The Study Of Protein High Sensitive And Rapid Detection Based On Microfluidic Chip

How to realize high sensitive and rapid detection of target protein from the biological samples with the property of wide varieties, complicated composition and low content has always been a worker’s pursuit and efforts to solve the one of the scientific problem. At present, a variety of large-scale testing equipment was developed, it can be used for efficient, accurate and sensitive detection of protein, but still cannot meet the current requirement of real-time, rapid detection in the environmental monitoring, food security, health care, drug testing and the other fields. Microfluidic chip has been developed into a potential analysis platform, it can integrate with the biotechnology and analysis methods(such as sampling, sample preparation, separation, enrichment and detection), finally formed a rapid detection method with the property of operation simple, portable, real-time, high throughput.By means of microfluidic chip technology, the paper was mainly focused on the three aspects:(1) The modification method of channel inner wall. By reducing the nonspecific adsorption of proteins, increased detection sensitivity of protein.(2) Developed two new temperature sensitive boric acid affinity microfluidic chip. By simple temperature adjustment to rapid capture and release of protein samples and improve the detection sensitivity.(3) Based on colloidal gold immunochromatographic strip technology, a new type of colloidal gold immunochromatographic microfluidic chip was developed, and realized the rapid, simultaneous, portable and visual detection of protein. The research content of the paper mainly includes the following aspects:1. Developed a simple coating technology of channel inner wall to inhibit the non-specific adsorption of proteins. In order to solve the sample adsorption, bovine serum albumin(BSA) coating — a new and simple coating was prepared in the microfluidic chip. Due to the capillary and the glass chip have the same material, and they can be directly used the basic protein as the research target, so the capillary electrophoresis(UV detector) was used for characterizing the coating effect. After optimization, the optimum coating conditions were obtained. The coating phosphate buffer pH is 4.2, the standing time is 12 h, BSA coating concentration is 1.5 mg/mL. Four basic proteins mixture of ribonuclease, lysozyme, trypsin and myoglobin was separated using the coated capillary. The coating process of the coating method is simple and rapid. The coating possesses the advantages of stability and reproducibility.2. Thermoresponsive boronate affinity PDMS microfluidic chip based on P(NIPAAm- co- AAPBA) was prepared for the sample enrichment and separation. P(NIPAAm- co-AAPBA) was grafted in the polydimethylsiloxane(PDMS) microfluidic chip channel by the UV induced graft polymerization reaction. By adjusting the temperature without changing the pH of the mobile phase, cis-diol-containing biological molecules were captured and released. By adjusting the temperature from 4 to 55 ℃, P(NIPAAm- co-AAPBA) grafted PDMS microchip was successfully used to capture and release cis-diol-containing biological molecules adenosine. With further development, the prepared PDMS microfluidic chip can be used as a potential tool for the capture and release of cis-diol-containing biological molecules,such as horseradish peroxidase and glycoprotein.3. Thermoresponsive boronate affinity glass microfluidic chip based on P(NIPAAm-co- VPBA) was prepared for the sample selective enrichment and separation. By the surface atom transfer radical polymerization(SI- ATRP), the glass microfluidic chip grafted P(NIPAAmco- VPBA) with thermoresponsive boronate affinity was successfully prepared. By the temperature regulation(from 4 ℃ to 55 ℃), microfluidic chip grafted P(NIPAAmco- VPBA) can specificity and selectively capture and release cis-diol-containing adenosine from the mixture of adenosine and deoxyadenosine. The time less than 10 min from sample injection to sample collection. The microfluidic chip grafted P(NIPAAmco-VPBA) will become a potential tool for high selectivity and reproducibility enrichment cis-diol-containing biological molecules, such as glycoprotein, carbohydrate, glycosylation peptides and nucleic acid.4. A colloidal gold immunochromatographic strip and colloidal gold immunochromatographic microfluidic chip were prepared for the rapid, simultaneous and visual detection of IL- 6 and TNF-α. The most commonly used detection method of interleukin 6(IL 6) and tumor necrosis factor(TNF-α) is enzyme linked immune sorbent assay(ELISA). However, this method is laborious and time-consuming. Compared with ELISA, colloidal gold immunochromatographic strip has the advantages of rapid detection, high sensitivity and specificity, simple preparation method, stored for a long time, low-cost and need not the large precision instrument and equipment. A new colloidal gold immunochromatographic strip and microchips were prepared for rapid, simultaneous and visual detection of IL- 6 and TNF-α, and the detection limit was 62.5 ng/mL and 30 ng/mL, respectively. Without using any instrument, the prepared strip and microchips can be used for simple, high specificity detection.