A three-in-one microfluidic droplet digital PCR platform for absolute quantitative analysis of DNA.

Droplet digital polymerase chain reaction (ddPCR) technology has found widespread applications in the ultrasensitive analysis of nucleic acids, where integrated ddPCR platforms with the capability of sample dispersion, followed by in situ amplification and data analysis, are highly expected. However, current integrated ddPCR platforms are usually limited by either difficultly mass-produced materials or lack of integrated control instruments, restricting their practical application. This paper proposes an integrated three-in-one ddPCR platform with high user-friendliness and practicability, which is composed of an easy-to-use chip and a matching control instrument. The chip was made of thermally resistant and easily mass-produced polycarbonate (PC) material, and the benchtop control instrument was designed to perform droplet generation, in situ amplification, and fluorescence reading. The droplet generation and in situ heating on the chip were well characterized. Finally, the performance of the platform was validated through the analysis of the EGFR L858R mutation in lung cancer. The proposed three-in-one ddPCR platform shows great practicability in ultrasensitive nucleic acid testing. By virtue of its sensitivity, practicability, and cost-effectiveness, the ddPCR can serve as a universal detection platform for monitoring nucleic acid in the fields of tumor diagnosis, pathogen detection, and prenatal diagnosis.

Regulation of probe density on upconversion nanoparticles enabling high-performance lateral flow assays.

Upconversion nanoparticles (UCNPs)-based fluorescence probes have shown great potential in point-of-care testing (POCT) applications, due to UCNPs' features of high photostability and background-free fluorescence. Ceaseless improvements of UCNPs-probes have been carried out to increase detection sensitivity and to broaden detection range of UCNPs-based POCT. In this paper, we optimized UCNPs-probes by regulating probe density. The optimization was verified by a traditional lateral flow assay (LFA) platform for C-reactive protein (CRP) detection. Further, the optimized UCNPs-LFA integrating with a home-made benchtop fluorescence analyzer holds the capability to achieve high-performance POCT. Finally, nearly a 20 times sensitivity enhancement with a limit of detection of 0.046 ng/mL and a broad detection range of 0.2-300 ng/mL for CRP detection was obtained. Moreover, the optimized UCNPs-LFA was applied to detecting CRP in clinical serum samples and the detection results were consistent with the clinical test, validating its clinical practicability. The proposed optimization method is also expected to optimize other nanoparticles-based bio-probes for wider POCT application.

Ball pen writing-without-ink: a truly simple and accessible method for sensitivity enhancement in lateral flow assays.

Lateral flow assays (LFAs), a popular point-of-care testing platform, have found widespread applications from laboratory to clinics. However, LFA-based testing is still subject to limited detection sensitivity, especially for classical gold nanoparticle-based LFAs. Inspired by traditional pen-based writing technologies, we developed a ball pen writing-without-ink method to amplify the detection signal of LFAs through controlling fluid flow rate. An enhancement of detection sensitivity by two times was obtained. Since the underlying mechanism of this method to improve detection sensitivity is to control the flow rate of the liquid on paper, it may be suitable for most paper-based platforms.

Upconversion fluorescence-based paper disc for multiplex point-of-care testing in water quality monitoring.

Water quality monitoring is of great significance for human health, which involves a large number of distinct targets detection, such as pathogens, heavy metal ions, and toxins. Traditional detection methods usually carried out in well-equipped central labs are subjected to time, labor, and expense consumption, thus not suitable for implementing water quality monitoring at point of care, where the detection implemented on a multiplexed, miniaturized, and simplified device with rapid and sensitive answer out is desired. To this end, we developed a paper disc relying on upconversion fluorescence signal and aptamer recognition probes for multiplex detection of water contaminants with high sensitivity and specificity. Finally, several different typical kinds of water contaminants have been successfully detected on our paper disc with limit of detection of 115 cfu/mL for Salmonella, 3 ng/mL for Ochratoxin A and Microcystin-LR, 20 nM for Hg2+ and 4 nM for Pb2+, respectively. Additionally, we designed a compact smartphone-based reading device to enable water quality monitoring at point-of-care. Furthermore, this paper disc-based detection platform can be extended to apply to multiplex detection for disease diagnosis and food safety monitoring.

Recombinase polymerase amplification integrated with microfluidics for nucleic acid testing at point of care.

Nucleic acid testing (NAT) implemented on a portable, miniaturized, and integrated device with rapid and sensitive results readout is highly demanded for pathogen detection or genetic screening at resource-limited settings, especially after the outbreak of coronavirus disease 2019 (COVID-19). The integration of recombinase polymerase amplification (RPA) with emerging microfluidics, classified by paper-based microfluidics and chip-based microfluidics, shows great potential to perform laboratory independent NAT assays at point of care with minimal labor, time and energy consumption. This review summarizes the state-of-the-art of RPA integrated with paper-based microfluidics and chip-based microfluidics, and discusses their pros and cons. Finally, existing challenges and possible ways for optimization of microfluidics-based RPA are proposed.