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.

A microfluidic biosensor architecture for the rapid detection of COVID-19

The lack of enough diagnostic capacity to detect severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) has been one of the major challenges in the control the 2019 COVID pandemic;this led to significant delay in prompt treatment of COVID-19 patients or accurately estimate disease situation. Current methods for the diagnosis of SARS-COV-2 infection on clinical specimens (e.g. nasal swabs) include polymerase chain reaction (PCR) based methods, such as real-time reverse transcription (rRT) PCR, real-time reverse transcription loop-mediated isothermal amplification (rRT-LAMP), and immunoassay based methods, such as rapid antigen test (RAT). These conventional PCR methods excel in sensitivity and specificity but require a laboratory setting and typically take up to six hours to obtain the results whereas RAT has a low sensitivity (typically at least 3000 TCID50/ml) although with the results with 15 mins. We have developed a robust micro-electro-mechanical system (MEMS) based impedance biosensor fit for rapid and accurate detection of SARS-COV-2 of clinical samples in the field with minimal training. The biosensor consisted of three regions that enabled concentrating, trapping, and sensing the virus present in low quantities with high selectivity and sensitivity in 40 minutes using an electrode coated with a specific SARS-COV-2 antibody cross-linker mixture. Changes in the impedance value due to the binding of the SARS-COV-2 antigen to the antibody will indicate positive or negative result. The testing results showed that the biosensor's limit of detection (LoD) for detection of inactivated SARS-COV-2 antigen in phosphate buffer saline (PBS) was as low as 50 TCID50/ml. The biosensor specificity was confirmed using the influenza virus while the selectivity was confirmed using influenza polyclonal sera. Overall, the results showed that the biosensor is able to detect SARS-COV-2 in clinical samples (swabs) in 40 min with a sensitivity of 26 TCID50/ml.

A Simple, Pipette-free, and Power-free Device for Virus Nucleic Acid Detection

A portable, inexpensive, and easy-to-manufacture microfluidic device is developed for the detection of SARS-CoV-2 dsDNA fragments. In this device, four reaction chambers separated by carbon fiber rods are pre-loaded with isothermal amplification and CRISPR-Cas12a reagents. The reaction is carried out by simply pulling the rods, without the need for manual pipetting. To facilitate power-free pathogen detection, the entire detection is designed to be heated with a disposable hand warmer. After the CRISPR reaction, the fluorescence signal generated by positive samples is identified by naked eye, using an inexpensive flashlight. This simple and sensitive device will serve as a new model for the next-generation viral diagnostics in either hospital or resource-limited settings. © 2023 SPIE.

Advanced flow cytometry for biomedical applications.

Flow cytometry (FC) is a versatile tool with excellent capabilities to detect and measure multiple characteristics of a population of cells or particles. Notable advancements in in vivo photoacoustic FC, coherent Raman FC, microfluidic FC, and so on, have been achieved in the last two decades, which endows FC with new functions and expands its applications in basic research and clinical practice. Advanced FC broadens the tools available to researchers to conduct research involving cancer detection, microbiology (COVID-19, HIV, bacteria, etc.), and nucleic acid analysis. This review presents an overall picture of advanced flow cytometers and provides not only a clear understanding of their mechanisms but also new insights into their practical applications. We identify the latest trends in this area and aim to raise awareness of advanced techniques of FC. We hope this review expands the applications of FC and accelerates its clinical translation.

High-throughput microbead assay system with a portable, cost-effective Wi-Fi imaging module, and disposable multi-layered microfluidic cartridges for virus and microparticle detection, and tracking.

In recent years biomedical scientific community has been working towards the development of high-throughput devices that allow a reliable, rapid and parallel detection of several strains of virus or microparticles simultaneously. One of the complexities of this problem lies on the rapid prototyping of new devices and wireless rapid detection of small particles and virus alike. By reducing the complexity of microfluidics microfabrication and using economic materials along with makerspace tools (Kundu et al. 2018) it is possible to provide an affordable solution to both the problems of high-throughput devices and detection technologies. We present the development of a wireless, standalone device and disposable microfluidics chips that rapidly generate parallel readouts for selected, possible virus variants from a nasal or saliva sample, based on motorized and non-motorized microbeads detection, and imaging processing of the motion tracks of these beads in micrometers. Microbeads and SARS-CoV-2 COVID-19 Delta variant were tested as proof-of-concept for testing the microfluidic cartridges and wireless imaging module. The Microbead Assay (MA) system kit consists of a Wi-Fi readout module, a microfluidic chip, and a sample collection/processing sub-system. Here, we focus on the fabrication and characterization of the microfluidic chip to multiplex various micrometer-sized beads for economic, disposable, and simultaneous detection of up to six different viruses, microparticles or variants in a single test, and data collection using a commercially available, Wi-Fi-capable, and camera integrated device (Fig. 1).

