Virtual Multiplexing Chamber-Based Digital PCR for Camel Milk Authentication Applications.

In this work, we proposed a chamber-based digital PCR (cdPCR) microfluidic device that is compatible with fluorescence imaging systems for milk adulteration detection. The device enables the digitalization of PCR reagents, which are loaded into microchambers, and subsequent thermocycling for DNA amplification. Then, fluorescence images of the microchambers are captured and analyzed to obtain the total number of positive chambers, which is used to calculate the copy numbers of the target DNA, enabling accurate quantitative detections to determine intentional milk adulteration from accidental contaminations. The validation of this device is performed by camel milk authentication. We performed 25,600-chamber virtual multiplexing cdPCR tests using 40 × 40 chamber devices for the detection of DNA templates extracted from pure or mixed milk with different dilutions. Then, the cdPCR chip was used to authenticate blind milk samples, demonstrating its efficacy in real biotechnical applications.

An integrated digital polymerase chain reaction chip for multiplexed meat adulteration detection.

Meat adulteration detection is a common concern of consumers. Here, we proposed a multiplex digital polymerase chain reaction method and a low-cost device for meat adulteration detection. Using a polydimethylsiloxane microfluidic device, polymerase chain reaction reagents could be pump-free loaded into microchambers (40 × 40 chambers) automatically. Due to the independence of multiplex fluorescence channels, deoxyribonucleic acid templates extracted from different animal species could be distinguished by one test. In this paper, we designed primers and probes for four types of meat (beef, chicken, pork, and duck) and labeled each of the four fluorescent markers (hexachlorocyclohexane [HEX], 6-carboxyfluorescein [FAM], X-rhodamine [ROX], and cyanine dyes 5 [CY5]) on the probes. Specific detection and mixed detection experiments were performed on four types of meat, realizing a limit of detection of 3 copies/µL. A mixture of four different species can be detected by four independent fluorescence channels. The quantitative capability of this method is found to meet the requirements of meat adulteration detections. This method has great potential for point-of-care testing together with portable microscopy equipment.

Easy-to-Operate Co-flow Step Emulsification Device for Droplet Digital Polymerase Chain Reaction.

Digital polymerase chain reaction (PCR) plays important roles in the detection and quantification of nucleic acid targets, while there still remain challenges including high cost, complex operation, and low integration of the instrumental system. Here, in this work, a novel microfluidic chip based on co-flow step emulsification is proposed for droplet digital PCR (ddPCR), which can achieve droplet generation, droplet array self-assembly, PCR amplification, and fluorescence detection on a single device. With the combination of single-layer lithography and punching operation, a step microstructure was constructed and it served as the key element to develop a Laplace pressure gradient at the Rayleigh-Plateau instability interface so as to achieve droplet generation. It is demonstrated that the fabrication of step microstructure is low cost, easy-to-operate, and reliable. In addition, the single droplet volume can be adjusted flexibly due to the co-flow design; thus, the ddPCR chip can get an ultrahigh upper limit of quantification to deal with DNA templates with high concentrations. Furthermore, the volume fraction of the resulting droplets in this ddPCR chip can be up to 72% and it results in closely spaced droplet arrays, makes the best of CCD camera for fluorescence detections, and is beneficial for the minimization of a ddPCR system. The quantitative capability of the ddPCR chip was evaluated by measuring template DNA at concentrations from 20 to 50 000 copies/µL. Owing to the characteristics of low cost, easy operation, excellent quantitative capability, and minimization, the proposed ddPCR chip meets the requirements of DNA molecule quantification and is expected to be applied in the point-of-care testing field.

Pre-Degassed Microfluidic Chamber-Based Digital PCR Device for Meat Authentication Applications.

Droplet digital polymerase chain reaction (ddPCR) suffers from the need for specific equipment and skilled personnel; thus, we here present a chamber-based digital PCR microfluidic device that is compatible with fluorescence image read-out systems and removes bubbles by a pre-degassed microfluidic device that consists of a pilot channel and micro chamber arrays. Digitalized PCR reagents are introduced into micro chambers, and thermocycles are taken to perform a DNA amplification process. Then, fluorescence images of a micro chamber array are read out and analyzed to obtain the total number of positive chambers. Thereby, the copy numbers of target DNA are calculated for quantitative detections. As a validation, this device is evaluated by the application of meat authentication. We performed dPCR tests using DNA templates extracted from a pure mutton DNA template with different dilutions. Then, the dPCR chip was used to identify the meat authentication using mutton-chicken mixtures with different mass ratios, showing its performance in real biotechnical applications.

Fluid mixing in droplet-based microfluidics with T junction and convergent-divergent sinusoidal microchannels.

To explore and utilize the advantages of droplet-based microfluidics, hydrodynamics, and mixing process within droplets traveling though the T junction channel and convergent-divergent sinusoidal microchannels are studied by numerical simulations and experiments, respectively. In the T junction channel, the mixing efficiency is significantly influenced by the twirling effect, which controls the initial distributions of the mixture during the droplet formation stage. Therefore, the internal recirculating flow can create a convection mechanism, thus improving mixing. The twirling effect is noticeably influenced by the velocity of the continuous phase; in the sinusoidal channel, the Dean vortices and droplet deformation are induced by centrifugal force and alternative velocity gradient, thus enhancing the mixing efficiency. The best mixing occurred when the droplet size is comparable with the channel width. Finally, we propose a unique optimized structure, which includes a T junction inlet joined to a sinusoidal channel. In this structure, the mixing of fluids in the droplets follows two routes: One is the twirling effect and symmetric recirculation flow in the straight channel. The other is the asymmetric recirculation and droplet deformation in the winding and variable cross-section. Among the three structures, the optimized structure has the best mixing efficiency at the shortest mixing time (0.25 ms). The combination of the twirling effect, variable cross-section effect, and Dean vortices greatly intensifies the chaotic flow. This study provides the insight of the mixing process and may benefit the design and operations of droplet-based microfluidics.