A 3D printed microfluidic PCR device towards detecting SARS-CoV-2

A 3D printed (3DP) microfluidic polymerase chain reaction (PCR) device was demonstrated by detecting synthetic SARSCoV-2 at 106 copies/μL. The microfluidic device was fabricated using stereolithography 3DP and had a reaction volume of ~22 nL. The microdevice showed PCR amplification with 85 base synthetic ssDNA targets and primers designed for a SARS-CoV-2-specific region. The device was 2.5 times faster compared to a qPCR instrument with >60,000 times smaller reagent volume. The 3DP microdevice is a promising technology to significantly reduce the manufacturing costs of microfluidic devices that could be used towards point-of-care applications. © 2023 SPIE.

Copper Sulfate-Induced Diphenylamine for Rapid Colorimetric Point-of-Care Detection of Contagious Pathogens Combined with Loop-Mediated Isothermal Amplification

Here, we developed a copper sulfate (CuSO4)-initiated diphenylamine (DPA)-based colorimetric strategy coupled with loop-mediated isothermal amplification (LAMP) for rapid detection of two critical contagious pathogens, SARS-CoV-2 and Enterococcus faecium. To detect the DNA, acid hydrolysis of LAMP amplicons was executed, enabling the development of a blue color. In the LAMP amplicons, the bond between the purines and deoxyribose is extremely labile. It can be broken using 70% sulfuric acid followed by phosphate group elimination, which generates a highly active keto aldehyde group. CuSO4 plays an imperative role inducing DPA to rapidly react with the keto aldehyde group, producing an intense blue color within 5 min. Moreover, low quantities such as 103 copies μL-1 of SARS-CoV-2 RNA and 102 CFU mL-1 of E. faecium were successfully detected, revealing the advantages of the introduced method. To confirm practical applicability, multiplex detection of pathogens was performed using a foldable microdevice comprising reaction and detection zones. Various reactions such as DNA extraction, LAMP, and acid hydrolysis occurred in the reaction zone. Then, colorimetric reagents (DPA, CuSO4, and ethylene glycol) contained in the detection zone were mixed with the keto aldehyde group by simply folding the microdevice, which was heated at 65 °C for 5 min for colorimetric detection. An intense blue color was developed where the target DNA was present. These results indicate that the method proposed in this study is highly suitable for point-of-care applications, especially in resource-limited settings for the rapid detection of harmful pathogens. © 2023 American Chemical Society.

Building Blood Vessel Chips with Enhanced Physiological Relevance

Blood vessel chips are bioengineered microdevices, consisting of biomaterials, human cells, and microstructures, which recapitulate essential vascular structure and physiology and allow a well-controlled microenvironment and spatial-temporal readouts. Blood vessel chips afford promising opportunities to understand molecular and cellular mechanisms underlying a range of vascular diseases. The physiological relevance is key to these blood vessel chips that rely on bioinspired strategies and bioengineering approaches to translate vascular physiology into artificial units. Here, several critical aspects of vascular physiology are discussed, including morphology, material composition, mechanical properties, flow dynamics, and mass transport, which provide essential guidelines and a valuable source of bioinspiration for the rational design of blood vessel chips. The state-of-art blood vessel chips are also reviewed that exhibit important physiological features of the vessel and reveal crucial insights into the biological processes and disease pathogenesis, including rare diseases, with notable implications for drug screening and clinical trials. It is envisioned that the advances in biomaterials, biofabrication, and stem cells improve the physiological relevance of blood vessel chips, which, along with the close collaborations between clinicians and bioengineers, enable their widespread utility. © 2023 Wiley-VCH GmbH.

Dual-mode visual detection strategies of viable pathogens for point-of-care testing

Here, we introduce a power-free foldable poly(methyl methacrylate) (PMMA) microdevice fully integrating DNA extraction, amplification, and visual detection, realized in novel dual modes – colorimetric and aggregate formation – using 4-Aminoantipyrine (4-AP) for monitoring pathogens. The microdevice contains two parts: reaction and detection zones. A sealing film was utilized to connect the two zones and make the device foldable. The FTA card was deposited in the reaction zone for DNA extraction, followed by loop-mediated isothermal amplification (LAMP) at 65 °C for 45 min. When the detection zone is folded toward the reaction zone, paper discs modified with 4-AP placed in the detection zone are delivered to the reaction zone. Specifically, in the presence of LAMP amplicons, 4-AP is oxidized into antipyrine red or generates aggregates by interacting with copper sulfate, forming copper hybrid nanostructure (Cu-hNs). In the absence of LAMP amplicons, 4-AP is not oxidized and maintains yellow color or fails to form aggregates. Furthermore, we introduced the ethidium homodimer-1 (EthD-1) to identify viable bacteria. EthD-1 penetrated the compromised membranes of nonviable cells and prevented further DNA amplification by intercalating with the DNA. In this way, only samples containing viable cells displayed color change or formed aggregates upon reaction with 4-AP. Using this method, SARS-CoV-2 RNA and Enterococcus faecium were identified by naked eye, with the limit of detection of 103 copies/μL and 102 CFU/mL, respectively, within 60 min. The introduced microdevice can be used for rapidly monitoring viable pathogens and controlling outbreaks of infectious disease in resource-limited settings. © 2022 Elsevier B.V.

Copper Sulfate-Induced Diphenylamine for Rapid Colorimetric Point-of-Care Detection of Contagious Pathogens Combined with Loop-Mediated Isothermal Amplification

Here, we developed a copper sulfate (CuSO4)-initiated diphenylamine (DPA)-based colorimetric strategy coupled with loop-mediated isothermal amplification (LAMP) for rapid detection of two critical contagious pathogens, SARS-CoV-2 and Enterococcus faecium. To detect the DNA, acid hydrolysis of LAMP amplicons was executed, enabling the development of a blue color. In the LAMP amplicons, the bond between the purines and deoxyribose is extremely labile. It can be broken using 70% sulfuric acid followed by phosphate group elimination, which generates a highly active keto aldehyde group. CuSO4 plays an imperative role inducing DPA to rapidly react with the keto aldehyde group, producing an intense blue color within 5 min. Moreover, low quantities such as 103 copies μL-1 of SARS-CoV-2 RNA and 102 CFU mL-1 of E. faecium were successfully detected, revealing the advantages of the introduced method. To confirm practical applicability, multiplex detection of pathogens was performed using a foldable microdevice comprising reaction and detection zones. Various reactions such as DNA extraction, LAMP, and acid hydrolysis occurred in the reaction zone. Then, colorimetric reagents (DPA, CuSO4, and ethylene glycol) contained in the detection zone were mixed with the keto aldehyde group by simply folding the microdevice, which was heated at 65 °C for 5 min for colorimetric detection. An intense blue color was developed where the target DNA was present. These results indicate that the method proposed in this study is highly suitable for point-of-care applications, especially in resource-limited settings for the rapid detection of harmful pathogens. © 2023 American Chemical Society

High-throughput and highly sensitive detection of SARS-CoV-2 spike protein in saliva without pretreatment by using immuno-wall microdevices

In this study, an immuno-wall microdevice was developed to detect the spike protein of SARS-CoV-2 virus in saliva. We performed the immunoassay of the SARS-CoV-2 spike protein in saliva within 30 min without pretreatment. The assay time was about 6-time shorter than commercial ELISA (5 h) and the limit of detection (LOD) was 5 ng/mL, which was close to the commercial ELISA kit. This immuno-wall microdevice has a high promising future to be applied to the massive, low-cost, and rapid test for COVID-19. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.