Fully Integrated Microfluidic Platform for Multiplexed Detection of Hunov by a Dynamic Confined-Space-Implemented One-Pot Rpa-Lamp System.

Human norovirus (HuNoV) is the leading cause of nonbacterial acute gastroenteritis, which is highly infectious, rapidly evolving, and easily transmitted through feces. The accurate and early detection of HuNoV subtypes is essential for effective treatment, early surveillance, risk assessment, and disease prevention. In this study, a portable multiplex HuNoV detection platform that combines integrated microfluidics and cascade isothermal amplification, using a streamlined protocol for clinical fecal-based diagnosis is presented. To overcome the problems of carryover contamination and the incompatibility between recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP), a Dynamic confined-space-implemented One-pot RPA-LAMP colorimetric detection system (DORLA) is developed by creating a hydrogen bond network. The DORLA system exhibits excellent sensitivity, with detection limits of 10 copies µL-1 and 1 copy µL-1 for HuNoV GI and GII, respectively. In addition, a portable diagnostic platform consisting of a thermostatic control module and an integrated 3D-printed microfluidic chip for specific HuNoV capture, nucleic acid pretreatment, and DORLA detection, which enables simultaneous diagnosis of HuNoV GI and GII is developed. A DORLA-based microfluidic platform exhibits satisfactory performance with high sensitivity and portability, and has high potential for the rapid point-of-care detection of HuNoV in clinical fecal samples, particularly in resource-limited settings.

Establishment and Validation of an Integrated Microfluidic Step Emulsification Chip Supporting Droplet Digital Nucleic Acid Analysis.

Uniform and stable droplet generation is critical for accurate and efficient digital nucleic acid analysis (dNAA). In this study, an integrated microfluidic step emulsification device with wide-range droplet generation capability, small device dimensions, convenient fabrication strategy, low contamination and high robustness was developed. A tree-shaped droplet generation nozzle distribution design was proposed to increase the uniformity of droplet generation by equating flow rates, and the flow field in the design was numerically simulated. Theoretical analysis and comparative experiments on droplet size were performed regarding the influences of nozzle dimensions and surface properties. With incubation and hydrophobic reagent treatment, droplets as small as 73.1 µm were generated with multiplex nozzles of 18 µm (h) × 80 µm (w). The droplets were then collected into a standard PCR tube and an on-chip monolayer droplet collection chamber, without manual transfer and sample contamination. The oil-to-sample volume ratio in the PCR tube was recorded during collection. In the end, the droplets generated and collected using the microfluidic device proved to be stable and uniform for nucleic acid amplification and detection. This study provides reliable characteristic information for the design and fabrication of a micro-droplet generation device, and represents a promising approach for the realization of a three-in-one dNAA device under a step emulsification method.

SERS-Based Immunoassay of Myocardial Infarction Biomarkers on a Microfluidic Chip with Plasmonic Nanostripe Microcones.

We developed a new plasmonic nanostripe microcone array (PNMA) substrate-integrated microfluidic chip for the simultaneous surface-enhanced Raman scattering (SERS)-based immunoassay of the creatine kinase MB isoenzyme (CK-MB) and cardiac troponin (cTnI) cardiac markers. The conventional immunoassay usually employs a microtiter plate as the solid capture plate to form the immunocomplexes. However, the two-dimensional (2D) surface of the microtiter plate limits the capture efficiency of the target antigens due to the steric hindrance effect. To address this issue, a gold film-coated microcone array with nanostripes was developed that can provide a large surface area for capture antibody conjugation and serve as a SERS-active substrate. This unique nano-microhierarchical structure showed an excellent light trapping effect and induced surface plasmon resonance to further enhance the Raman signals of the SERS nanoprobes. It significantly improved the sensitivity and applicability of SERS-based immunoassay on the microfluidic chip. With this integrated microfluidic chip, we successfully performed the simultaneous detection of CK-MB and cTnI, and the detection limit can reach 0.01 ng mL-1. It is believed that the PNMA substrate-integrated microfluidic chip would play a critical role in the rapid and sensitive diagnostics of cardiac diseases.

