A fully integrated nucleic acid analysis system for multiplex detection of genetic polymorphisms related to folic acid metabolism.

A sufficient intake of folic acid is essential during pregnancy, but several genetic polymorphisms reduce its absorption, threaten the lives of pregnant women and cause congenital disabilities in newborns. Traditional laboratory detection of genetic variants related to folic acid metabolism is time-consuming and labor-intensive. Microfluidics-based molecular diagnosis integrates sample pre-processing and nucleic acid amplification on-chip to achieve rapid, sensitive, high-throughput, and automated detection. Here, we developed a fully integrated microfluidic system for the detection of genetic polymorphisms related to folic acid metabolism in a "sample in-answer out" style. The system consists of nucleic acid extraction and amplification modules. During nucleic acid extraction, blood cells are lysed, and DNA is captured and eluted through a silica-gel membrane. After that, multiple gene loci are detected using loop-mediated isothermal amplification (LAMP) and the color of the reaction chamber indicates whether genetic mutations are present. The experimental results demonstrate that the system can accurately detect gene polymorphisms associated with folic acid metabolism in blood samples with high sensitivity and no cross-contamination between chambers. The blood samples of five patients were tested for mutant alleles on this system, and the test results were consistent with qPCR and DNA sequencing observations. The operation is fully automated, and the detection is completed in approximately 70 minutes. The proposed system has great potential in prenatal diagnosis and other types of nucleic acid detection.

An ultrasensitive and rapid "sample-to-answer" microsystem for on-site monitoring of SARS-CoV-2 in aerosols using "in situ" tetra-primer recombinase polymerase amplification.

Airborne transmissibility of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the urgent need for aerosol monitoring of SARS-CoV-2 to prevent sporadic outbreaks of COVID-19. The inadequate sensitivity of conventional methods and the lack of an on-site detection system limited the practical SARS-CoV-2 monitoring of aerosols in public spaces. We have developed a novel SARS-CoV-2-in-aerosol monitoring system (SIAMs) which consists of multiple portable cyclone samplers for collecting aerosols from several venues and a sensitive "sample-to-answer" microsystem employing an integrated cartridge for the analysis of SARS-CoV-2 in aerosols (iCASA) near the sampling site. By seamlessly combining viral RNA extraction based on a chitosan-modified quartz filter and "in situ" tetra-primer recombinase polymerase amplification (tpRPA) into an integrated microfluidic cartridge, iCASA can provide an ultra-high sensitivity of 20 copies/mL, which is nearly one order of magnitude greater than that of the commercial kit, and a short turnaround time of 25 min. By testing various clinical samples of nasopharyngeal swabs, saliva, and exhaled breath condensates obtained from 23 COVID-19 patients, we demonstrate that the positive rate of our system was 3.3 times higher than those of the conventional method. Combining with multiple portable cyclone samplers, we detected 52.2% (12/23) of the aerosol samples, six times higher than that of the commercial kit, collected from the isolation wards of COVID-19 patients, demonstrating the excellent performance of our system for SARS-CoV-2-in-aerosol monitoring. We envision the broad application of our microsystem in aerosol monitoring for fighting the COVID-19 pandemic.

A scalable microfluidic chamber array for sample-loss-free and bubble-proof sample compartmentalization by simple pipetting.

Sample compartmentalization is a pivotal technique in many bioanalytical applications, such as multiplex polymerase chain reaction (PCR) and digital PCR (dPCR). In this study, we successfully developed a novel self-compartmentalization device containing an array of microchambers, each of which is connected to a main microchannel with three capillary burst valves (CBVs) for fluid switching and partitioning. As these CBVs can be automatically opened in a predefined sequence, an incoming solution can be spontaneously directed into the chamber and held in place without further mixing. After that, either air or oil can be loaded into the main channel to isolate each chamber completely. By optimizing the relative burst pressures of the CBVs, a 100% sample utilization rate can be achieved even using a manual pipette and air bubbles in the sample cannot interfere with the loading. In addition, the number of the microchambers in an array can be easily scaled from a few to tens of thousands. To verify the feasibility of this self-compartmentalization method, we successfully conducted mock multiplex loop-mediated isothermal amplifications (LAMP) in an array that contains 144 microchambers, proving that our design method will provide a robust and versatile platform for various sample discretization purposes in the future.

A Fully Integrated In Vitro Diagnostic Microsystem for Pathogen Detection Developed Using a "3D Extensible" Microfluidic Design Paradigm.

Microfluidics is facing critical challenges in the quest of miniaturizing, integrating, and automating in vitro diagnostics, including the increasing complexity of assays, the gap between the macroscale world and the microscale devices, and the diverse throughput demands in various clinical settings. Here, a "3D extensible" microfluidic design paradigm that consists of a set of basic structures and unit operations was developed for constructing any application-specific assay. Four basic structures-check valve (in), check valve (out), double-check valve (in and out), and on-off valve-were designed to mimic basic acts in biochemical assays. By combining these structures linearly, a series of unit operations can be readily formed. We then proposed a "3D extensible" architecture to fulfill the needs of the function integration, the adaptive "world-to-chip" interface, and the adjustable throughput in the X, Y, and Z directions, respectively. To verify this design paradigm, we developed a fully integrated loop-mediated isothermal amplification microsystem that can directly accept swab samples and detect Chlamydia trachomatis automatically with a sensitivity one order higher than that of the conventional kit. This demonstration validated the feasibility of using this paradigm to develop integrated and automated microsystems in a less risky and more consistent manner.