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The Visual Colorimetric Detection of Multi-nucleotide Polymorphisms on a Pneumatic Droplet Manipulation Platform

A simple and visual method to detect multi-nucleotide polymorphism (MNP) was performed on a pneumatic droplet manipulation platform on an open surface. ...

National Taiwan University

We propose a novel approach to resolve simultaneously the distributions of velocities and concentration of multiple, submicron species in microfluidic devices using microparticle image velocimetry, and particle counting. Both two-dimensional measurement and three-dimensional analysis of flow fields, from the stacked images, are achieved on applying a confocal fluorescence microscope. The displacements of all seeding particles are monitored to determine the overall velocity field, whereas the multicolor particles are counted and analyzed individually for each color to reveal the distributions of concentration and velocity of each species. A particle-counting algorithm is developed to determine quantitatively the spatially resolved concentration. This simultaneous measurement is performed on a typical T-shaped channel to investigate the mixing of fluids. The results are verified with numerical simulation; satisfactory agreement is achieved. This measurement technique possesses reliability appropriate for a powerful tool to analyze multispecies mixing flows, two-phase flows, and biofluids in microfluidic devices.

Simultaneous measurement of concentrations and velocities of submicron species using multicolor imaging and microparticle image velocimetry.

Mobile hybridization is a concept proposed and verified herein. We have designed a microfluidic device that is capable of enhancing passive mixing through the morphology of micro-structures, positioned along the channels of the device. We investigated the capability of these structures to promote mobile hybridization of fluorophore-labeled target oligonucleotides to oligonucleotide gold-nanoparticle (Au-NP) probes. This process is monitored with fluorescence through the quenching of the fluorescent signal within the device as the target oligonucleotides become bound to the Au-NP probes. We evaluated the fluorescent intensity of a sample image that showed enhanced probability of mobile hybridization of the samples, which was completed in about 7.2 s. Mobile hybridization is thus much more effective than traditional static hybridization (reaction overnight) limited by molecular diffusion. This approach promises an improved hybridization of samples with these probes, and is beneficial for microfluidic-based systems for biomedical detection.

Enhanced mobile hybridization of gold nanoparticles decorated with oligonucleotide in microchannel devices.

For the diagnosis of biochemical reactions, the investigation of microflow behavior, and the confirmation of simulation results in microfluidics, experimentally quantitative measurements are indispensable. To characterize the mixing and reaction of fluids in microchannel devices, we propose a mixing quality index (M(qi)) to quantify the cross-sectional patterns (also called mixing patterns) of fluids, captured with a confocal-fluorescence microscope (CFM). The operating parameters of the CFM for quantification were carefully tested. We analyzed mixing patterns, flow advection, and mass exchange of fluids in the devices with overlapping channels of two kinds. The mixing length of the two devices derived from the analysis of M(qi) is demonstrated to be more precise than that estimated with a commonly applied method of blending dye liquors. By means of fluorescence resonance-energy transfer (FRET), we monitored the hybridization of two complementary oligonucleotides (a FRET pair) in the devices. The captured patterns reveal that hybridization is a progressive process along the downstream channel. The FRET reaction and the hybridization period were characterized through quantification of the reaction patterns. This analytical approach is a promising diagnostic tool that is applicable to the real-time analysis of biochemical and chemical reactions such as polymerase chain reaction (PCR), catalytic, or synthetic processes in microfluidic devices.

Characterization of microfluidic mixing and reaction in microchannels via analysis of cross-sectional patterns.

We propose a novel technique that allows oligonucleotides with specific end-modification within a plug in a plug-based microfluidic device to undergo a locally enhanced concentration at the rear of the plug as the plug moves downstream. DNA was enriched and detected in situ upon exploiting a combined effect underlain by an entropic force induced through fluid shear (i.e. a hydrodynamic-repellent effect) and the interfacial adsorption (aqueous/oil interface) attributed to affinity. Flow fields within a plug were visualized quantitatively using micro-particle image velocimetry (micro-PIV); the distribution of the fluid shear strain rate explains how the hydrodynamic-repellent effect engenders a dumbbell-like region with an increased concentration of DNA. The concentration of FAM (6-carboxy-fluorescein)-labeled DNA (FC-DNA) and of TAMRA (tetramethyl-6-carboxyrhodamine)-labeled DNA (TC-DNA), respectively, and the hybridization of probe DNA (modified with FAM) with target DNA (modified with TAMRA) were investigated in devices; a confocal fluorescence microscope (CFM) was utilized to monitor the processes and to resolve the corresponding 2D patterns and 3D reconstruction of the DNA distribution in a plug. TC-DNA, but not FC-DNA, concentrating within a plug was affected by the combined effect so as to achieve a concentration factor (C(r)) twice that of FC-DNA because of the lipophilicity of TAMRA. Using fluorescence resonance-energy transfer (FRET), we characterized the hybridization of the DNA in a plug; the detection limit of a system, improved by virtue of the proposed technique (the locally enhanced concentration), for DNA detection was estimated to be 20-50 nM. This technique enables DNA to concentrate locally in a nL-pL free-solution plug, the locally enhanced concentration to profit the hybridization efficiency and the detection of DNA, prospectively serving as a versatile means to accomplish a rapid DNA detection in a small volume for a Lab-on-a-Chip (LOC) system.