Droplet magnetic-controlled microfluidic chip integrated nucleic acid extraction and amplification for the detection of pathogens and tumor mutation sites.

Traditional nucleic acid extraction and detection is based on open operation, which may cause cross-contamination and aerosol formation. This study developed a droplet magnetic-controlled microfluidic chip integrated nucleic acid extraction, purification and amplification. The reagent is sealed in oil to form a droplet, and the nucleic acid is extracted and purified by controlling the movement of the magnetic beads (MBs) through a permanent magnet, ensuring a closed environment. This chip can automatically extract nucleic acid from multiple samples within 20 min, and can be directly placed in the in situ amplification instrument for amplification without further transfer of nucleic acid, characterized by simple, fast, time-saving and labor-saving. The results showed that the chip was able to detect <10 copies/test SARS-CoV-2 RNA, and EGFR exon 21 L858R mutations were detected in H1975 cells as low as 4 cells. In addition, on the basis of the droplet magnetic-controlled microfluidic chip, we further developed a multi-target detection chip, which used MBs to divide the nucleic acid of the sample into three parts. And the macrolides resistance mutations A2063G and A2064G, and the P1 gene of mycoplasma pneumoniae (MP) were successfully detected in clinical samples by the multi-target detection chip, providing the possibility for future application in the detection of multiple pathogens.

Vessel-on-a-Chip: A Powerful Tool for Investigating Endothelial COVID-19 Fingerprints.

Coronavirus disease (COVID-19) causes various vascular and blood-related reactions, including exacerbated responses. The role of endothelial cells in this acute response is remarkable and may remain important beyond the acute phase. As we move into a post-COVID-19 era (where most people have been or will be infected by the SARS-CoV-2 virus), it is crucial to define the vascular consequences of COVID-19, including the long-term effects on the cardiovascular system. Research is needed to determine whether chronic endothelial dysfunction following COVID-19 could lead to an increased risk of cardiovascular and thrombotic events. Endothelial dysfunction could also serve as a diagnostic and therapeutic target for post-COVID-19. This review covers these topics and examines the potential of emerging vessel-on-a-chip technology to address these needs. Vessel-on-a-chip would allow for the study of COVID-19 pathophysiology in endothelial cells, including the analysis of SARS-CoV-2 interactions with endothelial function, leukocyte recruitment, and platelet activation. "Personalization" could be implemented in the models through induced pluripotent stem cells, patient-specific characteristics, or genetic modified cells. Adaptation for massive testing under standardized protocols is now possible, so the chips could be incorporated for the personalized follow-up of the disease or its sequalae (long COVID) and for the research of new drugs against COVID-19.

All-in-one microfluidic chip for 30-min quantitative point-of-care-testing of nucleic acids

Nucleic acid testing (NAT) is directly oriented to determining the genetic material of pathogens and is characterized by its high sensitivity and specificity, which are indispensable qualities in disease diagnosis. However, standard laboratory NAT methods require joint testing by highly trained inspectors using multiple instruments in professional laboratories. The entire process requires many manual steps, and the total testing time may range from 3 to 5h, indicating that these methods cannot be used to realize the demands of on-site rapid testing. In this study, we propose a microfluidic chip for the on-site and rapid detection of nucleic acids. We utilize dynamic sealing, ultrasound, and advanced control methods and integrate the entire process of reagent pre-storage, extraction, Real-time Quantitative polymerase chain reaction (qPCR), and fluorescence detection. The sensitivity of this system is in line with current clinical standards, and the nucleic acid quantification process is completed fully automated within 30min. Compared with conventional microfluidic chips, the proposed system has the advantages of high integration, low cost, and it may be produced at a high volume. Moreover, it can be used in a wide range of screening cases in the context of the COVID-19 pandemic and exhibits broad clinical application prospects.

