocrystal/magnetite nanoparticle hybrid system was proposed as a potential hyperglycaemia monitor because magnetite nanoparticles can decompose hydrogen peroxide, which is a by-product of glucose degradation. Glucose oxidase and 2,2'-azino-bis[3-ethylbenzothiazoline-6-sulphonic acid] (ABTS) were incorporated in the system to create a self-indicating testing strip capable of detecting glucose concentrations as low as 5 mmol/L. The system shows very high catalytic activity at pH 2 and at body temperature (37 °C); therefore, it can be used to monitor glucose levels in sweat and saliva, which tend to be slightly acidic. The system made from sulphated cellulose nanocrystals was more catalytically active, which was attributed to the presence of sulphate groups on the surface. Compared to other glucose monitoring systems, the cellulose nanocrystal/magnetite nanoparticle hybrid system is an inexpensive, quick, painless, self-indicating method for monitoring hyperglycaemia.

0 10 20 30 40 50 Glucose concentration (mM)

Figure S10 - Dependence of 5 wt.% Fe3O4NP N-CNC/Fe3O4NP (grey) and S-CNC/Fe3O4NP (red) catalytic activity on glucose concentration.

During AFM analysis, the cellulose nanocrystal/gold nanoparticle hybrid system showed high hygroscopicity; thus, it was proposed as a potential humidity sensor. The hygroscopic nature of the hybrid was attributed to the gold nanoparticles that were prepared from chloroauric acid, which is famously hygroscopic. The laser diffraction method showed that the cellulose matrix is active and participates in sensing. Here, water molecules adsorb onto and desorb from the surface of the cellulose matrix as humidity increases and decreases. Laser diffraction also showed that the system is reversible and autonomous.

Figure S11 - Laser diffraction pattern of 3 wt.% AuNP CNC/AuNP hybrid film (a) at 0 min, (b) 1 min into 90% humidity treatment, (c) 2 min into 90% humidity treatment, (d) 1 min after humidity treatment, and (e) 2 min after humidity treatment. The size of each square was 17 x 17 mm.

Fluorescence quenching was used to determine the effect of relative humidity on sensing. The optical signal showed a linear dependence on humidity, which is preferable for obtaining sensors due to ease of calibration and less uncertainty in scaling the output signal. The hybrid was determined to have an operating range between 35% and 80% relative humidity, which is typical for humidity sensors.

Figure S12 - Decrease in fluorescence intensity with increasing humidity for the N-CNC/AuNP hybrid (grey) and pure CNC (red) films doped with fluorescent Rhodamine B dye.

Finally, the cellulose nanocrystal/copper nanoparticle and chitin nanocrystal/copper nanoparticle hybrid systems were proposed as potential antibacterial agents. The hybrids were effective against both gram-negative Escherichia coli and gram-positive Bacillus subtilis bacteria, with a minimum inhibitory concentration of 0.4 ^g/mL and a minimum bactericidal concentration of 1.6 ^g/mL. The hybrids inhibited the growth of E. coli within 18 h and B. subtilis within 36 h. Efficacy against gramnegative bacteria was also shown 24 h after treatment; however, efficacy decreased after 24 h against gram-positive bacteria. SEM analysis was used to postulate a potential mechanism of action. Several studies have shown that copper nanoparticles tend to release copper (II) ions in solution. The micrographs showed pitting in the bacteria cells and completely lysed cells after treatment with the hybrids. Thus, it was hypothesised that the nanoparticles penetrate the bacteria cell wall and membrane while copper (II) ions are transported across the membrane via channel proteins, disrupting bioprocesses, damaging biomolecules, and resulting in eventual cell lysis.

Figure S13 - Potential mechanism for PNP/CuNP bactericidal activity.

The chitin nanocrystal/copper nanoparticle hybrid system appeared to be more active compared to the cellulose nanocrystal/copper nanoparticle system, which was expected since several studies have shown that chitin has antibacterial properties. However, the sulfated cellulose nanocrystal system was more potent than the acetylated cellulose nanocrystal system due to the presence of highly active sulphate groups on its surface. Compared to other metal/metal oxide nanoparticle bactericides, the hybrid is a potent antibacterial agent, with competitive minimum inhibitory and minimum bactericidal concentrations.

Thus, this thesis is devoted to the fabrication of functional polysaccharide nanoparticle/metallic nanoparticle hybrid systems for practical applications. In the dissertation, semiempirical calculations are used to determine particle pair interaction energies between similarly and oppositely charged polysaccharide and metal/metal oxide nanoparticles, predictive models are proposed to determine appropriate

hybridisation methods, the effect of organic-inorganic component ratio and electrolyte concentration on hybrid sol stability are investigated, the physicochemical properties of the resulting hybrid films and freeze-dried powders are analysed, and potential applications of the resulting hybrid systems are suggested.

Summary of the main results:

A physicochemical model that predicts the formation of hybrid structures due to interparticle interactions between polysaccharide and metal/metal oxide nanoparticles is described. The model is based on semiempirical calculations of particle interaction energies during the coagulation process, which allow the probability of interaction between metal/metal oxide and polysaccharide nanoparticles to be estimated. The decisive role of long-range forces in the solution has been established, which are based on the occurrence of disjoining pressure and distribution of nanoparticles in the solution volume. The surface charges of metal/metal oxide and polysaccharide nanoparticles were found to determine the synthetic method. The interaction between oppositely charged metal/metal oxide and polysaccharide nanoparticles is accompanied by the absence of a potential energy barrier. It is shown that interparticle interaction between oppositely charged nanoparticles is determined by electrostatic attraction. For hybrid systems based on metal/metal oxide and polysaccharide nanoparticles, the areas of aggregative stability and component ratios for the formation of hybrid structures due to electrostatic interactions and hydrogen bonding are determined. Conversely, interaction between similarly charged metal/metal oxide and polysaccharide nanoparticles is accompanied by a high potential energy barrier due to electrical repulsive forces. Therefore, a methodical approach was used to form hybrid systems based on similarly charged metal/metal oxide and polysaccharide nanoparticles - hydrothermal treatment was used to overcome the energy barrier and facilitate heteroaggregation. In one case, hydrothermal treatment was also used to stabilise metal nanoparticles and prevent oxidation. Hybrid systems based on cellulose nanocrystals/magnetite nanoparticles, cellulose nanocrystals/gold nanoparticles, cellulose nanocrystals/copper nanoparticles, and chitin nanocrystals/copper nanoparticles were obtained using the proposed models.

The physicochemical properties of hybrid systems based on oppositely charged magnetite nanoparticles and cellulose nanocrystals were studied. The results showed that the synthesis method had minimal effect on the components of the system and that the resulting material retained the properties of its components. Magnetite nanoparticles are biomimetic and mimic the action of peroxidase enzymes and cellulose is biocompatible; thus, the system is proposed for use as a potential means of monitoring high glucose levels in biological fluids. The system can be used at body temperature (37 °C) and low pH, and has a glucose concentration detection limit of 5 mmol/L, indicated by an almost instantaneous colour change from white to blue. Blood glucose levels greater than 11.6 mmol/L are considered high; however, other body fluids such as sweat and saliva typically have lower glucose levels, and glucose levels in saliva and sweat greater than 5 mmol/L are considered high. Compared to other glucose monitoring devices, the proposed system has a high detection limit (5 mmol/L versus < 2 ^mol/L); however, these devices are designed to monitor hypo- and hyperglycemia, whereas the proposed system based on cellulose and magnetite nanocrystals only monitors high glucose levels. Furthermore, other glucose monitoring devices require expensive specialized test strips that produce electrical signals that can only be interpreted by reader devices, whereas cellulose/magnetite nanocrystal-based strips are inexpensive and self-indicating. Thus, the proposed system is a suitable alternative for monitoring glucose levels in non-blood biological fluids such as sweat and saliva, which tend to be slightly acidic and have lower glucose concentrations than blood or urine.

The physicochemical properties of hybrid systems based on similarly charged gold nanoparticles and cellulose nanocrystals after treatment at elevated temperature and pressure were studied. This approach makes it possible to overcome the energy barrier caused by repulsive forces and increase the contact area between the system components due to the formation of hydrogen bonds. Physicochemical analysis showed that while hydrothermal treatment stabilized the gold nanoparticles, it had a negligible effect on the system components. The system was also found to be highly hygroscopic, which prompted it to be proposed for use as a humidity sensor. The sensor, which is optically active, showed a linear dependence of the optical signal on humidity and can operate

between 35% and 80% relative humidity. The sensor is autonomous and reversible; therefore, it can operate independently and monitor increases and decreases in ambient humidity. Compared to other humidity sensors, the operating range of the system based on cellulose nanocrystals and gold nanoparticles is typical, and the response/recovery time is comparable to conventional systems. The undoubted advantage of the system is that it is reversible and autonomous.

Interactions between oppositely charged cellulose nanocrystals and copper nanoparticles as well as similarly charged chitin nanocrystals and copper nanoparticles were predicted using the model proposed in this thesis. Hydrothermal treatment was used to overcome the high potential energy barrier that exists between similarly chitin nanocrystals and copper nanoparticles. While an electrostatic attraction existed between the oppositely charged copper nanoparticles and the cellulose nanocrystals, the copper nanoparticles were unstable and readily oxidized to copper (I) oxide. Therefore, hydrothermal treatment was also used to treat the cellulose nanocrystal/copper nanoparticle hybrid system. The results showed that hydrothermal treatment helped to stabilise the copper nanoparticles. The physicochemical properties of hybrid systems based on chitin nanocrystals/copper nanoparticles and cellulose nanocrystals/copper nanoparticles were studied. The results showed that hydrothermal treatment stabilised the copper nanoparticles and had little effect on the components. Copper nanoparticles are known for their antibacterial activity, they inhibit bacterial growth; thus, the hybrid systems are proposed as a potential antibacterial agents and bactericides. The hybrids prevent visible growth of both gram-negative and gram-positive bacteria and are potent with minimum inhibitory concentrations of 0.4 ^g/mL. The hybrid systems showed inhibitory activity against gram-negative and gram-positive bacteria for 18 and 36 h, respectively. The systems were also potent bactericides with minimum bactericidal concentrations of 1.6 ^g/mL. The nanoparticles penetrated the cell walls and cell membranes of both gram-negative and gram-positive bacteria and caused cell lysis within 36 h. Subculturing showed that the hybrid systems killed 99.9% of gram-negative bacteria and prevented their growth within 24 h; however, although 99.9% of grampositive bacteria were observed to die, its effectiveness decreased within 24 h, and 0.1%

of the bacteria that were not destroyed began to multiply. Compared with other metal/metal oxide nanoparticle-based antibacterial agents, these hybrid systems exhibit very strong bactericidal and inhibitory effects against both gram-negative and grampositive bacteria, and have minimum inhibitory and bactericidal concentration comparable to those of silver nanoparticles. Publications.

The key research results are described in four publications, all of which were in first quartile international journals indexed in Scopus and Web of Science. A1. Torlopov, M.A., Martakov, I.S., Mikhaylov, V.I., Legki, P.V., Golubev, Y.A., Krivoshapkina, E.F., Tracey, C.T., Sitnikov, P.A., Udoratina, E.V. Manipulating the colloidal properties of (non-)sulfated cellulose nanocrystals via stepwise cyanoethylation/carboxylation. European Polymer Journal 115. p. 225-233. (2019).

