| Nucleic acid plays crucial roles in the growth, development and differentiation of cells. For a long time, Oligonucleotide and cDNA hybridization microarrays,as important methods to detect nucleic acid, have a huge influence on the field of high-throughput DNA analysis. Microarrays can detect hundreds of genes in one reaction by using oligonucleotide probes with special chemical modification, however they cannot meet the demand for detection of low content of nucleic acid due to low sensitivity. Polymerase chain reaction (PCR),as a method for amplification of nucleic acid in vitro, is characterized by high sensitivity, accuracy, efficiency and thus becomes the most widely used means for detecting nucleic acid. Multiplex PCR is a technique which can perform amplification of multiple targets simultaneously in a single reaction. However, conventional multiplex PCR has some limitations such as the imbalanced and inefficient amplification caused by interference and competition among primers,so it is difficult to detect more than ten targets.In recent years, droplet manipulation technology has developed rapidly. Compared to conventional reaction in a tube, reaction in droplet has some unique advantages. Firstly, the volume of reaction in droplet is as small as picoliters or nanoliters,so the consumption of reagents can be decreased by more than three order of magnitudes. Secondly,droplet segregated by immiscible phase acts as a micro-reactor,so droplet can perform parallel high-throughput analysis for multiple targets. Thirdly, droplet can realize fast biochemical reactions due to rapid blend of reactant and high efficiency of heat and mass transfer,which dramatically reduces the reaction time. Fourthly, the size of droplet comparable to that of a cell enables analysis for single cell. Lastly, miniaturized reactors contribute to the combination of a series of operations including purification, mix,amplification, detection of samples, which makes automation and portability of system more likely to come true. Currently, droplet technology has been widely used in biological detection technology such as protein detection, kinetics of enzyme reaction, PCR and single cell analysis.1. Preparation of micro-droplet array based on hydrophilic-hydrophobic patternsA facile and rapid method was proposed to fabricate microarrays of separated, spatially organized droplets on a hydrophilic surface patterned with hydrophobic moieties. The droplets can encapsulate any substance dissolved or suspended in an aqueous solution, and drying the droplets results in the homogeneous deposition of the substance in the hydrophilic microspots. The small volumes of the droplets require fewer reagents compared to microplates and can enhance the detection of low abundance molecules or signals. The simple approach presented here for making hydrophilic patterns on hydrophobic surfaces will find numerous applications for biological, medical, and diagnostic purposes.2. A universal multiplex real-time PCR system using the micro-droplet arrayA middle throughput micro-droplet array was developed for performing multiplex PCR assays. By imaging the micro-droplet array after each cycle, the real time quantitative PCR (RT-PCR) could be realized. The specific primers for PCR were pre-loaded accompanying the aqueous solution in which the primers dissolved. Using the micro-droplet array, more than 96 PCR assays could be run for the size of a standard microscope slide. RT-PCR was demonstrated with an accuracy and precision equivalent to the same assay in a PCR tube but in a 40-fold smaller reaction volume and a workflow compatible with standard microplate protocols. The system has a dynamic range spanning five orders of magnitude, and could detect as low as one copy of DNA per reaction, which allows for the digital PCR research.The system was applied for HPV typing, which results in high specificity and accuracy. We envision the micro-droplet array based RT-PCR system could be a universal and low-cost platform for gene quantification in routine biological laboratories.3. A universal multiplex nucleic acid detection combined with isothermal amplificationA unique micro-droplet array, was produced to perform multiple gene detection. Combining with the HRCA technology, amplification of nucleic acid with high efficiency and outstanding specificity is achieved. It was successfully applied for the SNP detection with a detection limit lowered to 5 amol per droplet. The consumption of reagents for each droplet is only 0.5μl, which was much lower than that of conventional in-tube assays. Moreover, this method, in conjunction with the isothermal amplification of nucleic acid, does not need a precise thermal cycler, which would facilitate its use as a universal platform in ordinary biological laboratories. All the features provide the method a promising diagnostic approach for disease detection and prevention. |
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