Мультиплексная микрочиповая система с иммобилизованными реактивами для молекулярно-генетического анализа тема диссертации и автореферата по ВАК РФ 02.00.02, кандидат химических наук Наволоцкий, Денис Васильевич
Наволоцкий, Денис Васильевич
кандидат химических наук
2010
- Специальность ВАК РФ02.00.02
- Количество страниц 161
Заключение диссертации по теме «Аналитическая химия», Наволоцкий, Денис Васильевич
выводы
1. Разработана схема анализа методом мультиплексной ПЦР-РВ с использованием микрочипов с иммобилизованными реактивами. Разработана аналитическая система, обеспечивающая детектирование флуоресценции двух красителей, в которой для термоциклирования используется элемент Пельтье с управлением по ПИД-алгоритму и корректировкой температуры по АЮС-модели. Разработанная система легла в основу серийного ПЦР-анализатора.
2. В качестве материалов для изготовления микрочипов выбраны кремний и алюминий, обладающие высокой теплопроводностью. Из этих материалов разработаны микрочипы с микрореакторами открытого типа, позволяющие проводить одновременно 16—48 мультиплексных ПЦР-РВ. Стоимость разработанного микрочипа по сравнению с микрофлюидным чипом снижена в 12 раз в случае применения кремния, и в 80 раз — в случае алюминия.
3. Разработаны способы модификации поверхности кремния и алюминия, заключающиеся в создании двумерного распределения гидрофильных и гидрофобных зон путем последовательной обработки поверхности 81 пластины [3-(2,3-эпоксипропокси)-пропил]-триметоксисиланом и этиленгликоль-диметиловым эфиром, и путем обработки А1 пластины в смеси Н3РО4 и ЬШОз и в растворе ПВС для создания гидрофильного покрытия, с нанесением слоя ПММС для создания гидрофобного покрытия. Такая обработка обеспечила высокую эффективность ПНР (94±6% - для 81, 94±5% - для А1), позволила предотвратить ингибирование ПЦР, исключить взаимное смешение образцов.
4. Разработан способ иммобилизации ПЦР-реактивов путем высушивания растворов в микрореакторах и оптимизирован состав стабилизирующих добавок (ЮОтМ трегалоза, 1% Т\уееп20, 2% ПВП), что позволило достигнуть стабильности при хранении микрочипов при комнатной температуре в течение 4 мес.
5. Показано, что для достижения высокого быстродействия и селективности ПЦР-РВ анализа оптимальная длина амплифицируемого участка ДНК должна находиться в диапазоне от 80 до 150 п.о.
6. Разработаны условия качественного и количественного анализа суспензий микроорганизмов в диапазоне концентраций 102-Ч07 КОЕ/мл методом мультиплексной ПЦР-РВ. Достигнута близкая к максимальной эффективность ПЦР (94+100%). Разработана методика скрининга и количественного определения ГМО с использованием микрочипов с иммобилизованными реактивами. Время анализа составило 23 мин, а предел обнаружения — 0,05% от ГМ растения.
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