A2. Koroleva, M.S., Tracey, C.T., Sidunets, Y.A., Torlopov, M.A., Mikhaylov, V.I., Krivoshapkin, P.V., Martakov, I.S., Krivoshapkina, E.F. Environmentally friendly Au@CNC hybrid systems as prospective humidity sensors. RSC Advances 10(58). p. 35031 - 35038. (2020). A3. Tracey, C.T., Torlopov, M.A., Martakov, I.S., Vdovichecko, E.A., Zhukov, M., Krivoshapkin, P.V., Mikhaylov, V.I., Krivoshapkina, E.F. Hybrid cellulose nanocrystal/magnetite glucose biosensors. Carbohydrate Polymers 247. p. 116704. (2020).

A4. Tracey, C.T., Predeina, A.L., Krivoshapkina, E.F., Kumacheva, E. A 3D printing approach to intelligent food packaging. Trends in Food Science & Technology. (2022).

Introduction

Relevance of the topic.

The search for environmentally friendly strong, lightweight, active materials has led to the development of hybrid materials. Hybrid materials are composite materials with at least one inorganic and one organic component that both occur on the nanoscale or at the molecular level. Since the components are on the nanoscale, hybrid materials exhibit physical and chemical properties atypical of bulk materials. Furthermore, the inorganic and organic components interact in such a manner that enhanced or entirely new physicochemical properties occur. The unique synergistic effects between the organic and inorganic components have prompted researchers to study hybrid materials and exploit their physicochemical properties in applications across several industries.

Inorganic metallic nanoparticles respond to electric and magnetic fields and are often optically and catalytically active; as such they have major roles in sensing, imaging, and optoelectronics. However, metallic nanoparticles tend to aggregate, which results in reduced activity. Materials such as silica, titania, and synthetic polymers are often used to stabilise metallic nanoparticles. Recently, research has shifted to more environmentally friendly alternative supports, namely biopolymers. Biopolymers are polymers that originate from living organisms and include nucleic acids, lipids, proteins, and polysaccharides. Because of their abundance, highly replenishable resources, and low environmental impact, polysaccharide nanoparticles have been widely researched. Due to their high tensile strengths, high elastic moduli, high specific surface area, optical transparency, and chemical reactivity, cellulose and chitin nanocrystals have been used as matrices in many hybrid materials. This unique combination leads to the development of biodegradable, biocompatible, active hybrid systems.

The relevance of this dissertation lies in the development of functional metallic nanoparticle/polysaccharide nanoparticle hybrid systems that can find real-world application. This study investigates the interactions between the metallic and polysaccharide nanoparticles and uses the Derjaguin-Verwy-Landau-Overbeek (DLVO) theory to explain how factors such as surface functional groups, surface charge, metallic nanoparticle mass fraction, and electrolyte concentration affect the colloidal-chemical properties of metallic nanoparticle/polysaccharide nanoparticle hybrid systems. The impact of hybridisation on the structures of the components is also investigated, and the physicochemical properties of the resulting hybrids are analysed and characterised. Finally, several areas of application for the developed hybrid systems are proposed. Goal of research.

The aim of the thesis is to develop physical and chemical bases for obtaining hybrid systems based on polysaccharide nanoparticles and magnetite, gold or copper; to study the mechanisms of interparticle interaction of similarly and oppositely charged nanoparticles in colloidal systems; to establish the relationship between the structure of the obtained materials and optical, catalytical, and biochemical properties. Research objectives.

In order to achieve the goal in the framework of the thesis, the following objectives have been established:

• To develop synthetic methods for the fabrication of hybrid systems that ensure the interaction of similarly and oppositely charged metallic and polysaccharide nanoparticles.

• To conduct a comprehensive study of the colloidal-chemical properties of pure and hybrid dispersions based on metallic and polysaccharide nanoparticles and their aggregative stability as well as study the mechanisms of formation of hybrid systems.

• To assess interparticle interactions between metallic and polysaccharide nanoparticles in colloidal systems based on classical and generalized interaction theories that allow the presence or absence of particle heteroaggregation to be predicted.

• To study the composition, morphology, and physicochemical properties of the obtained hybrid materials and to establish the peculiarities of their formation processes, including those under hydrothermal treatment.

• To establish the relationship between the composition and structure of hybrid materials and their optical, catalytic and biochemical properties.

The novelty of research

For the first time, the regularities of the formation of hybrid systems based on metallic and polysaccharide nanoparticles were established, depending on the ratio of components, surface charge, and the composition of the dispersion medium.

It has been shown that electrostatic attraction governs interparticle interactions between oppositely charged nanoparticles. For hybrid systems based on metallic and polysaccharide nanoparticles, the areas of aggregative stability and component ratios in the formation of hybrid structures due to electrostatic interactions and the formation of hydrogen bonds have been determined.

A mechanism for the formation of hybrid systems based on similarly charged particles by overcoming the energy barrier caused by repulsive forces via hydrothermal treatment is proposed, which makes it possible to predict the properties of systems with specified parameters.

A physicochemical model that predicts the formation of hybrid structures based on interparticle interactions between metallic and polysaccharide nanoparticles is proposed. The model is based on semi-empirical calculations of particle pair interaction energies during coagulation, which allow the probability of the interaction between metallic and polysaccharide nanoparticles to be estimated.

The mechanisms of heteroaggregation between oppositely charged magnetite nanoparticles and cellulose nanocrystals have been studied. The catalytic activity of the resulting hybrid material in the glucose oxidation reaction was investigated, and the effect of temperature, pH, time, and glucose concentration were examined. Due to high catalytic activity at low pH and body temperature as well as a 5 mM limit of detection, a non-invasive biosensor for determining the level of glucose in biological fluids such as sweat and saliva was obtained.

The physicochemical properties of hybrid systems based on similarly charged gold nanoparticles and cellulose nanocrystals after processing at elevated temperature and pressure have been studied. This approach allows the energy barrier caused by repulsive forces to be overcome and increases the area of contacts due to the formation of hydrogen bonds. Due to the hygroscopic nature of the hybrid, it was proposed for application as a humidity sensor. The sensor shows a linear dependence on humidity and can operate between 35% and 80% relative humidity.

The interactions between oppositely charged cellulose nanocrystals and copper nanoparticles as well as similarly charged chitin nanocrystals and copper nanoparticles were predicted using the model proposed in this thesis. Hydrothermal treatment was used to overcome the high potential energy barrier that exists between chitin nanocrystals and copper nanoparticles. While electrostatic attraction existed between the oppositely charged copper nanoparticles and cellulose nanocrystals, the copper nanoparticles oxidized to copper (I) oxide. Therefore, hydrothermal treatment was also used to process the cellulose nanocrystal/copper nanoparticle hybrid system. The results showed that hydrothermal treatment helped to stabilise copper nanoparticles.

The physicochemical properties of the chitin nanocrystal/copper nanoparticle and cellulose nanocrystal/copper nanoparticle hybrid systems were studied. The hybrids are effective against gram-negative and gram-positive bacteria and are very potent (minimum inhibitory concentration = 0.4 ^g/mL; minimum bactericidal concentration = 1.6 ^g/mL). The hybrids can be used to inhibit the visible growth of and eradicate gramnegative and gram-positive bacteria.

Theoretical and practical significance of the research

The theoretical significance of the conducted research lies in the evidence that the surface charges of the metallic and polysaccharide nanoparticles, metallic nanoparticle mass fraction, and electrolyte concentration affect the colloidal-chemical properties of metallic nanoparticle/polysaccharide nanoparticle hybrid systems and that they can be manipulated and optimised in order to obtain functional hybrid systems.

The practical significance of the conducted research lies in the development of functional metallic nanoparticle/polysaccharide nanoparticle hybrid systems that can

find application as sensors and bactericides in the biomedical, pharmaceutical, and food industries. This shows that metallic and polysaccharide nanoparticles can be hybridized together to create functional materials. There is a plethora of metallic and polysaccharide nanoparticles, which can lead to the development of countless new functional materials.

Assertations presented for the defence

1. A physicochemical model has been proposed to estimate the probability of interaction of metallic nanoparticles with the surface of polysaccharide nanoparticles. The decisive role of long-range forces in solution has been established which are based on the occurrence of disjoining pressure and nanoparticles distribution in solution volume.

2. The interaction between oppositely charged metallic and polysaccharide nanoparticles is accompanied by the absence of a potential energy barrier and takes place by the mechanism of irreversible heteroaggregation.

3. The interaction of similarly charged metallic and polysaccharide nanoparticles is accompanied by the presence of a high potential energy barrier due to electric double layer repulsive forces.

4. A methodological approach to the formation of hybrid systems from similarly charged metallic and polysaccharide nanoparticles through hydrothermal treatment to overcome the energy barrier and facilitate heteroaggregation.

5. Physicochemical basis for the production of hybrid materials intended for monitoring high blood glucose levels, humidity sensing, and application as an antibacterial agent and/or bactericide.

Approbation of research results.

Key research results were presented and discussed at the following conferences: Congress of Young Scientists. ITMO University, Russia (2019); XX International SolGel Conference, Russia (2019); XIII International Competition-Conference of Scientific Works for Students named A.A. Yakovkin, Russia (2019); Congress of Young Scientists, ITMO University, Russia (2020); Congress of Young Scientists, ITMO

University, Russia (2021); International Conference on Advanced Plasmonics, Magnetics & Magneto-Optical Technologies, Canada (2022).

The accuracy of the obtained results is achieved through the use of verifiable and reliable synthetic methods, characterisation techniques, and theoretical and computational models that are recognised by and accepted throughout the scientific community. Additionally, the results were validated during the publication process where scientific peers examined and reviewed the work that was presented. Publications.

A1. Torlopov, M.A., Martakov, I.S., Mikhaylov, V.I., Legki, P.V., Golubev, Y.A., Krivoshapkina, E.F., Tracey, C.T., Sitnikov, P.A., Udoratina, E.V. Manipulating the colloidal properties of (non-)sulfated cellulose nanocrystals via stepwise cyanoethylation/carboxylation. European Polymer Journal 115. p. 225-233. (2019)

A2. Koroleva, M.S., Tracey, C.T., Sidunets, Y.A., Torlopov, M.A., Mikhaylov, V.I., Krivoshapkin, P.V., Martakov, I.S., Krivoshapkina, E.F. Environmentally friendly Au@CNC hybrid systems as prospective humidity sensors. RSC Advances 10(58). p. 35031 - 35038. (2020) A3. Tracey, C.T., Torlopov, M.A., Martakov, I.S., Vdovichecko, E.A., Zhukov, M., Krivoshapkin, P.V., Mikhaylov, V.I., Krivoshapkina, E.F. Hybrid cellulose nanocrystal/magnetite glucose biosensors. Carbohydrate Polymers 247. p. 116704. (2020)

A4. Tracey, C.T., Predeina, A.L., Krivoshapkina, E.F., Kumacheva, E. A 3D printing approach to intelligent food packaging. Trends in Food Science & Technology. (2022)

Thesis structure and number of pages

This thesis is a total of 225 pages and consists of an introduction, a literature review, six chapters, a conclusion, and a list of references. There are a total of 79 figures, 6 tables, and 163 references.

Chapter 1. Literature review

1.1. Hybrid materials

Hybrid materials are a special type of composite material that consist of at least one organic and one inorganic component that both occur on the nanoscale or at the molecular level [68, 69, 122]. The combination of organic and inorganic compounds typically has a synergistic effect, resulting in novel materials with entirely new or enhanced exploitable physicochemical properties [69]. The inorganic component of a hybrid material is active while the organic component is biodegradable, biocompatible, non-toxic, replenishable, and may be active or passive. Due to the characteristics of the organic component, hybrid materials often find applications in the medical and biomedical fields as well as in the food and cosmetics industries.