Internet-of-medical-things integrated point-of-care biosensing devices for infectious diseases: Toward better preparedness for futuristic pandemics.

Microbial pathogens have threatened the world due to their pathogenicity and ability to spread in communities. The conventional laboratory-based diagnostics of microbes such as bacteria and viruses need bulky expensive experimental instruments and skilled personnel which limits their usage in resource-limited settings. The biosensors-based point-of-care (POC) diagnostics have shown huge potential to detect microbial pathogens in a faster, cost-effective, and user-friendly manner. The use of various transducers such as electrochemical and optical along with microfluidic integrated biosensors further enhances the sensitivity and selectivity of detection. Additionally, microfluidic-based biosensors offer the advantages of multiplexed detection of analyte and the ability to deal with nanoliters volume of fluid in an integrated portable platform. In the present review, we discussed the design and fabrication of POCT devices for the detection of microbial pathogens which include bacteria, viruses, fungi, and parasites. The electrochemical techniques and current advances in this field in terms of integrated electrochemical platforms that include mainly microfluidic- based approaches and smartphone and Internet-of-things (IoT) and Internet-of-Medical-Things (IoMT) integrated systems have been highlighted. Further, the availability of commercial biosensors for the detection of microbial pathogens will be briefed. In the end, the challenges while fabrication of POC biosensors and expected future advances in the field of biosensing have been discussed. The integrated biosensor-based platforms with the IoT/IoMT usually collect the data to track the community spread of infectious diseases which would be beneficial in terms of better preparedness for current and futuristic pandemics and is expected to prevent social and economic losses.

Recent applications of microfluidic immunosensors

[Display omitted] • Materials have an important effect on the reliability of microfluidic systems. • Magnetic particles are widely used in the fabrication of microfluidics immunosensors. • Near field communication-integrated microfluidics will more use in the future studies. The fast diagnosis of diseases is vital in the early stages of the cure of illnesses. Although conventional procedures have been broadly employed in clinics, newly presented microfluidic microchips are becoming more attractive. The benefits of the new microfluidic system involve more fast diagnosis, the need for low patient samples and reagents, user-friendly application, and high repeatability in the quantification of biomolecules. The primary aim of this review is to offer a summary of the effect of the applied nanomaterials in the fabrication of novel immunosensor-based microfluidic sticks and to carefully explore different applications of microfluidic systems in the determination of bioagents. New kinds of immunosensor-based microfluidic systems for coronavirus disease and HIV are also explored. The next types of biomedical diagnosis will mainly rely on point-of-care (POC) methods, which propose rapid and sensitive detections. However, microfluidic systems propose a high potential to fabricate reliable POC devices. [ FROM AUTHOR] Copyright of Microchemical Journal is the property of Elsevier B.V. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)

A novel isothermal digital amplification system and its application for absolute quantification of respiratory infectious virus

Herein, we establish a novel isothermal digital amplification system termed digital nicking and extension chain reaction system-based amplification (dNESBA) by utilizing the isothermal NESBA technique and the newly developed miniaturized fluorescence monitoring system (mFMS). dNESBA enables parallel isothermal NESBA reactions in more than 10,000 localized droplet microreactors and read the fluorescence signals rapidly in 150 s by mFMS. This system could identify the genomic RNA (gRNA) extracted from target respiratory syncytial virus A (RSV A) as low as 10 copies with remarkable specificity. The practical applicability of dNESBA was also successfully verified by reliably detecting the gRNA in the artificial sputum samples with excellent reproducibility and accuracy. Due to the intrinsic advantages of isothermal amplifying technique including the elimination of the requirement of thermocycling device and the enhanced portability of the miniaturized read-out equipment, the dNESBA technique equipped with mFMS could serve as a promising platform system to achieve point-of-care (POC) digital molecular diagnostics, enabling absolute and ultra-sensitive quantification of various infectious pathogens even in an early stage.Copyright © 2023

Microfluidic Devices for Biosensing

This article focuses on recent developments in microfluidic, lab on a chip technologies to enable point of care (POC) medical diagnostic devices, which can detect and monitor diseases outside of hospital settings. We provide a summary of the techniques to interface biological samples from macro-world to micro environments, on-chip processing steps to extract, isolate and transfer biomarkers of interest, and recent approaches to integrate advanced detection technologies in portable and easy to use devices. We highlight different applications of the proposed technologies, and review microfluidic methods proposed for the detection of infectious diseases such as COVID-19 caused by the novel Corona Virus. © 2023 Elsevier Ltd. All rights reserved

A Silicon-Based PDMS-PEG Copolymer Microfluidic Chip for Real-Time Polymerase Chain Reaction Diagnosis.