Locally enhanced concentration and detection of oligonucleotides in a plug-based microfluidic device.

A particle-based display medium and a driving mechanism insensitive to the charge polarity of those particles, based on the transformation of particle chains, are developed for reflective electronic paper displays. Particle chains are formed by dipole-dipole interactions between polarized particles with an appropriate electric field applied across the tested display medium, i.e. the solution that regulates the light in the field of display technology, containing neutral polystyrene (PS) particles dispersed in water. Formation of the particle chains results in a large change in optical transmittance and reflectance of the display medium. The performance of the particle chain displays (PCD) was evaluated according to macroscopic (device), microscopic (particle) and optical (reflectance) points of view. A display medium (thickness 100 μm) containing colored PS particles (3 μm, 2.5% w/v) was polarized to display the fixed images of the directly driven electrodes and programmable images of arrayed (5 × 5) electrodes with electric fields (0.48 MV m(-1) and 0.09 MV m(-1), 500 kHz, respectively). The formation of particle chains under electric fields (0.2 MV m(-1) and 0.4 MV m(-1), 500 kHz) was observed in the microscopic images of a display medium (thickness 100 μm) with fluorescent PS particles (5 μm, 1%). Images recorded with a confocal microscope demonstrated the particle chains. The opacity, a common parameter serving to characterize a display medium, was derived by measuring the reflectance ratio of a black background to a white background of the display medium with varied thickness and particle concentration. The temporal response of a display medium (thickness 50 μm) with black PS particles (3 μm, 5%) was tested. When an electric field (0.6 MV m(-1), 500 kHz) was applied, the reflectance increased twice at the first data point in 0.7 s, attaining a contrast ratio of 2. Application of a voltage (20 s) yielded a contrast ratio of 10. The performance of a tested display medium, composed of simple PS particles and water and driven to form particle chains by polarization, is reported.

Particle chain display--an optofluidic electronic paper.

[Point massage and cupping therapys for 31 cases of esophageal achalasia].

A novel aggregation-based biosensing method to achieve detection of oligonucleotides in a pinched-flow fractionation (PFF) microseparator was developed. Employing functionalized polystyrene microspheres, this method is capable of the direct detection of the concentration of a specific DNA sequence. The label-free target DNA hybridizes with probe DNA of two kinds on the surface of the microspheres and causes the formation of an aggregate, thus increasing the average size of the aggregate particles. On introducing the sample into a PFF microseparator, the aggregate particles locate at a specific position depending on the size of the aggregate. Through a multi-outlet asymmetric PFF microseparator, the aggregate particles become separated according to outlets. Because the size of the aggregate particles is proportional to the concentration of the target DNA, a rapid quantitative analysis is achievable with an optical microscope. A biological dose-response curve with concentration in a dynamic range 0.33-10nM has been achieved; the limit of detection is between 33 and 330 pM. The specificity of the method and the potential to detect single-nucleotide polymorphism (SNP) of known concentration were examined. The method features simple, direct and cheap detection, with a prospect of detecting other biochemical samples with distinct aggregation behavior, such as heavy-metal ions, bacteria and proteins.

Wei-Feng Fang

Department of Mechanical Engineering

National Taiwan University

The Visual Colorimetric Detection of Multi-nucleotide Polymorphisms on a Pneumatic Droplet Manipulation Platform

Wei-Feng Fang

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Mechanical Engineering

National Taiwan University

The Visual Colorimetric Detection of Multi-nucleotide Polymorphisms on a Pneumatic Droplet Manipulation Platform

Department of Mechanical Engineering