Hybrid materials are both naturally occurring (e.g., pearls, bones, and teeth) and manmade, and include materials consisting of a matrix and (sub)micron-level dispersions as well as submicron-level mixtures of various materials formed through physical interactions or chemical bonds [102]. Hybrid materials are highly varied and range from crystalline solids to amorphous sol-gel compounds.

There are two main approaches to categorising hybrid materials: hybrids can be grouped based on the type of modification/structural properties or based on the interactions between the organic and inorganic components. When grouped according to type of modification, hybrid materials can be classified as organic molecule-modified inorganic materials (Figure 1.1) and inorganic modified organic materials (Figure 1.2). Organic molecule-modified inorganic materials can be further divided into two groups: inorganic structures that have been modified by organic molecules and colloidal inorganic nanoparticles that have been stabilised through functionalisation with organic molecules. Regardless of the type, within these hybrids, the organic moiety contains a functional group that allows its attachment to the inorganic network [68].

Figure 1.1 - Organic molecule-modified inorganic nanoparticle. Reproducedfrom [26].

Inorganic-modified organic materials, however, are those where active inorganic nanoparticles are dispersed throughout an organic matrix [12]. The organic matrix is passive or active, flexible, biodegradable, and biocompatible while the inorganic nanoparticles contribute mechanical strength as well as optical, magnetic, and/or conductive properties to the hybrid material. Inorganic-modified organic materials are studied in this dissertation. Hybrid materials can also be categorised according to the type of interaction. Depending on the type of interactions that exist between the inorganic and organic components, hybrid materials may be classified as Class I hybrids or Class II hybrids (Figure 1.2).

Figure 1.2 - Class I (top) and Class II (bottom) hybrids. Reproduced from [93].

Class I hybrids are either blends or interpenetrating networks (Figure 1.2, top). In a typical blend, inorganic nanoparticles are trapped within a crosslinked organic polymer matrix. Contrarily, interpenetrating networks are formed when there is simultaneous crosslinking between the organic components and the inorganic components. Here, as the organic matrix forms, the inorganic particles form their own network simultaneously, resulting in the organic matrix and the inorganic network being intertwined. Class I hybrids have weak interactions between the organic and inorganic components. These weak interactions are usually physical in nature and include forces such as van der Waal forces, hydrogen bonding, and electrostatic attraction (Figure 1.3).

Figure 1.3 - Weak physical forces that exist between Class I hybrids: (A) van der Waals interactions, (B) hydrogen bonding, and (C) electrostatic attraction.

When highly electronegative atoms are in proximity with less electronegative atoms, otherwise neutrally charged particles form induced dipoles. In some instances, the significant difference in electronegativity of covalently bound atoms form permanent dipoles. Van der Waals forces are weak physical interactions that exist between the induced or permanent dipoles of molecules in proximity. There are three types: London

dispersion forces, Debye forces, and Keesom forces (Figure 1.3A). London dispersion forces are those in which two induced dipoles interact with each other; when induced dipoles interact with permanent dipoles, it is known as Debye forces; and Keesom forces describe two permanent dipoles interacting with each other. Regardless of the type of dipole-dipole interaction, van der Waals forces can be attractive or repulsive, with the nature of the force depending on the distance between the two interacting molecules. Usually, the intermolecular forces are attractive but, as the distance between the molecules lessens, and if there are no other forces present, the similarly charged electron clouds surrounding each atom being too close to each other results in repulsion. Hydrogen bonding (Figure 1.3B) is another type of physical interaction that occurs in hybrid materials. A special type of dipole-dipole interaction, hydrogen bonding is a primarily electrostatic attractive force that occurs between a positively charged hydrogen atom in one molecule and a very electronegative atom in another molecule. Hydrogen bonds are, in general, stronger than van der Waals forces. Finally, electrostatic attraction (Figure 1.3C) occurs when the inorganic and organic nanoparticles are oppositely charged. The positively charged and negatively charged particles are attracted to each other, leading to hybridisation.

In Class I hybrid materials, both the organic and inorganic constituents possess some specific functional group that cause the components to physically interact with each other. These physical interactions influence how the particles arrange themselves within a hybrid material in a process known as self-assembly. Self-assembly occurs when nanoparticles and/or molecules spontaneously convene into well-organised larger units [61, 105]. How particles interact with their environment also influences their arrangement. During self-assembly, the particles organise themselves into the configuration with the lowest energy. A well-known and deeply studied example of self-assembly is the phospholipid bilayer of animal cell membranes (Figure 1.4). Although it is generally an intrinsic process, external forces (e.g., applied electrical field, applied magnetic field) may be used to influence self-assembly. Regarding hybridisation, these physical interactions lead to the formation of homogeneous hybrid materials. The strength of the intermolecular forces impacts the structural order of the hybrid. If there

are no functional groups present on the organic and/or inorganic constituents, incomplete homogeneity occurs and there is a phase separation between the organic and inorganic components.

Figure 1.4 - Self-assembled phospholipid bilayer of an animal cell membrane.

Reproducedfrom [162].

Class II hybrids, however, are formed when discrete inorganic blocks are bonded to organic polymers or when inorganic and organic polymers are (usually) covalently connected to each other (Figure 1.2, bottom). The development of chemical bonds between the organic and inorganic components in Class II hybrid materials is analogous to the formation of hybrid molecular orbitals. Unlike Class I hybrid materials, Class II hybrids have strong interactions between the organic and inorganic phases. While the interactions in Class I hybrids are physical, the interactions in Class II hybrids are chemical in nature and include interactions such as ionic bonding, coordinate bonding, and, mainly, covalent bonding (Figure 1.5).

Figure 1.5. - Strong chemical interactions that exist between Class II hybrids: (A) ionic bonding, (B) covalent bonding, and (C) coordinate (covalent) bonding.

During ionic bonding, electrons are transferred from one atom to another. An exceptionally large electron affinity disparity exists between the atoms involved and atoms with low electron affinity donate electrons to atoms with high electron affinity, forming cations (positively charged ions) and anions (negatively charged ions), respectively (Figure 1.5A). When atoms have similar electron affinity, however, they tend to share electrons instead, forming covalent bonds where each atom donates a single electron to the shared electron pair (Figure 1.5B). There is a special type of covalent bonding known as coordinate covalent bonding. In this type of chemical bond, the atoms involved share an electron pair but, unlike a typical covalent bond, one atom donates both electrons (Figure 1.5C).

Chemical interactions between the organic and inorganic components are preferred because this leads to the hybrid being very stable and highly homogeneous [69]. The organic and inorganic constituents usually have at least two distinct functional groups: alkoxy groups (R-OM bonds) which allow hydrolysis-condensation reactions in the presence of water and allow for the formation of the polymer network; and metal-to-carbon links which are stable during these hydrolysis-condensation reactions [63].

However, in the case of many hybrids, weak physical interactions exist between the organic and inorganic nanoparticles, leading to less stable hybrids.

Organic-inorganic hybrid materials are more than a simple combination of organic and inorganic materials, and are an entirely new category of materials whose properties are governed by the interactions between the constituents [102]. These interactions result in hybrid materials displaying excellent properties on the macroscopic scale. Inorganic nanoparticles/molecules are hard (i.e., strong and brittle), and have high refractive indices, high densities, high conductivities, high thermal stabilities, and good magnetic properties [105]. Organic nanoparticles/molecules tend to be soft (i.e., flexible and elastic), and have low refractive indices, low densities, poor conductivities, low thermal stabilities, and poor magnetic properties [61]. The noticeably dissimilar properties of the organic and inorganic components can be favourably combined to produce a virtually unlimited number of hybrid materials with a wide range of potential properties and possible applications. This is important because the materials available today (i.e., ceramics, metals, synthetic polymers, and biopolymers) can no longer cultivate technological advancement. The need for more flexible, stronger, more durable, yet lighter functional materials has fostered the development of hybrid materials [9]. Another major advantage of hybrid materials is the possibility of creating multifunctional materials through the incorporation of inorganic clusters and nanoparticles with specific optical, electronic, and/or magnetic properties into organic polymer matrices [68].

Due to current environmental constraints, naturally occurring biopolymers have become increasingly popular organic constituents of hybrid materials instead of synthetic polymers derived from petroleum [49]. Biopolymers, which include nucleic acids, lipids, proteins, and polysaccharides, have several environmentally friendly properties. These polymers are obtained from living organisms (i.e., plants, animals, bacteria, and fungi) and therefore have highly replenishable natural resources. They are also biodegradable, biocompatible, and non-toxic, properties that are passed on to materials that are made from them. Polysaccharides are of particular interest because of their excellent physicochemical properties, namely low thermal expansion coefficients,

high mechanical strengths, high tensile strengths, high specific surface areas, high elastic moduli, optical transparency, and high chemical reactivities [16].

1.2. Polysaccharides

Polysaccharides are the most abundant biopolymers in the biosphere and include glucose, starch, glycogen, phytoglycogen, cellulose, chitin, and their derivatives [5]. Also known as carbohydrates, polysaccharides consist of thousands of monosaccharide units joined via glycosidic bonds, resulting in polymers with structures ranging from linear to highly branched. Polysaccharides are important biopolymers as they have several functions in living organisms, usually structure- or storage-related. Glucose, starch, glycogen, and phytoglycogen are energy storage materials while cellulose and chitin are structural polysaccharides. Due to their high tensile strengths, high Young's moduli, excellent dispersibility in water, hydrophilicity, and good stabilities, cellulose and chitin nanoparticles were hybridised with metallic nanoparticles (MNPs) in this dissertation.

1.2.1. Cellulose nanocrystals

Although primarily associated with plants, cellulose is also produced by bacteria (e.g., Gluconacetobacter sp., Sarcina sp., and Agrobacterium sp.), fungi (e.g., Ascomycetes, Basidiomycetes, and Deuteromycetes), and animals (e.g., Urochordates sp.), making it the most abundant biopolymer, and therefore the most abundant polysaccharide, in the biosphere [72]. Tough and insoluble yet hydrophilic, cellulose plays a vital role in maintaining the structure of plant cell walls and animal exoskeletons [45]. However, it also serves as a nutrition source for fungi and helps bacteria to attach to other organisms, float, and maintain very specific ambient environments [10]. An organic compound with molecular formula (C6H10O5)n, cellulose is a long-chain polysaccharide that has glucopyranose (D-glucose) monomers joined together by P-1,4-glycosidic bonds, with every other monomer being inverted 180° with respect to its neighbour (Figure 1.6) [45]. As Figure 1.6 shows, each monomer unit has three hydroxyl groups, which gives cellulose some of its more notable properties, including chirality, biodegradability, hydrophilicity, and high chemical reactivity [72].

Furthermore, the physical interactions between these hydroxyl groups and adjacent oxygen atoms, namely hydrogen bonding, hold the polymer chains together to form elementary fibrils which then aggregate to form microfibrils with high tensile strength [54].

Figure 1.6. - Cellulose structural unit.

Cellulose-based materials such as cotton, wood, and hemp have found several engineering and societal applications for millennia. However, today, due to knowledge of the quantum effect and the distinctly different physicochemical properties of nanoparticles from their bulk counterparts, the use of nanocellulose, particularly cellulose nanocrystals, has become more ubiquitous. Cellulose fibrils are highly crystalline, especially compared to other polysaccharide biopolymers such as starch and glucose. There are regions where cellulose chains are highly ordered (crystalline) and other regions where the arrangement of cellulose chains is highly disordered (amorphous-like) within cellulose fibrils [99]. To obtain cellulose nanocrystals (CNC), the crystalline regions of the cellulose nanofibrils are extracted (Figure 1.7).

