•The stacking chip was improved from our previous work.•The stacking chip combined the passive PDMS pumping with serial operations.•The enzymatic kinetics of alkaline phosphatase was studied with the stacking chip.•The stacking chip is applicable in high throughput and multiplex assays.This paper describes a microwell-based microdevice for performing quantitative bioanalysis. This microdevice combined the passive pumping by degassed polydimethylsiloxane (PDMS) with serial operations including solution dispensing, plates splitting and plates stacking. We name this microdevice “stacking chip”. To use the stacking chip in quantitative bioanalysis, nanoliter solutions were first dispensed into the microwells through the degassed PDMS microchannels. Next, we split the microwell and microchannel plates assisted by the application of one drop of silicone oil, which resulted in a microwell array containing the reagent solutions. Microreactor arrays were formed by stacking the two microwell arrays containing the reagent solutions. With this microdevice, the enzymatic kinetics of alkaline phosphatase during the dissociation of the fluorescein diphosphate was measured and analyzed by the Michaelis-Menten model. The stacking chip is simple to fabricate and operate, and amenable to automation for high throughput analysis.The stacking chip facilitates a series of operation including solution dispensing, plates splitting and plates stacking for bioanalysis.Download high-res image (193KB)Download full-size image

•A droplet microfluidic platform was constructed to measure both rapid and slow polymerase kinetics with matching and mismatching primers.•Kinetics based detection was applied with the increased discrimination ability.•Single nucleotide variation in the target sequences could be discriminated at room temperature with large discrimination factors.In this paper we presented a droplet-based microfluidic platform for the detection of single nucleotide variation by measuring the kinetics of Klenow Fragments (exo-) based extension reaction. In the platform, nanoliter droplets containing the DNA sequence, Klenow Fragments (exo-), buffer and dNTPs or rNTPs mixture were formed in the microchannel, which acted as microreactors. Target DNA sequences with and without single nucleotide variation were used as primers during the extension. For the rapid kinetics with matching template-primer duplex, the reaction time was directly related to the droplets’ traveling distance. For the slow kinetics with mismatching template-primer duplex, the flow was stopped after the formation of the droplets, and then the selected droplets were monitored to measure the kinetics. The droplet-based platform presented a dead time of sub-seconds and a wide range of observation time from sub-seconds to hundreds of minutes. The platform allowed the measurement of reaction kinetics ranging from s−1 to 10−4 s−1 and achieved large discrimination factors ranging from 100 to 550 for the detection of single nucleotide variation.

We herein report an ultralow background substrate for protein microarrays. Conventional protein microarray substrates often suffer from non-specific protein adsorption and inhomogeneous spot morphology. Consequently, surface treatment and a suitable printing solution are required to improve the microarray performance. In the current work, we improved the situation by developing a new microarray substrate based on a fluorinated ethylene propylene (FEP) membrane. A polydopamine microspot array was fabricated on the FEP membrane, with proteins conjugated to the FEP surface through polydopamine. Uniform microspots were obtained on FEP without the application of a special printing solution. The modified FEP membrane demonstrated ultralow background signal and was applied in protein and peptide microarray analysis.

A series of organic reactions proceed dramatically faster in a heterogeneous mixture of the reactants and water than in a homogeneous mixture. Currently it is unclear whether the rate acceleration is due to the free OH groups at the organic–water interface, or due to the hydrodynamic effects caused by vigorous stirring, vortexing, or ultrasonication. Herein we produced static droplets in microfluidic devices to answer this question. In the work, a series of organic droplets containing diethyl azodicarboxylate (DEAD) and quadricyclane surrounded by water were produced, which were transferred to and confined in glass capillaries to minimize the hydrodynamic effects. The cycloaddition process of DEAD with quadricyclane was recorded by a CCD camera. The results showed the reaction proceeded in three steps, and the organic–water interface alone was catalytically efficient enough to enhance the reaction rate to the same level as in the bulk emulsion reaction, indicating that the hydrodynamic effects were negligible.