Polydimethylsiloxane (PDMS) has been widely used to make lab-on-a-chip devices, such as reactors and sensors, for biological research. Real-time nucleic acid testing is one of the main applications of PDMS microfluidic chips due to their high biocompatibility and transparency. However, the inherent hydrophobicity and excessive gas permeability of PDMS hinder its applications in many fields. This study developed a silicon-based polydimethylsiloxane-polyethylene-glycol (PDMS-PEG) copolymer microfluidic chip, the PDMS-PEG copolymer silicon chip (PPc-Si chip), for biomolecular diagnosis. By adjusting the modifier formula for PDMS, the hydrophilic switch occurred within 15 s after contact with water, resulting in only a 0.8% reduction in transmittance after modification. In addition, we evaluated the transmittance at a wide range of wavelengths from 200 nm to 1000 nm to provide a reference for its optical property study and application in optical-related devices. The improved hydrophilicity was achieved by introducing a large number of hydroxyl groups, which also resulted in excellent bonding strength of PPc-Si chips. The bonding condition was easy to achieve and time-saving. Real-time PCR tests were successfully conducted with higher efficiency and lower non-specific absorption. This chip has a high potential for a wide range of applications in point-of-care tests (POCT) and rapid disease diagnosis.

A Multiplexed SERS Microassay for Accurate Detection of SARS-CoV-2 and Variants of Concern.

Severe acute respiratory syndrome coronavirus 2 variants play an important role in predicting patient outcome during postinfection, and with growing fears of COVID-19 reservoirs in domestic and wild animals, it is necessary to adapt detection systems for variant detection. However, variant-specific detection remains challenging. Surface-enhanced Raman scattering is a sensitive and multiplexing technique that allows the simultaneous detection of multiple targets for accurate identification. Here we propose the development of a multiplex SERS microassay to detect both the spike and nucleocapsid structural proteins of SARS-CoV-2. The designed SERS microassay integrates gold-silver hollow nanobox barcodes and electrohydrodynamically induced nanomixing which in combination enables highly specific and sensitive detection of SARS-CoV-2 and the S-protein epitopes to delineate between ancestral prevariant strains with the newer variants of concern, Delta and Omicron. The microassay allows detection from as low as 20 virus/µL and 50 pg/mL RBD protein and can clearly identify the virus among infected versus healthy nasopharyngeal swabs, with the potential to identify between variants. The detection of both S- and N-proteins of SARS-CoV-2 and the differentiation of variants on the SERS microassay can aid the early detection of COVID-19 to reduce transmission rates and lead into adequate treatments for those severely affected by the virus.

Advanced Microfluidic Vascularized Tissues as Platform for the Study of Human Diseases and Drug Development.

The vascular system plays a critical role in human physiology and diseases. It is a complex subject to study using in vitro models due to its dynamic and three-dimensional microenvironment. Microfluidic technology has recently become a popular technology in various biological fields for its advantages in mimicking complex microenvironments to an extent not achievable by more conventional platforms. Microfluidic technologies can reproduce different vascular system-related structures and functions that can be utilized for drug development and human diseases studies. Herein we first review the relevant structural and functional vascular biology systems of various organ systems and then the fabrication methods to reproduce these vascular districts. We provide a thorough review of the latest achievement in vascular organ-on-chip modeling specific to lung, heart, and the brain microvasculature for drug screening and the study of human disorders.

A Microfluidic Biosensor for Rapid Detection of Covid-19

We have designed, fabricated, and tested a MEMS-based impedance biosensor for accurate and rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) using of clinical samples. The device consists of focusing region that concentrate low quantities of the virus present in the samples to a detectable threshold, trap region hat maximize the captured virus, and detection region to detect the virus with high selectivity and sensitivity, using an array of interdigitated electrodes (IDE) coated with a specific antibody. Changes in the impedance value due to the binding of the SARS-COV-2 antigen to the antibody will indicate positive or negative result. The device was able to detect inactivated SARS-COV-2 antigen present in phosphate buffer saline (PBS) with a concentration as low as 50 TCID50/ml in 30 minutes. In addition, the biosensor was able to detect SARS-COV-2 in clinical samples (swabs) with a sensitivity of 84 TCID50/ml, also in 30 minutes. © 2023 IEEE.