Due to the extraction method, CNC simultaneously undergo surface functionalisation. The most common CNC synthetic method is sulphuric acid hydrolysis; however, in recent years, other acids such as hydrochloric, phosphoric, hydrobromic, and glacial acetic acids have been used [142]. Depending on the type of acid used during extraction, the CNC surface will have different functional groups, which will affect physical properties such as colloidal and thermal stability, morphology, density, and crystallinity [142]. Additionally, post-synthesis surface modification can be performed

due to the presence of the -OH groups. Regardless of preparation method, CNC are biodegradable, biocompatible, non-toxic, and very hydrophilic. They also tend to have large surface area-to-volume ratios, high tensile strength, high Young's modulus, and excellent dispersibility, making them ideal for use in many areas that require such properties, including drug delivery, tissue scaffolding, wound healing, and food packaging.

Figure 1.7 - Separation of crystalline and amorphous phases in cellulose microfibrils via acid hydrolysis during cellulose nanocrystal synthesis. Reproduced from [94].

1.2.2. Chitin nanoparticles

Chitin is the second most abundant polysaccharide in the biosphere [128]. Chitin is found in the exoskeletons of invertebrates and is the material responsible for crustaceans and insects having tough outer bodies. Like cellulose, chitin is also found in the cell walls of some fungi (e.g., Aspergillus, Agaricus, Boletus, and Phycomyces) [28]. An organic compound with molecular formula (C8H13O5N)n, chitin is a long-chain polysaccharide that has N-acetylglucosamine monomers joined together by P-1,4-glycosidic bonds, with every other monomer being inverted 180° with respect to its neighbour (Figure 1.8). N-acetylglucosamine is an amide derivative of glucose. As Figure 1.8 shows, each monomer unit has two hydroxyl groups, which gives chitin some

of its more notable properties, including chirality, biodegradability, hydrophilicity, and high chemical reactivity. Furthermore, the physical interactions between these hydroxyl groups and adjacent oxygen atoms, namely hydrogen bonding, hold the polymer chains together to form elementary fibrils which then aggregate to form microfibrils with high tensile strength [103]. Figure 1.8 also shows that the N-acetylglucosamine monomer has an amino group, which is also highly reactive and is often used as a point of modification. However, it is believed that this amino group confers antibacterial properties to chitin; the cationic amino groups likely bind to anionic groups in microorganisms to prevent bacterial growth [36].

Figure 1.8 - Chitin structural unit.

Like cellulose, chitin forms fibrils that have highly ordered (crystalline) and highly disordered (amorphous-like) regions [103]. To obtain chitin nanocrystals (ChNC), the crystalline regions of the chitin nanofibrils are extracted under reflux via hydrochloric acid hydrolysis (Figure 1.9). The chitin nanocrystal surface can be modified post synthesis due to the presence of the -OH and amino groups. ChNC is biodegradable, biocompatible, non-toxic, and very hydrophilic. It also has a large surface area-to-volume ratio, high tensile strength, high Young's modulus, excellent dispersibility, and good antibacterial properties, making it ideal for use in many areas, including drug delivery, tissue scaffolding, wound healing, and food packaging.

CH3

CH3

n

A Prawn shells B Chitin microfibrils C Chitin nanocrystals

Figure 1.9 - Separation of crystalline and amorphous phases in chitin microfibrils via acid hydrolysis during chitin nanocrystal synthesis and the self-assembly of chitin nanocrystals. Reproduced from [103].

1.3. Metallic nanoparticles

Metallic nanoparticles (MNPs) are nanoscale moieties (1 - 100 nm) composed of pure metals or their oxides, chlorides, phosphides, and sulphides. Scaling metals and their derivatives down to the nanoscale introduces quantum effects that, like with polysaccharides and polysaccharide nanoparticles (PNPs), allow MNPs to exhibit physicochemical properties atypical of bulk materials [8, 108]. MNPs tend to have large surface area-to-volume ratios, large surface energies, short range ordering, plasmon excitation, quantum confinement, and a significant number of low-coordination sites. They also tend to have many dangling bonds, which causes them to have specific exploitable chemical properties. Due to their large surface area-to-volume ratios, MNPs are considered excellent catalysts [8]. These physicochemical properties allow them to be used across several fields, including the medical, biomedical, food, and cosmetics industries. In this thesis, magnetite, gold, and copper nanoparticles were used to create hybrid systems.

Gao et al. and Wei & Wang [44, 147, 148] showed that magnetite can mimic peroxidase activity. In living organisms, peroxidases oxidise substrates in the presence of peroxides - particularly hydrogen peroxide [148]. The authors of [148] verified that magnetite nanoparticles have their own peroxidase activity and exhibit important potential applications in numerous fields. Magnetite nanoparticles are also

biocompatible and non-toxic in low doses, allowing their use in the medical and biomedical industries [42].

Due to their unique properties, gold nanoparticles have found applications in medicine [155], optoelectronics [121], and sensing [51, 130], and have attracted considerable attention over the last few decades. Like cellulose nanocrystals and magnetite nanoparticles, gold nanoparticles are biocompatible and have very low toxicity, resulting in them having many biological and chemical applications. Furthermore, gold nanoparticles are one of the most effective catalysts for several important chemical reactions [65]. However, colloidal gold is highly unstable and, due to the low repulsive forces that exist between them, gold nanoparticles tend to aggregate, thereby reducing their catalytic activity and effectiveness. To overcome this and maintain its efficacy, gold nanoparticles are generally supported by more colloidally stable materials such as carbon nanomaterials, silicon oxides, and metal oxides. They are also sometimes encapsulated in synthetic polymers. However, as science and technology continue to embrace more sustainable and environmentally friendly processes, methods for improving colloidal gold stability have also become more ecological. One such method is the use of nanocellulose. In this hybrid, the cellulose nanocrystals act as stabilizing agents, resulting in the formation of stable hybrid systems.

Of the metal and metal oxide nanoparticles that exhibit antimicrobial activity, silver nanoparticles are the most potent [40, 110]. However, silver is very expensive, making its large-scale use impractical. Copper nanoparticles have been found to exhibit comparable antimicrobial activity to that of silver nanoparticles [23]. While copper is an inexpensive and abundant metal, it is highly unstable and easily oxidises to copper (I) oxide, which exhibits reduced antimicrobial activity [29]. As is typical of MNPs, copper nanoparticles also tend to aggregate, which contributes to lower antimicrobial activity [66]. Therefore, copper nanoparticles are often stabilised with other materials to slow oxidation, reduce aggregation, and maintain antimicrobial activity. For example, copper nanoparticle-doped zinc oxide has many dermatological and biomedical applications and is an active ingredient in creams, ointments, and lotions [120]; and Ti-Cu alloys, which have a 90% antibacterial rate, are used as orthopaedic implants [157]. Biocompatible

polymers have also been used to stabilise copper nanoparticles, forming hybrid materials which have several medical and biomedical applications such as dressings for accelerated wound healing, tissue scaffolding, and protective clothing [67, 84, 146].

1.4. Approaches to hybridisation

There are two main approaches to the fabrication of organic-inorganic hybrid materials: the building block approach and in-situ hybridisation. In the building block approach, template building blocks are used as starting units for obtaining hybrid organic-inorganic structures. Although there are several building block approaches to hybridisation, what is consistent throughout is that preformed 'blocks' are made to react with each other to create a hybrid that consists of precursors that either partially or completely maintain their original integrity. A typical block, whose surface structure and composition are known, consists of a well-defined molecule or nanoparticle with well-defined shape and size [96]. Using highly sophisticated methods (e.g., atom substitution), the building block approach allows hybrid materials to be designed at the molecular level [69]. Also known as the bottom-up approach, building blocks are used to hierarchically create complex structures. Using the building block approach, chemists can precisely design a hybrid material with exact properties [63]. However, the building block approach to hybridisation is very complicated, time-consuming, and expensive, resulting in its infrequent use.

The main approach to the fabrication of hybrid materials is in-situ hybridisation. In this approach, well-defined discrete molecules are chemically transformed into complex multidimensional structures. As such, hybrid materials created via the in-situ hybridisation method can have completely different properties from their precursors or possess enhanced precursor properties. Reaction conditions and precursor composition determine the internal structure of the hybrid. Controlling the reaction conditions is therefore vital, since even a slight variation in the methodology can result in completely different hybrids with completely different physicochemical properties. In-situ hybridisation encompasses three approaches: emulsion polymerisation, the blending method, and the sol-gel method. Regarding emulsion polymerisation, this approach to

in-situ hybridisation is an industrial process where inorganic 'seeds' are introduced to the organic component as stabilisers. This results in the formation of composite colloids. The blending and sol-gel methods are more often used.

Blending is the physical mixing of organic and inorganic precursors to form a hybrid. This is usually done under stirring or using sonication. There are three types of blending: solution blending, melt blending, and powder blending [96]. Solution blending is one of the simplest approaches to material hybridisation. Here, the organic material is dissolved in a suitable solvent before the inorganic component is introduced. While solution blending has many advantages including simplicity and cost-effectiveness, the hybrid may possess inferior physical, chemical, and mechanical properties due to particle agglomeration if the inorganic component is not properly dispersed throughout the organic component [96]. While very similar to solution blending, melt blending does not involve organic solvents. Rather, in a more environmentally-friendly process, the inorganic component is dispersed throughout a melt of the organic component [80]. In the powder blending approach, solid-state powders of the organic and inorganic constituents are milled together through a series of energy transfers [21]. During milling, the inorganic powder is broken down into smaller particles which are then incorporated into the emerging organic matrix. This method is suitable for hybrids formed with organic components that have poor solubility or very high melting points, making them unsuited to solution blending and melt blending.

In the sol-gel in-situ approach, a solid-like material is formed from a precursor mix (a sol or a solution) through evaporation, dehydration, and/or chemical crosslinking between the solid particles or dissolved precursors [111]. This method provides a versatile way to prepare functional materials under mild conditions and includes the water-catalysed sol-gel method, nonhydrolytic sol-gel method, and the interpenetrating polymer network approach [25, 96]. In the water-catalysed sol-gel method, organic functional groups and metal alkoxide groups interact to simultaneously form an inorganic network and a polymer chain in the presence of water (Figure 1.2, bottom right). The nonhydrolytic sol-gel approach is remarkably similar to the water catalysed approach, except that the reaction takes place in the absence of a solvent. In this method,

ligand exchange occurs in the presence of a halide or an organic molecule with an oxygen donor moiety. The interpenetrating polymer network approach involves the simultaneous crosslinking of organic and inorganic groups to create the hybrid network (Figure 1.2, top right). Given the advantages and disadvantages of each approach to hybridisation, the sol-gel method is the most viable, which is reflected in how often this particular method is used. In this dissertation, the sol-gel approach was used to obtain both pure and hybrid materials.

1.5. Advantages of polysaccharide nanoparticle/metallic nanoparticle hybridisation

When compared to the materials currently available, hybrid materials have superior physicochemical properties as well as new, unique, and enhanced functions [96]. The physicochemical properties of a hybrid material can either lie between those of its organic and inorganic components or can be entirely new. New physicochemical properties are due to the manifestation of quantum size effects that occur as systems approach the nano- and molecular scale. Sometimes, hybridisation can have a synergistic effect. This occurs when the organic and inorganic nanoparticles/molecules interact in such a manner that an overall effect greater than the sum of their combined individual effects is realised [116].

