Fluorescence Colorimetry-Based Droplet Microfluidic Chip For Nucleic Acid And Protein Detection

Droplet-based microfluidics is a new technology for manipulation of small liquid volume, which has been widely applied in biology, chemistry and medicine due to its unique characteristics, such as small volume, free of the dispersion, immobilization, incubation time and cross-contamination of reagents, rapid reagent mixing, etc.. Up to now, it provides a new platform for automatic, miniaturization, simple analysis, favorable for the development of biochemical assay. However, it is still a challenge for droplet-based microfluidic to develop low-cost, high throughput and quantitative method for biochemical field, as well expand application scope. On the basis of these, we focus on droplet micro fluidics for developing new methods of biochemical analysis. The following summarize the conclusions of four aspects.1. A new fluorescence colorimetry-based droplet microfluidic chip for the determination of double-stranded DNAs (dsDNA) by using two separate output fluorescent signals of Ru(bpy)2dppx2+(Ru) and quantum dots (QDs). In the experiment, Ru serves a quencher for QDs and a reporter for dsDNA simultaneously. In the absence of dsDNA, the electrostatic interaction makes Ru close to QDs and suppress efficiently its fluorescence. As a result, a dark green appears in droplet. With the addition of dsDNA, the quenching process is prevented because of the competition of dsDNA with QDs for Ru. Meanwhile, the luminescence of QDs is restored and the red fluorescence of Ru is emitted, causing the droplet in a mixed color. According to the droplet variation, a colorimetric strategy for dsDNA is established in droplet. Under the optimum conditions, this biosensing system exhibits not only good sensitivity and specificity for calf thymus DNA with the detection limit of1.0pg, but also coincident performances in diluted human serum with the detection limit of0.9pg. The droplet biosensor is highly efficient, simple and rapid.2. A droplet-based biosensor for multiplexed DNA analysis is developed with a common imaging device via the different absorption between graphene nanoprobe (GO) and single-stranded DNA or double-stranded DNA. In the absence of target DNA, the DNA probes labeled carboxyfluorescein (FAM) and6-carboxy-X-rhodamine (ROX) are quenched by GO. As a result, the fluorescence of FAM and ROX become weak, leading to the droplet in dark color. While the droplet changes from dark to bright color when the DNA probes form double helix with the specific target DNAs leading to the dyes far away from GO and the fluorescence recovery of FAM and ROX. This colorimetric droplet biosensor exhibits a quantitative capability for simultaneous detection of two different target DNAs with the detection limits of9.46and9.67×108M, respectively. The incorporation of graphene nanoprobe and droplet technique drive the biosensor field one more step to some extent.3. A cooperation of stepwise reagent introduction and droplet techniques are developed for multiplexed DNA sensing. Five different DNA probes labeled FAM are sequentially introduced into chip channel with gas and buffer as separating medium. Under free of interferences, probe droplet is formed one by one, fused orderly into target DNAs and graphene oxide droplets. When the first DNA probe droplet is formed and fused into GO droplet, the DNA probe is absorbed on the surface of GO. As a result, the fluorescence quenching of DNA probe occurs by FRET between FAM and GO. Correspondingly, the droplet is imaged in dark color. While the DNA probe droplet is fused with target DNAs and GO droplets, the FRET is broken due to the formation of dsDNA. Therefore, the droplet shows the green color of FAM. Compare the droplet color variation in the absence and presence of target DNA, the qualitative and quantitative analysis of target DNA can be realized. According to the same expeimental principle, more DNA sensing can be realized by increasing the number of DNA probes introduced. This strategy has great potential for the development of high-throughput DNA sensors without complicated label of fluorescent material and chip design.4. A microscale immunoassay for a-Fetoprotein (AFP) is developed on droplet-based microfluidics by the incorporation of (Ru(bpy)2(mcbpy-O-Su-ester)(PF6)2(Ru) and green quantum dots (QDs). In this work, Ru is coupled with the monoclonal antibody (Ab) of AFP to form a stable red Ru-Ab complex as both an effective quencher for QDs and a capture probe for AFP, as well as color reference. In the absence of AFP, the fluorescence of QDs is quenched, leading to droplet in red color. With the addition of AFP, the color of droplet changed from red to green because of the competition of AFP with QDs for Ru-Ab. This biosensor exhibits not only good sensitivity and specificity for AFP with a detection limit of0.06ng/ml, but also satisfactory performances in diluted human sera with a detection limit of0.4ng/ml. This method can be applied in the AFP analysis of clinic sample, which has huge promising for the detection of expensive and rare samples.