Large-scale single-cell encapsulation in microgels through metastable droplet-templating combined with microfluidic-integration.

Current techniques for the generation of cell-laden microgels are limited by numerous challenges, including poorly uncontrolled batch-to-batch variations, processes that are both labor- and time-consuming, the high expense of devices and reagents, and low production rates; this hampers the translation of laboratory findings to clinical applications. To address these challenges, we develop a droplet-based microfluidic strategy based on metastable droplet-templating and microchannel integration for the substantial large-scale production of single cell-laden alginate microgels. Specifically, we present a continuous processing method for microgel generation by introducing amphiphilic perfluoronated alcohols to obtain metastable emulsion droplets as sacrificial templates. In addition, to adapt to the metastable emulsion system, integrated microfluidic chips containing 80 drop-maker units are designed and optimized based on the computational fluid dynamics simulation. This strategy allows single cell encapsulation in microgels at a maximum production rate of 10 ml h-1of cell suspension while retaining cell viability and functionality. These results represent a significant advance toward using cell-laden microgels for clinical-relevant applications, including cell therapy, tissue regeneration and 3D bioprinting.

Multiplexed colorimetric detection of SARS-CoV-2 and other pathogens in wastewater on a 3D printed integrated microfluidic chip.

Severe acute respiratory coronavirus 2 (SARS-CoV-2) pandemic has become a global public health emergency. The detection of SARS-CoV-2 and human enteric pathogens in wastewater can provide an early warning of disease outbreak. Herein, a sensitive, multiplexed, colorimetric detection (termed "SMCD") method was established for pathogen detection in wastewater samples. The SMCD method integrated on-chip nucleic acid extraction, two-stage isothermal amplification, and colorimetric detection on a 3D printed microfluidic chip. The colorimetric signal during nucleic acid amplification was recorded in real-time and analyzed by a programmed smartphone without the need for complicated equipment. By combining two-stage isothermal amplification assay into the integrated microfluidic platform, we detected SARS-CoV-2 and human enteric pathogens with sensitivities of 100 genome equivalent (GE)/mL and 500 colony-forming units (CFU)/mL, respectively, in wastewater within one hour. Additionally, we realized smart, connected, on-site detection with a reporting framework embedded in a portable detection platform, which exhibited potential for rapid spatiotemporal epidemiologic data collection regarding the environmental dynamics, transmission, and persistence of infectious diseases.

An Evaluation Approach of Cell Viability Based on Cell Detachment Assay in a Single-Channel Integrated Microfluidic Chip.

Due to the heterogeneity of cancer cell populations, the traditional evaluation approach of cell viability based on the cell counting assay is quite inaccurate for the dose-response test of anticancer drugs, cell toxicology assays, and other biochemical stimulations. In this paper, an evaluation approach of cell viability based on the cell detachment assay in a single-channel integrated microfluidic chip is proposed to improve the accuracy of cell viability assessment. The electrodes are coated by fibronectin for specific cell adhesion, and it is biologically significant to study the cell detachment assay in vitro. The maximum number of cells that can be detected by this sensor is about 105 cells (overgrowing), while the minimum is about 100 cells. This method is calibrated with the half-maximal inhibitory concentration assay, and the results show that the cell viability calculated by adhesion strength is more accurate than that evaluated using the cell counting assay. Meanwhile, the shear rate is transformed into shear stress for the comparability among the results in other papers. The most sensitive frequency is also determined as 1 kHz according to normalized impedance. Besides, the impedance of cell adhesion affected by different shear stresses is monitored to study the optimized plan for long-term culture of cells in the integrated microfluidic chip prepared for the cell detachment assay. Adhesion strength τ25, which is the magnitude of shear stress needed to detach 75% of cell population, is introduced to describe the cell adhesion forces. It is calculated and normalized based on the cell detachment assay to evaluate cell viability. The relative errors of the cell detachment method compared with those of the cell counting method decrease by 0.637 (0% FBS), 0.586 (0.5% FBS), and 0.342 (2% FBS).