Like other inorganic nanoparticles, MNPs tend to aggregate in solution, causing them to lose their unique nanoscale properties [35]. In this study, CNC and ChNC are used as stabilising agents. The MNPs are primarily optically, magnetically, and catalytically active while the PNPs make the hybrid biocompatible, biodegradable, and non-toxic. As such, these systems can be used across many industries. However, as mentioned earlier, several reaction conditions, such as the organic-to-inorganic component ratio, pH, temperature, and surface functionalisation, can affect the colloidal-chemical and physicochemical properties of the hybrid system; therefore, these parameters must be carefully chosen and monitored. The physicochemical properties of hybrid films and coatings heavily depend on the colloidal stability of the hybrid system during synthesis.

1.6. Disperse system colloidal stability: the DLVO Theory

In 1941, Derjaguin, Verwy, Landau, and Overbeek postulated that the stability of a colloidal system depends on its total potential energy function U [109]. In a colloidal system, both attractive and repulsive forces exist between particles. The total potential energy of a system is a function of the distance between particles (h) and depends on whether attraction or repulsion is dominant; greater attraction results in aggregation and eventual sedimentation while greater repulsion leads to highly stable systems. Known as the DLVO theory, the theory proposed by Derjaguin, Verwy, Landau, and Overbeek explains the aggregation of particles in aqueous dispersions and describes the interactive forces between charged surfaces interacting through a liquid medium. Simply, the DLVO theory is used to quantify the relationship between U and h, and is mathematically expressed as:

U = Ue + Um + Us Equation 1.1

where Um and Ue are the attractive and repulsive forces, respectively, and Us is the potential energy due to the solvent. While the contribution of Us to the total potential energy is marginal over the last few nanometres of separation and is therefore negligible, the contributions from Ue and Um are much larger because they operate over much larger distances.

According to the DLVO theory, colloidal stability is determined by the sum of the electric double layer repulsive (Ue) forces and the van der Waals attractive (Um) forces that exist between particles as they approach each other. Van der Waals forces were defined earlier in this chapter. However, van der Waals attraction between two particles in a colloidal system depends on the size and shape of the particles, and is the sum of the dispersion, polarity, and induced coupling forces between particles. Um is a function of the distance between particles. For a colloidal system, Um can be expressed as:

tj A ( 2r1r2 2r1r2 , , h2 + 2r1h+2r2h 1 „ . _

Um=- -—--+ -—--+ In—-1-2-} Equation 1.2

m 6 lh2+2r1h+2r2h h2+2r1h+2r2h+4r1r2 h2+2r1h+2r2h+4r1r2) n

where A is the Hamaker constant of particles interacting through a water layer; rt is the particle radius, nm; and h is the distance between particle surfaces, nm. For a hybrid system, A is expressed as ^123, and is determined using the following equation:

Equation 1.3

where A11, ^22, and ^123 are the Hamaker constants of the particles in the hybrid system and A33 is the Hamaker constant of water.

The electric double layer, however, is a structure that appears on the surface of an object dispersed in a liquid (Figure 1.10). As its name implies, the structure consists of two parallel layers: a first layer, the Stern layer, which is a positive or negative surface charge, that consists of ions adsorbed onto the object due to chemical interactions; and a second layer composed of ions attracted to the Stern layer via Coulomb forces.

Figure 1.10 - Electrical double layer of a solid dispersed substance within a fluid.

Reproduced from [112].

The second layer electrically screens the first layer and is only loosely associated with the dispersed substance [101]. Also called the diffuse layer, the second layer consists of free ions that are governed by thermal motion and electric/electrostatic attraction and, instead of being firmly bonded to the dispersed substance surface, they move freely throughout the fluid.

Like Um, Ue is also a function of the distance between particles. For hybrid systems, Ue can be expressed as:

Ue = n^rMvl+ri^ ln[1 + e-2kh]} Equati0n 1.4

rl+r2 +(Pl J

where £ is the dielectric permittivity of the medium; £0 is the dielectric permittivity of a vacuum, F-m-1; rt is the particle radius, nm; ^ is the zeta (Z) potential of an interacting particle, mV; k is the Debye parameter, nm-1; and h is the distance between the particle surfaces, nm.

The thickness of the double layer is known as the Debye length, K-1, and can be expressed as:

K-1 = Equa«on 1.5

where £ is the dielectric permittivity of the medium; £0 is the dielectric permittivity of a vacuum; KB is Boltzmann's constant; NA is Avogadro's number; e is the charge; and I is ion concentration for a symmetric electrolyte.

When two dispersed particles in a colloidal system are in proximity, their diffusion layers overlap, upsetting their ion distribution, and disrupting the electrical screening effect. For particles with similarly charged Stern layers, this leads to electrostatic repulsion. The attractive and repulsive interaction energies between particles can be graphically expressed as functions of the distance between the particle surfaces (Figure 1.11). According to DLVO theory, repulsive forces prevent the particles in a colloidal system from approaching each other, as shown by the net barrier, which is the apex of the sum of these forces (dashed line) [81]. Nevertheless, if enough energy is provided (e.g., an increase in temperature) and the particles manage to surmount that energy

barrier (i.e., primary minimum), attractive van der Waals forces will strongly and irreversibly join them together. Therefore, for colloidal systems to be stable, strong repulsive forces are desirable. However, if repulsion is weak and attraction is strong, the dispersion will be highly unstable and the particles will aggregate and phase separation will occur, causing particle flocculation or sedimentation [3].

Figure 1.11 - Interactive energy resulting in attraction or repulsion between solid particles dispersed in a colloidal system. Reproduced from [81].

A particle in suspension has a zeta (Z) potential, a physical property that is a readily measurable indicator of colloidal stability and is often used to optimise the formulations of colloidal systems, predict surface interactions, and optimise the formation of films and coatings [88]. The Z potential is the net electrical charge at the slipping plane in the electrical double layer (Figure 1.10); simply put, it is the potential difference between the dispersion medium and the layer of fluid surrounding the dispersed particle. The magnitude of the Z potential, which can be either positive or negative and is measured in mV, indicates the degree of electrostatic repulsion between similarly charged particles

in a suspension. A Z potential of 0 - 5 mV typically results in rapid coagulation or flocculation; Z potentials between 10 and 30 mV indicate incipient instability, which means that instability has already begun and it may be too late to take corrective measures to prevent coagulation; colloidal systems with measured Z potentials between 30 and 40 mV are considered moderately stable; and systems with Z potentials between 40 and 60 mV have good stability [79]. Colloidal systems with Z potentials exceeding 61 mV have excellent stability. Therefore, when studying the stability of a colloidal system, both its Z potential and average particle size must be considered. An ideal colloid has high Z potential and small particle size. A large particle size can indicate low Z potential and, therefore, low sol stability; however, if the system has a high Z potential, the particles may simply be large [79]. This is undesirable because low sol stability affects the physicochemical properties and activities of colloidal systems.

1.7. Conclusion

Because the materials available today can no longer foster technological development, researchers have created new types of materials. One such type is hybrid materials, which are materials with one organic and one inorganic component, both of which occur on the nanoscale or at the molecular level. Hybrid materials can be classified according to type of modification and type of interactions that exist between components.

Due to the focus on environmental protection, biopolymers have been used as the organic component of hybrid materials in recent years. In this chapter, polysaccharides were proposed as viable organic components due to their biodegradability, biocompatibility, and low toxicity. As for the inorganic components, metallic nanoparticles were proposed. This is because they are very catalytically active nanoparticles that also have many exploitable characteristics such as optical activity, electronic conductivity, and magnetic susceptibility. Therefore, it was hypothesised that metallic nanoparticles can be hybridised with polysaccharide nanoparticles to create functional environmentally friendly hybrid systems. There are different hybridisation methods, namely the building block approach to hybridisation and in-situ hybridisation

methods, which include the sol-gel and blending methods and emulsion polymerisation. The main approach to hybridisation is sol-gel synthesis, which results in the formation of colloids.

The colloidal-chemical properties of a colloid are believed to have an important role in the physicochemical properties of any subsequent films, coatings, and gels. One approach to understanding the colloidal-chemical properties of a system is the DLVO theory, which states that the interaction energy between particles depends on the distance between said particles. The theory uses the zeta potential and apparent size of particles in a system to determine the magnitude of the interaction energies and explain colloidal stability, which is affected by several factors.

In this dissertation, the DLVO theory is used to obtain predictive models for the interactions between charged polysaccharide and metallic nanoparticles. Also, the dependence of the regularities of the formation of polysaccharide nanoparticle/metallic nanoparticle hybrid systems on organic-to-inorganic component ratio, surface charge, and electrolyte concentration are established. The effect of these factors on the synthetic method for hybrid formation is investigated, and the colloidal-chemical and physicochemical properties of the resulting hybrid systems are examined. Finally, potential applications of the developed hybrid systems based on their physicochemical characteristics are determined.

Chapter 2. Experimental section

2.1. Materials

Cotton microcrystalline cellulose (>99.8% Glc); sodium hydroxide (NaOH, >98.0%); copper(II) acetate monohydrate (Cu(CO2CH3>H2O, >99.0%); iron(II) chloride tetrahydrate (FeCl2-4H2O, >99.0%); iron(III) chloride hexahydrate (FeCl3-6H2O, 97.0-102.0%); glucose oxidase from Aspergillus niger Type X-S (100,000-250,000 units/g solid); 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS, >98.0%); Rhodamine B dye; copper (II) chloride dihydrate (CuC^^O, >99.0%); potassium chloride (KCl, 99.0 - 100.5%); chitin (from shrimp shells; practical grade); and trisodium citrate dihydrate (Na3C6H5O7-2H2O >99.0%) were purchased from Sigma-Aldrich.

Crystalline glucose; concentrated nitric acid (HNO3, 65%); acrylonitrile (C3H3N); ethanol (C2H5OH, reagent grade); phosphotungstic acid (H3PW12O40, PTA); and hydrogen peroxide (H2O2, 30%) were purchased from LenReactiv (Russia). Trilon B was obtained from NevaReactiv (Russia), and glacial acetic acid (CH3OOH, anhydrous) was purchased from Vekton (Russia). Sulfuric acid (H2SO4, 95.0-96.0%); hexadecyltrimethylammonium bromide [(C16H33)N(CH3)3]Br (CTAB); hydrazine (N2H4); and hydrochloric acid (HCl, >99.0%) were purchased from Sigma-Tech (Russia). Gold foil was purchased from Chemmed (Russia). Luria Bertani agar (Lennox, microbiology grade) was purchased from Diaem and agar (microbiology grade) was purchased from Helicon.

Unless otherwise stated, all reagents were used without further purification. The water (DI) used in the syntheses and experiments was ultrapure (Millipore, 18.2 MQ-cm).

2.2. Synthetic methods

2.2.1. Cellulose nanocrystal synthesis

Sulphated cellulose nanocrystals

Sulphuric acid hydrolysis was used to obtain sulphated CNC (S-CNC) [132].

Briefly, 25 mL deionised (DI) water was added to 24.4 mL laboratory grade sulphuric acid (H2SO4). The mixture was cooled to 60 °C using an ice bath. The diluted H2SO4 was then placed in a one-neck round bottom flask and its temperature maintained at 60 °C. Microcrystalline cellulose (MCC, 5 g) was then added to the acid and the mixture stirred for 2 h at 60 °C. The mixture was poured into 1000 mL ice-cold water to quench the reaction and allowed to sediment overnight. The following morning, the supernatant was decanted and the sediment centrifuged at 4000 rpms for 10 min. The supernatant was discarded and the sediment redispersed in DI water. Using concentrated sodium hydroxide (NaOH) and H2SO4 solutions, the pH was increased to approximately 7/8. The mixture was then centrifuged (4000 rpms, 20 min) and the supernatant discarded. The sediment was redispersed in DI water, centrifuged (4000 rpms, 20 min), and the sediment collected. The sediment was redispersed in DI water then dialysed for 3 days with the water being changed every 2 - 3 h. After dialysis, the mixture was sonicated (30 min), centrifuged (4000 rpms, 30 min), and the supernatant collected to obtain the S-CNC sol.