Mosaic Patterned Surfaces toward Generating Hardly‐Volatile Capsular Droplet Arrays for High‐Precision Droplet‐Based Storage and Detection

Mosaic Patterned SurfacesIn article number 2206274, Yanjun Hu, Lin Li, and co‐workers report a mosaic patterned surface‐based chip that acquires mutually independent and hardly‐volatile capsular droplet arrays. The concept shows high compatibility and practicability, paving the way for the new microfluidic chips used in COVID‐19 diagnosis and other high‐precision detection.

An Aluminum-Based Microfluidic Chip for Polymerase Chain Reaction Diagnosis.

Real-time polymerase chain reaction (real-time PCR) tests were successfully conducted in an aluminum-based microfluidic chip developed in this work. The reaction chamber was coated with silicone-modified epoxy resin to isolate the reaction system from metal surfaces, preventing the metal ions from interfering with the reaction process. The patterned aluminum substrate was bonded with a hydroxylated glass mask using silicone sealant at room temperature. The effect of thermal expansion was counteracted by the elasticity of cured silicone. With the heating process closely monitored, real-time PCR testing in reaction chambers proceeded smoothly, and the results show similar quantification cycle values to those of traditional test sets. Scanning electron microscope (SEM) and atomic force microscopy (AFM) images showed that the surface of the reaction chamber was smoothly coated, illustrating the promising coating and isolating properties. Energy-dispersive X-ray spectroscopy (EDS), X-ray photoelectron spectroscopy (XPS), and inductively coupled plasma-optical emission spectrometer (ICP-OES) showed that no metal ions escaped from the metal to the chip surface. Fourier-transform infrared spectroscopy (FTIR) was used to check the surface chemical state before and after tests, and the unchanged infrared absorption peaks indicated the unreacted, antifouling surface. The limit of detection (LOD) of at least two copies can be obtained in this chip.

Application of microfluidic technologies on COVID-19 diagnosis and drug discovery.

The ongoing coronavirus disease 2019 (COVID-19) pandemic has boosted the development of antiviral research. Microfluidic technologies offer powerful platforms for diagnosis and drug discovery for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnosis and drug discovery. In this review, we introduce the structure of SARS-CoV-2 and the basic knowledge of microfluidic design. We discuss the application of microfluidic devices in SARS-CoV-2 diagnosis based on detecting viral nucleic acid, antibodies, and antigens. We highlight the contribution of lab-on-a-chip to manufacturing point-of-care equipment of accurate, sensitive, low-cost, and user-friendly virus-detection devices. We then investigate the efforts in organ-on-a-chip and lipid nanoparticles (LNPs) synthesizing chips in antiviral drug screening and mRNA vaccine preparation. Microfluidic technologies contribute to the ongoing SARS-CoV-2 research efforts and provide tools for future viral outbreaks.

Microfluidics for COVID-19: From Current Work to Future Perspective.

Spread of coronavirus disease 2019 (COVID-19) has significantly impacted the public health and economic sectors. It is urgently necessary to develop rapid, convenient, and cost-effective point-of-care testing (POCT) technologies for the early diagnosis and control of the plague's transmission. Developing POCT methods and related devices is critical for achieving point-of-care diagnosis. With the advantages of miniaturization, high throughput, small sample requirements, and low actual consumption, microfluidics is an essential technology for the development of POCT devices. In this review, according to the different driving forces of the fluid, we introduce the common POCT devices based on microfluidic technology on the market, including paper-based microfluidic, centrifugal microfluidic, optical fluid, and digital microfluidic platforms. Furthermore, various microfluidic-based assays for diagnosing COVID-19 are summarized, including immunoassays, such as ELISA, and molecular assays, such as PCR. Finally, the challenges of and future perspectives on microfluidic device design and development are presented. The ultimate goals of this paper are to provide new insights and directions for the development of microfluidic diagnostics while expecting to contribute to the control of COVID-19.