This paper describes a centrifugation-based method of quantifying the adhesion strength of a thin film to a substrate. Normally thin films possess extremely small mass, and the centrifugal force cannot dislodge the thin films from the substrates even with the most powerful centrifuge. To solve this problem, we synthesized hydrogel particles on top of the thin film. The hydrogel particle was bound to the thin film through covalent bonds. The centrifugal force was therefore substantially increased, and the thin film could be dislodged from the substrate together with the hydrogel particles in a tabletop centrifuge. We validated this method by measuring the adhesion strength of adhesive tape to Teflon and compared it with the pull-off test result. We applied this method to measuring the adhesion strength of a polydopamine (PDA) thin film to Teflon, which has not yet been characterized. The measurement of the PDA adhesion strength would help in studying the mechanism of PDA adhesion and develop stronger PDA adhesive. The centrifugation-based method is simple and applicable to a broad range of thin films and substrates.

Conventional mapping of a phase diagram of a polymer in a solvent requires a substantial amount of polymer (e.g., at least of the order of ∼100 mg of narrowly distributed samples with different molar masses) and may take months or even years to reach the true two phase equilibrium at each given temperature, especially when the polymer concentration is high. This is why good phase diagrams of polymer solutions are rare in the literature. To solve such a problem, we developed a Teflon microfluidic device to prepare and store a series of droplets (∼10 nL) at different polymer concentrations inside a glass capillary. The phase transition inside each polymer solution droplet sealed and isolated in immiscible fluorohydrocarbon could be quickly and precisely monitored by a newly developed small angle laser light scattering detector. Using poly(vinyl acetate) (PVAc) in isobutyl alcohol and in benzene as two examples, we demonstrated that a combination of microfluidic device and small angle light scattering enables us to map the phase diagram of a polymer in a given solvent within hours by using only a few mg of the sample because (1) each droplet contains no more than ∼10 μg polymer and (2) the phase-transition induced interchain association inside each droplet can be quickly and sensitively detected. We have demonstrated that two sets of a total of eight precisely mapped phase diagrams of four PVAc fractions in the two solvents can be reasonably scaled together to form a master curve.

We present a laser scanning confocal microscopy (LSCM) and continuous flow microreactor (CFMR)-based platform to study the Belousov–Zhabotinsky (BZ) oscillators. We demonstrated that the scanning laser light below a certain power had no detectable influence on the BZ reaction. The CFMR consisted of the poly(methyl methacrylate) (PMMA) microwell and the polydimethylsiloxane (PDMS) microchannel and maintained the oscillation with a continuous supply of the catalyst-free BZ mixture. The synchronization of the two nonidentical oscillators was studied by the platform. The coupling intensity was controlled by changing the distance between the two oscillators. Results showed that the synchronization occurred as the oscillators were closer than a critical distance. The transition from desynchronization to synchronization was observed when the distance between the oscillators was near a critical value. The results of the numerical simulation by COMSOL agreed qualitatively with the experimental observation.

Droplets containing RNA and Mg2+ were generated in microfluidic channels. By integrating a group of pneumatic valves and phase separation channels in the microfluidic system, the rapid RNA–Mg2+ binding kinetics was studied by measuring the Mg2+ ion concentration using an ion-selective electrode.

In the work presented, rehydratable polyacrylamide gel is introduced as a medium to uptake and store nanoliter protein solutions in microwells for multiplex bioanalysis. The polyacrylamide gel, produced and stored in the microwells, shrank by 97% upon dehydration and could be reversibly rehydrated to 95% of the initial volume by absorbing aqueous solution. We employed the rehydratable gel to load aqueous solutions of different proteins with molecular weights in the range of 14.7–250 kDa. The protein loading occurred simultaneously with the gel rehydration and reached saturated state in 5 min. The relative protein concentrations in the gel ranged from 92% to 53%, depending on the molecular weight of the proteins. Particularly, the rehydratable gel had a much higher protein loading efficiency than the fresh gel. We applied the protein-carrying gel to the crystallization of four model proteins in the microwells and produced diffraction-quality protein crystals. The rehydratable gel is simple to fabricate, efficient to load with protein, and has good capacity for storing the protein solutions in microwells with minimal dilution effects on the protein solution. The rehydratable gel incorporated microwell chip should be useful in multiplex analysis that requires small sample consumption and high throughput.

Droplets containing RNA and Mg2+ were generated in microfluidic channels. By integrating a group of pneumatic valves and phase separation channels in the microfluidic system, the rapid RNA–Mg2+ binding kinetics was studied by measuring the Mg2+ ion concentration using an ion-selective electrode.