Acetylated cellulose nanocrystals

A Fenton-like system was used to obtain acetylated CNC [138]. Acetylated

cellulose nanocrystals have a structure very similar to that of native cellulose; therefore, it was designated as N-CNC. Briefly, glacial acetic acid (100 mL) and copper (II) acetate (0.4031 g) were brought to a boil in a two-neck round bottom flask under reflux. MCC (10 g) was added to the reaction vessel and the mixture was stirred under reflux for 40 min. Then, every 5 min for 1 h, 30% hydrogen peroxide (H2O2, 1 mL) was added to the reaction. The reaction was quenched by pouring it into 1000 mL ice-cold DI water and allowed to sediment overnight. The following morning, the supernatant was decanted and the sediment centrifuged at 4000 rpms for 10 min. The supernatant was discarded

and the sediment redispersed in DI water. Using concentrated NaOH and H2SO4 solutions, the pH was increased to approximately 7/8. Trilon B (0.6882 g) was stirred into the mixture, which was left undisturbed overnight. The blue supernatant was decanted the following day and the sediment centrifuged at 4000 rpms for 10 min. The sediment was washed by redispersing it in DI water and centrifugating until the supernatant was colourless. The clean sediment was redispersed in DI water, centrifuged (4000 rpms, 20 min), and the sediment collected. The sediment was redispersed in DI water then dialysed for 3 days with the water being changed every 2 - 3 h. After dialysis, the mixture was sonicated (30 min), centrifuged (4000 rpms, 30 min), and the supernatant collected to obtain the N-CNC sol.

2.2.2. Chitin nanocrystal synthesis

ChNC was fabricated via acid hydrolysis [145]. Briefly, chitin from shrimp shell (5 g) was boiled in DI water (168 mL) under reflux for 1 h. At the 1 h mark, concentrated hydrochloric acid (HCl, 56 mL) was added to the reaction vessel and the mixture boiled under reflux for 4 h. The reaction was quenched by pouring the mixture into ice-cold DI water (1000 mL) and allowed to sediment overnight. The following morning, the supernatant was decanted and the sediment centrifuged at 6000 rpms for 10 min. The supernatant was discarded and the sediment redispersed in DI water then dialysed for 3 days with the water being changed every 2 - 3 h. After dialysis, the mixture was sonicated (30 min) then collected to obtain the ChNC sol.

2.2.3. Gold nanoparticle synthesis

Gold nanoparticles were synthesised using the citrate method [31]. Briefly, chloroauric acid (HAuCU) was synthesised from gold foil. Gold (0.0739 g) was introduced to a one-neck round bottom flask. Laboratory-grade hydrochloric acid (1 mL) and 3 drops of laboratory-grade nitric acid were added to the flask, which was then sealed. The sealed flask was heated until boiling, after which it was open and deionised water (1 mL) was added to remove the excess acid. The solution was then diluted to 25 mL to obtain a 0.015 M HAuCl4 stock solution. The stock solution (32 mL) was then diluted to obtain 35 mL of 0.001 M HAuCU, which was heated until boiling under

stirring. Once boiling, sodium citrate (0.0034 M, 1.75 mL) was added to the solution and the stirring speed increased. The mixture was stirred for 10 min until a wine-red solution was obtained. The suspension was wrapped in aluminium foil and stored in the refrigerator for further use.

2.2.4. Magnetite nanoparticle synthesis

Magnetite nanoparticles were prepared by dissolving FeCl2-4H2O (2.5 g) and FeCl3-6H2O (5 g) in DI water (100 mL) under constant stirring [33]. Aqueous ammonia solution (25%, 12 mL) was then added to the reaction vessel and the mixture stirred at 25 °C for 10 min. The magnetite nanoparticles were collected using a magnet and then repeatedly washed with DI water until the sol had a pH of 7. The washed magnetite nanoparticles were redispersed in DI water (100 mL) then sonicated for 120 min.

2.2.5. Copper nanoparticle synthesis

Copper nanoparticles were synthesised by reducing copper (II) chloride dihydrate (CuCl2-2H2O) with hydrazine [119]. Briefly, ammonium hydroxide (2 mL) was added to 20 mL 0.01 M CuCl2-2H2O solution and stirred. Cetyltrimethylammonium bromide (CTAB, 0.01 M, 10 mL) was added and the mixture was slowly stirred to prevent frothing. Meanwhile, in a separate beaker, 670 ^L hydrazine was added to 31.330 mL 0.01 M CTAB. The solutions were then mixed together and left undisturbed for at least 1 h to allow copper nanoparticle formation.

2.2.6. Cellulose nanocrystal/magnetite nanoparticle (CNC/Fe3O4NP) hybrid synthesis

CNC/Fe3O4NP hybrids were synthesised by mixing the constituent sols in the appropriate ratios under constant stirring. Briefly, 0.1 g CNC and the pre-determined mass of Fe3O4 NP were added to a beaker and stirred for 30 min.

S-CNC/Fe3O4NP hybrid sols (10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 wt.% Fe3O4) were synthesised. Immediately after synthesis, the Z potentials and particle sizes of each hybrid was measured on the Photocor Compact-Z particle size and zeta potential analyser. Aqueous KCl (0.001 M) was added to each hybrid in a 1:1 ratio and the mixture

stirred for 30 min, after which the Z potentials and particle sizes were measured. To determine the effect of background electrolyte concentration on colloidal stability, the process was repeated with 0.01 M KCl. The experiment was repeated with 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 wt.% Fe3O4 N-CNC/Fe3O4NP hybrid sols.

After investigating the colloidal-chemical properties of the hybrid, 1, 5, and 10 wt.% Fe3O4NP CNC/Fe3O4NP hybrids were fabricated for physicochemical characterisation and application testing. CNC/Fe3O4NP films were obtained by casting the treated sols in Petri dishes and air drying at room temperature for 12 h.

2.2.7. Cellulose nanocrystal/gold nanoparticle (CNC/AuNP) hybrid synthesis

CNC/AuNP hybrids were synthesised by mixing the constituent sols in the appropriate ratios and then subjecting the mixed sol to hydrothermal treatment. Briefly, 0.1 g CNC and the pre-determined mass of AuNP were added to a beaker and stirred for 30 min. Deionised water was added to the mixture to increase the volume to 80% that of the autoclave used in hydrothermal treatment. The mixture was placed in the autoclave then hydrothermally treated in the oven at 140 °C for 12 h. To determine the reaction temperature, CNC and CNC sols were hydrothermally treated at 100, 110, 120, 130, 140, and 150 °C. CNC was stable at 130 °C.

CNC/AuNP hybrid sols (10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 wt.% AuNP) were synthesised. Immediately following hydrothermal treatment, the Z potentials and particle sizes of each hybrid was measured on a Compact-Z particle size and zeta potential analyser (Photocor, Russia). Aqueous KCl (0.001 M) was added to each hybrid in a 1:1 ratio and the mixture stirred for 30 min, after which the Z potentials and particle sizes were measured. To determine the effect of background electrolyte concentration on colloidal stability, the process was repeated with 0.01 M KCl.

After investigating the colloidal-chemical properties of the hybrid, 1, 3, 5, and 7 wt.% AuNP CNC/AuNP hybrids were fabricated for physicochemical characterisation and application testing. CNC/AuNP films were obtained by casting the treated sols in Petri dishes and oven-drying at 60 °C for 12 h. The sols were also freeze-dried to obtain powdered samples for characterisation.

2.2.8. Cellulose nanocrystal/copper nanoparticle (CNC/CuNP) and chitin nanocrystal/copper nanoparticle (ChNC/CuNP) hybrid synthesis

CNC/CuNP and ChNC/CuNP (1, 3, and 5 wt.% CuNP) hybrid sols were synthesised by mixing the constituent sols in the appropriate ratios under constant stirring and then hydrothermally treating the mixtures. Briefly, 0.1 g S-CNC and the predetermined mass of CuNP were added to a beaker and stirred for 30 min. Deionised water was added to the mixture to increase the volume to 80% that of the autoclave used in hydrothermal treatment. The mixture was placed in the autoclave then hydrothermally treated in the oven at 140 °C for 12 h. The procedure was repeated with N-CNC and ChNC to obtain N-CNC/CuNP and ChNC/CuNP hybrid sols. The hybrids were freeze-dried for characterisation and application testing.

2.3. Methods for investigating physicochemical properties

Atomic force microscopy (AFM) was used for nanomorphology characterisation. A 15 ^L drop of 0.1 wt.% suspension was placed on a newly cleaved mica substrate, allowed to set at room temperature for 15 min, then air-dried at ambient conditions. Sample surface images (256 x 256 pi) were obtained in semi-contact mode at 25 °C on an Integra Prima atomic force microscope (NT-MDT, Russia). Super sharp SSS-NCH silicon cantilevers (Nanosensors) with 10 - 15 ^m pyramidal tips were used. The tip radius was 2 - 5 nm. Nova software (NT-MDT) was used for analysis.

XRD analysis was performed over an angular range of 20 = 5° - 80° with 0.1° increments on an XRD-6000 diffractometer (Shimadzu, Japan) using CuKa radiation (X = 1.541 A) at the anode. The voltage and current were 30 kV and 30 mA, respectively. The crystallinity index (CI) of cellulose was determined using Equation 2.1 [41]:

CI = /(200)-/(^ Equation 2.1

^(200)

where Z(200) is the integrated peak intensity attributed to crystalline regions (20 = 22.6°); and I(am) is the minimum intensity between the peaks at (200) and (110) (20 = 18.5°).

Freeze-dried samples were mixed with crystalline KBr in a ratio of 2 mg sample per 10 mg KBr and pressed into pellets. The FTIR spectra were obtained on a Prestige 21 FTIR spectrometer (Shimadzu, Japan) equipped with a DLATGS detector within the 4000 - 400 cm-1 range at 4 cm-1 increments. The data was processed using manufacturer software.

Thermogravimetric analysis was performed on an STA 409 PC/PG Luxx instrument (NETZSCH, Germany) in argon at a heating rate of 10 °C/min. Differential scanning calorimetry (DSC) was performed on the same instrument to examine the thermal properties of hybrid thin films.

Photometric measurements were performed on a Cary 60 UV/Vis spectrophotometer (Agilent, USA) at X560 nm. In the coagulation experiments, KCl was used as the coagulating electrolyte. KCl concentration was varied between 1.0 to 75.0 mM.

The hydrodynamic diameter of the particles and the zeta potentials for 0.01 wt.% sample sols were measured by dynamic light scattering (DLS) and laser Doppler electrophoresis (LDE) on a Malvern ZetaSizer Nano ZS (Malvern Panalytical, United Kingdom) instrument (4 mW He/Ne laser, 633 nm) at 25 °C in a DTS1070 disposable capillary cell.

Scanning electron microscopy (SEM) and energy-dispersive X-ray (EDX) spectroscopy were conducted on a VEGA3 scanning electron microscope (Tescan, Czech Republic) equipped with an X-act detector (Aztec Energy, India).

The viscosities of the CNC and hybrid sols were analysed using a BV-III Ultra rheometer (Brookfield, USA) equipped with a small mount adapter. All measurements were conducted at 20.0±0.1 °C.

Optical photographs were taken with a Nikon D3200 camera.

Transmission electron microscopy (TEM) was used to study the morphologies of the nanoparticles. Investigation was conducted on a JEM 1400 transmission electron microscope (JEOL, Japan).

The textural properties of the samples were estimated from isotherms of low temperature (77 K) nitrogen physical adsorption-desorption via volumetry on a

Quantachrome Nova 1200e surface area and porosity analyser. The specific surface areas were determined by the Brunauer-Emmett-Teller (BET) method. The cumulative adsorption volume and the average pore diameter were calculated via the Barrett-Joyner-Halenda (BJH) method. Before the analysis, samples were held in a vacuum at 180 °C for 4 h.

X-ray photoelectron spectroscopy (XPS) analysis was conducted on a K-Alpha X-ray photoelectron spectrometer (ThermoFisher Scientific).

2.4. Application testing

2.4.1. Glucose sensing

Colorimetry was used to evaluate the glucose sensing ability of the CNC/Fe3O4NP

hybrids. The detection of H2O2 and glucose based on CNC/Fe3O4NP peroxidase-like activity was performed in the presence of ABTS.

The peroxidase-like activity of each sample was determined by adding 24 ^L 60 mM ABTS, 20 ^L 5 mg/mL glucose oxidase (GOx), 800 ^L H2O, 200 ^L of glucose water solution with varying concentration to 10 ^L of the sample and incubating the mixture at 40 °C for 5 min. The optical density (OD) of the resulting solution was measured using a Cary 60 UV-Vis spectrophotometer (Agilent Technologies, USA) to determine catalytic activity.

Temperature, pH, H2O2 concentration, incubation time, sample concentration, and glucose concentration were tested to determine optimal reaction conditions. For these reactions, hydrogen peroxide (24 ^L 100 mM H2O2) was used instead of GOx and glucose solution.

2.4.2. Humidity sensing

Hybrid films were subjected to the laser diffraction method and fluorescence measurements to determine the mechanism of action of humidity sensing. Laser diffraction was performed using the method described here [60]. Briefly, the 3 wt.% AuNP CNC/AuNP hybrid film was coated in holographic paper. Then, the laser diffraction pattern obtained when a laser was pointed at the coated hybrid was taken as

the default. Ambient humidity was gradually increased to 90% and then gradually lowered. The laser patterns at various humidity levels and periods were photographed.

Prior to hydrothermal treatment, a 3 wt.% AuNP hybrid sol was doped with fluorescent Rhodamine B dye. A pure N-CNC film doped with Rhodamine B was used as a control. The degree of florescence quenching was determined by gradually increasing ambient humidity to 80% and measuring the fluorescence intensity.

2.4.3. Antibacterial activity testing Zone of inhibition

Luria-Bertani (LB) agar medium was prepared by adding 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, and 15 g agar to 953.1 mL DI water and stirring. The LB agar medium was autoclaved for 20 min at 121 °C then allowed to cool to 55 °C. The medium was then poured into two 100 mm Petri dishes and allowed to set. Gram negative Escherichia coli (E. coli) was inoculated on the agar plates and a cup borer was used to make a well at the centre of each half of the agar. The S-CNC/CuNP (0, 1, 3, and 5 wt.% CuNP, 100 ^L) were added to the wells and labelled. The process was repeated for the N-CNC/CuNP and ChNC/CuNP hybrids.

The medium was poured into two 100 mm Petri dishes and allowed to set. Gram positive Bacillus subtilis (B. subtilis) was inoculated on the agar plates and a cup borer was used to make a well at the centre of each half of the agar. The S-CNC/CuNP (0, 1, 3, and 5 wt.% CuNP, 100 ^L) were added to the wells and labelled. The process was repeated for the N-CNC/CuNP and ChNC/CuNP hybrids.

Positive (bacteria, no sample) and negative (sample, no bacteria) controls were also prepared. All Petri dishes were incubated for 24 h at 37 °C. After incubation, the zones of inhibition were measured.

Minimum inhibitory concentration (MIC)

LB broth was prepared by adding 10 g tryptone, 5 g yeast extract, 10 g sodium

chloride, and 15 g agar to 953.1 mL DI water and stirring. The LB agar broth was autoclaved for 20 min at 121 °C then allowed to cool to 55 °C. The broth (5 mL) was then added to labelled Erlenmeyer tubes. E. coli inoculum (50 ^L), which was prepared

according to 0.5 McFarland standard (OD600 = 0.08 - 0.1; ~1e8 colony forming units (CFU)/mL), was inoculated into each tube. Pure S-CNC (25, 50, 75, and 100 ^L) was added to four different E. coli-inoculated test tubes. The process was repeated for the 1, 3, and 5 wt.% CuNP S-CNC/CuNP hybrids and the 0, 1, 3, and 5 wt.% CuNP N-CNC/CuNP and ChNC/CuNP hybrids.

The broth (5 mL) was added to labelled Erlenmeyer tubes. B. subtilis inoculum (50 ^L), which was prepared according to 0.5 McFarland standard (OD600 = 0.08 - 0.1; ~1e8 CFU/mL), was inoculated into each tube. Pure S-CNC (25, 50, 75, and 100 ^L) was added to four different B. subtilis-inoculated test tubes. The process was repeated for the 1, 3, and 5 wt.% CuNP copper nanoparticle/S-CNC hybrids and the 0, 1, 3, and 5 wt.% CuNP N-CNC/CuNP and ChNC/CuNP hybrids.

Positive (bacteria, no sample) and negative (sample, no bacteria) controls were also prepared. All Erlenmeyer tubes were incubated for 18, and 36 h at 37 °C. After each incubation period, OD600 was measured for each sample on a Cary UV/Vis spectrophotometer.

Minimum bactericidal concentration (MBC)

After the MIC assay, the 18 and 36 h 5 wt% CuNP ChNC/CuNP hybrid samples

were used to determine the minimum bactericidal concentration against gram-negative E. coli and gram-positive B. subtilis. Briefly, 100 ^L of the samples were introduced to agar plates then spread using a cell spreader. The plates were incubated at 37 °C for 24 h. After incubation, the number of CFUs were counted.

Chapter 3. Synthesis and characterisation of polysaccharide and metallic

nanoparticles

3.1. Introduction

Materials on the nanoscale exhibit many new exploitable physicochemical properties atypical of bulk materials, atoms, and molecules. Hybrid materials, which consist of at least one organic and one inorganic component that coexist and interact in such a manner that enhanced or entirely new physicochemical properties arise, are primary examples of the advantages associated with taking materials to the nanoscale. Nanomaterials have had a major impact on human health in areas such as detecting bacteria in food and food (flavour, colour, and texture) enhancement [124] as well as drug delivery, bioimaging, wound healing, and antibiotics [32]. Polysaccharide nanoparticles (PNPs) and metallic nanoparticles (MNPs) are often used for these applications. MNPs are used due to their high biocompatibility in small doses, high stability, and the opportunity for large-scale production [71]. PNPs are of interest because of their green chemistry association; polysaccharides are the most abundant biopolymers in the biosphere, which allows for their large-scale production. They are also biodegradable; biocompatible; and non-toxic, allowing them to find application in consumer- and health-centric industries such as the food and biomedical industries [5, 32, 124].

This dissertation explores the synthesis, characterisation, and potential applications of metallic nanoparticle/polysaccharide nanoparticle hybrid systems. MNPs experience a phenomenon known as quantum confinement, which results in the manifestation of several unique optical and electronic properties [104, 127, 163]. MNPs also have high surface areas, which makes them ideal for use in areas such as sensing and catalysis [114]. Herein, magnetite, gold, and copper nanoparticles are studied. However, these optically and catalytically active MNPs tend to aggregate in liquid environments, which diminishes their properties. As such, they are often supported by other materials, including polysaccharide particles, in an effort to prevent aggregation and maintain these desirable characteristics. In this thesis MNPs are hybridised with

cellulose and chitin nanocrystals to create functional materials. In this chapter, magnetite, gold, and copper nanoparticles as well as cellulose and chitin nanocrystals for eventual hybridisation were fabricated and characterised.

3.2. Metallic nanoparticles Magnetite nanoparticles

Ammonia was used to obtain Fe3O4NPs by co-precipitating iron (II) and iron (III) chlorides, as described in Chapter 2. The Z potential and hydrodynamic diameter were measured using LDE and DLS. The Fe3O4NP sol was found to have a Z potential of+36 mV, which can be attributed to the formation of iron (II) hydroxide [Fe(OH)2] on the magnetite surface during synthesis due to excess Fe2+ ions in solution. In neutral environments, Fe(OH)2 has a positive surface charge [34]. DLS showed that the particles had an average hydrodynamic diameter of 52.5±0.3 mV. However, TEM analysis of a freeze-dried sample (Figure 3.1) showed that the actual size of the Fe3O4NPs was between 5 and 20 nm, with an average diameter of 9 nm. The disparity between the apparent and actual sizes is because aggregation is highly likely in liquid environments and DLS includes the solvate shell during measurement. TEM analysis also showed that the magnetite nanoparticles have a spherical morphology.

Figure 3.1 - TEM micrograph of spherical magnetite nanoparticles.

FTIR spectroscopy was used to analyse the freeze-dried Fe3O4NP sample, and the spectrum is shown below in Figure 3.2. The bands at 3373 cm-1 and 3211 cm-1 can be attributed to water molecule stretching vibrations and the bands at 1625 cm-1 and 1419 cm-1 can be assigned to -OH vibrations of Fe(OH)2 on the magnetite surface; the very strong band at 629 cm-1 is due to Fe-O stretching vibrations [129].

Wavenumber (cm-1) Figure 3.2 - Fe3O4NP FTIR spectrum.

The freeze-dried Fe3O4NP sample was also characterised by XRD analysis. As shown in the XRD pattern (Figure 3.3), the main diffraction peaks are found at 20cu = 18.05°, 30.4°, 35.7°, 43.55°, 50.8°, 57.15°, 62.8°, and 79.3°, corresponding to the (111), (220), (311), (400), (422), (511), (440), and (444) reflection indices of the crystalline planes of magnetite.

-1-1-1-1-1-1-1-1---1---1---r

10 20 30 40 SO 60 70 SO

29 (degrees)

Figure 3.3 - Fe3O4NP XRD pattern.

Gold nanoparticles

As described in Chapter 2, the citrate method was used to obtain gold nanoparticles; consequently, the particles that were obtained had a negative Z potential, which was determined to be -20.7 mV via LDE. The size of the AuNPs was also measured using UV/Vis spectroscopy. The spectrum (Figure 3.4) showed a Vax of 520 nm, which corresponded to a particle size of 30 nm [52].

400 500 600 Wavelength (nml

Figure 3.4 - AuNP sol UV/Vis spectrum.

XRD analysis was also used to characterise the nanoparticles that were obtained. The XRD pattern showed peaks characteristic of gold nanoparticles. As shown in Figure 3.5, the main diffraction peaks occurred at 20Cu = 38.3°, 45.6°, 56.6°, and 64.8°, which

corresponded to the (111), (200), (220), and (311) reflection indices of the crystalline planes of gold [113].

(111)

3

«

(fl

(200)

*v vL (22o) (3iii

ÜIJI _

40 50 GO

26 (degrees)

70

Figure 3.5 - AuNP XRD pattern.

Copper nanoparticles

As described in Chapter 2, hydrazine was used to reduce Cu2+ ions in the presence of CTAB in order to obtain CuNPs; consequently, the particles that were obtained had a positive Z potential, which was determined to be +32.1±0.9 mV via LDE. The size of the CuNPs was also measured. UV/Vis spectroscopy showed that the CuNP sol had an absorption wavelength of 573 nm (Figure 3.6), which is characteristic of copper nanoparticles and corresponds to a particle size of approximately 64 nm [153].

Figure 3.6 - CuNP sol UV/Vis spectrum.

The UV/Vis spectrum also showed that the copper nanoparticles were unoxidised, evidenced by the absence of the absorbance peak characteristic of copper(I) oxide (~333 nm) [70]. The resulting sol was stored in an airtight container. Further characterisation of CuNP proved difficult due to them oxidising to copper oxide. CuNP remained stable if left undisturbed.

3.3. Cellulose nanocrystals

As stated in Chapter 1, due to its natural abundance, highly replenishable

resources, high tensile strength, and high Young's modulus, cellulose can be used to stabilise MNPs, resulting in the creation of new materials for application in various industries [85]. As described in Chapter 2, cellulose nanocrystals were obtained via two synthetic methods. In the first approach, conventional sulphuric acid hydrolysis was used to separate the crystalline and amorphous regions of microcrystalline cellulose, resulting in the fabrication of cellulose nanocrystals that had sulphate groups on their surface (S-CNC) [39]. In the second method, a Fenton-like system was used to obtain cellulose nanocrystals with a surface very similar to that of native cellulose (N-CNC) [139]. Acetyl groups are on the N-CNC surface. The N-CNC and S-CNC suspensions were found to be stable for a minimum of four months when stored at 4 °C.

LDE and DLS were used to determine the Z potentials and particle sizes of N-CNC and S-CNC. The Z potentials of N-CNC and S-CNC were -36 and -44 mV, respectively. The strongly negative charges were attributed to the hydroxyl groups on the N-CNC surface and the hydroxyl and sulphate groups on the S-CNC surface [140]. The more negative Z potential of S-CNC was attributed to the sulphate groups on its surface. DLS showed that N-CNC and S-CNC had average hydrodynamic diameters of 330 and 270 nm, respectively. However, TEM showed the actual sizes: N-CNC was 170 nm long and 46 nm wide, whereas S-CNC was 180 nm in length and had a width of 28 nm. TEM analysis also showed that |N-CNC and S-CNC both had rod-like morphologies (Figure 3.7)

A V / ' < A ^Hr f HVfV I! hk i ^ . ,, B

200 nm f_ 200 nm

Figure 3.7 - TEM micrographs of rod-like (A) N-CNC and (B) S-CNC.

The samples were freeze-dried for further characterisation. FTIR analysis showed that both samples had spectra characteristic of cellulose. For N-CNC (Figure 3.8A), the adsorption bands at 3452 cm-1 and 3323 cm-1 can be attributed to hydroxyl group stretching; the 2906 cm-1 and 1373 cm-1 are due to C-H and C-C vibrations; the band at 1087 cm-1 is assigned to the C-O-C bonds in secondary alcohols and ethers in the cellulose backbone; and the signal at 896 cm-1 is characteristic of the P-1,4-glycosidic bonds between glucose units [4]. Similar bands were observed in the S-CNC FTIR spectrum. However, there is a peak at 1728 cm-1 in the N-CNC spectrum that is absent from that of S-CNC due to the presence of acetate groups on the N-CNC surface.

4000 3500 3000 2500 2000 1500 1000 500 4000 3500 3000 2500 2000 1500 1000 500 Wavenumber (cm1) Wavenumber (cm"1)

Figure 3.8 - FTIR spectra of (A) N-CNC and (B) S-CNC.

N-CNC and S-CNC were also subjected to XPS analysis (Figure 3.9), which showed a very strong peak S2p(1/2) at ~169.2 eV in the S-CNC S2p spectrum that is not observed in the N-CNC S2p spectrum, confirming the presence of sulphate groups on the S-CNC surface.

N-CNC S-CNC

s2p(1/2)

180 178 176 174 172 170 ' 168 166 164 ' 162 Binding energy (eV)

Figure 3.9 - S2p XPS spectra of N-CNC (black) and S-CNC (red).

XRD analysis of both types of cellulose nanocrystals (Figure 3.10) confirmed the high degree of order of N-CNC and S-CNC, with crystallinity indices of 0.87 and 0.86, respectively, which were calculated using Equation 2.1. The main diffraction peaks for both types of CNC were located at 20Cu= 14.9°, 16.5°, 22.6°, and 34.2° corresponding to reflection indices (1-10), (110), (200), and (004), respectively, which is characteristic of the allomorphic structure of cellulose I. These results indicate that only surface functional groups differentiate S-CNC from N-CNC, and their crystalline structures are very similar.

N-CNC S-CNC

5 10 15 20 25 30 35 40 28(degrees)

Figure 3.10 - XRD patterns of N-CNC (grey) and S-CNC (red).

3.4. Chitin nanocrystals

Like cellulose, chitin is a naturally abundant polysaccharide with highly

replenishable resources, high tensile strength, and high Young's modulus; as such, it too can be used to support MNPs. As described in Chapter 2, chitin nanocrystals (ChNC) were obtained via the acid hydrolysis of shrimp shell chitin under reflux. The ChNC suspension was found to be stable for a minimum of four months when stored at 4 °C.

LDE and DLS were used to determine the Z potential and particle size of ChNC. The Z potential was +48 mV. Chitin is the only positively charged naturally occurring polysaccharide, which is due to the presence of N-amino-D-glucosamine units in its backbone [117]. DLS showed that ChNC had an average hydrodynamic diameter of 376 nm. However, TEM showed that ChNC was approximately 300 nm long and 45 nm wide. TEM analysis also showed that ChNC had a rod-like morphology (Figure 3.11).

M

F ' "

AJy j if ^m^, mm: i - it *

F&M 4 O y f rfhf * S

/ Jfl SOD nn

Figure 3.11 - TEM micrograph of rod-like ChNC.

The sample was freeze-dried for subsequent analysis. FTIR spectroscopy showed bands characteristic of chitin. The bands at 3459 cm-1 and 3287 cm-1 can be attributed to N-H, O-H, and NH2 vibrations; the peak at approximately 2925 cm-1 can be ascribed to -CH2 and -CH3 groups; an amide I peak is observed at 1660 cm-1 and the peak at 1586 cm-1 can be attributed to amide II N-H bending; and the peak at 1374 cm-1 can be ascribed to the C-H bending vibrations of -CH2 groups [73].

Wavenumber (cm-1) Figure 3.12 - ChNC FTIR spectrum.

The freeze-dried sample was also analysed using X-ray diffraction. The main diffraction peaks at 20Cu = 9.5°, 13°, 19.7°, 21.3°, 23.2°, and 27° correspond to the (020),

(021), (110), (120), (130), and (013) reflection indices of the crystalline planes of chitin, respectively [73].

(110}

5 10 15 20 25 30 28 (degrees)

Figure 3.13 - ChNC XRD pattern. 3.5. Conclusion

Magnetite, gold, and copper metallic nanoparticles and cellulose and chitin polysaccharide nanocrystals were synthesised for the design and fabrication of functional hybrid materials. Co-precipitation with ammonia was used to obtain positively charged spherical magnetite nanoparticles; the citrate method was used to obtain negatively charged gold nanoparticles; and reducing Cu2+ ions in solution with hydrazine in the presence of CTAB proved suitable for obtaining positively charged copper nanoparticles. Different acid hydrolysis techniques were used to obtain cellulose and chitin nanocrystals. LDE, DLS, UV/Vis spectroscopy, FTIR spectroscopy, TEM, and XRD analysis were used to characterise the materials and confirm that the relevant nanoparticles were obtained.

Chapter 4. Colloidal-chemical basis of interactions between metallic and

polysaccharide nanoparticles

4.1. Introduction

As mentioned in Chapter 1, the sol-gel approach to hybridisation is the most popular method for forming hybrid systems. This approach was used in the synthesis of magnetite, gold, and copper nanoparticles, as well as (non-)sulphated cellulose and chitin nanocrystals. As was also stated in Chapter 1, solid particles in liquid media exhibit a physical property known as their zeta (Z) potential. The Z potential of a suspension, colloid, emulsion, or sol reflects how stable the dispersion system is due to how the individual particles interact with each other. Similarly charged particles in suspension tend to repel each other, thereby preventing the formation of aggregates larger than colloidal dimensions [27]. Therefore, a large negative or positive Z potential indicates that a system is highly stable and strong repulsive forces exist between the particles, whereas systems with Z potentials close to zero tend to be very unstable because repulsion between particles is weak. Ideal colloidal systems have very high stabilities.

A sol is formed when hybrid nanoparticles are dispersed throughout a solvent and can be considered a colloidal system. Colloidal systems are very useful and have found applications in major fields such as the food, cosmetics, medicinal, and biomedical industries [74]. Colloidal stability is adversely affected under unfavourable conditions, and the particles in the dispersion adhere to each other and form aggregates. Aggregation tends to be successive, with aggregates accumulating to form even larger aggregates that then either precipitate or flocculate out of solution, resulting in phase separation. This renders the system useless, which is undesirable. Since the colloidal-chemical properties of a colloidal system affect the physicochemical properties of any subsequent materials (i.e., coatings, films, or gels), it is important to study them and ensure that highly stable colloidal systems are obtained.

As previously defined, a hybrid system consists of at least one inorganic and one organic nanoparticle or molecule interacting with each other. The DLVO theory can be used to explain the interactions between these particles. As stated in Chapter 1, there are two main forces acting on two particles in proximity: van der Waals attraction and

electric double layer repulsion. For a mixture of oppositely charged organic and inorganic nanoparticles, van der Waals attraction is the dominant force over short to mid-range distances and the organic and inorganic nanoparticles irreversibly interact via electrostatic attraction to form hybrid organic/inorganic nanoparticles. However, for a mixture of similarly charged organic and inorganic nanoparticles, as the particles approach each other, electric double layer repulsion is the dominant force and the particles must overcome a potential energy barrier (primary minimum interaction energy) for organic/inorganic hybrid particles to be obtained. Once hybridisation occurs, the system is considered a hybrid system.

The hybrid systems investigated are classified as lyophobic colloids [27]. Although the name suggests otherwise, the dispersed substance does interact with the dispersion medium in a lyophobic system and strong interactions exist between the colloid and 1 - 2 monolayers of the suspending medium; however, lyophobic systems are thermodynamically unstable and the measured particle size is usually of aggregates rather than individual particles [27]. This is due to the presence of a secondary minimum interaction energy, as shown in Figure 1.11 (Chapter 1). Overcoming the secondary minimum results in much weaker and potentially reversible adhesion between particles, and the particles can be redispersed under an external applied force such as agitation. At the secondary minimum, several factors can affect hybrid particle interaction. One of the main approaches to manipulating the colloidal stability of a lyophobic system is by adding a salt (background electrolyte) to the suspension. Another factor that affects colloidal stability is organic-inorganic component ratio.

In this chapter, the colloidal-chemical properties of hybrid polysaccharide nanoparticle/metallic nanoparticle systems are studied. Different approaches to the hybridisation of metallic nanoparticles with polysaccharide nanoparticles are investigated. The impact of surface charge on hybridisation method as well as the effect of metallic nanoparticle mass fraction and electrolyte concentration on sol stability are examined. Energy interaction models for the hybridisation between oppositely and similarly charged metallic and polysaccharide nanoparticles are proposed, as well as the dependence of the Z potential of the system on wt.% MNP. Additionally, the effect of

hybridisation on metallic and polysaccharide nanoparticles is explored, and the physicochemical properties of the hybrid systems determined.

4.2. Cellulose nanocrystal/magnetite nanoparticle hybrid systems