Analytical and biomedical applications of microfluidics in traditional Chinese medicine research

Traditional Chinese medicine (TCM) has significant benefits in the prevention and treatment of diseases due to its unique theoretical system and research techniques. However, there are still key issues to be resolved in the full interpretation and use of TCM, such as vague active compounds and mechanism of action. Therefore, it is promising to promote the research on TCM through innovative strategies and advanced cutting-edge technologies. Microfluidic chips have provided controllable unique platforms for biomedical applications in TCM research with flexible composition and large-scale integration. In this review, the analysis and biomedical applications of microfluidics in the field of TCM are highlighted, including quality control of Chinese herbal medicines (CHMs), delivery of CHMs, evaluation of pharmacological activity as well as disease diagnosis. Finally, potential challenges and prospects of existing microfluidic technologies in the inheritance and innovation of TCM are discussed.Copyright © 2022 Elsevier B.V.

Development of a Lymphoid Organ-Chip to Evaluate Covid Vaccine Boosting Strategies

Background: Secondary lymphoid organs provide the adequate microenvironment for the development of antigen (Ag)-specific immune responses. The tight collaboration between CD4+ T cells and B cells in germinal centers is crucial to shape B cell fate and optimize antibody maturation. Dissecting these immune interactions remains challenging in humans, and animal models do not always recapitulate human physiology. To address this issue, we developed an in vitro 3D model of a human lymphoid organ. The model relies on a microfluidic device, enabling primary human cells to self-organize in an extracellular matrix (ECM) under continuous fluid perfusion. We applied this Lymphoid Organ-Chip (LO chip) system to the analysis of B cell recall responses to SARS-CoV-2 antigens. Method(s): We used a two-channel microfluidic Chip S1 from Emulate, where the top channel is perfused with antigen (spike protein or SARS-CoV-2 mRNA vaccine), while the bottom channel contains PBMC (n = 14 independent donors) seeded at high-density in a collagen-based ECM. Immune cell division and cluster formation were monitored by confocal imaging, plasmablast differentiation and spike-specific B cell amplification by flow cytometry, antibody secretion by a cell-based binding assay (S-flow). Result(s): Chip perfusion with the SARS-CoV-2 spike protein for 6 days resulted in the induction CD38hiCD27hi plasmablast maturation compared to an irrelevant BSA protein (P< 0.0001). Using fluorescent spike as a probe, we observed a strong amplification of spike-specific B cell (from 3.7 to 140-fold increase). In line with this rapid memory B cell response, spike-specific antibodies production could be detected as early as day 6 of culture. Spike perfusion also induced CD4+ T cell activation (CD38+ ICOS+), which correlated with the level of B cell maturation. The magnitude of specific B cell amplification in the LO chip was higher than in 2D and 3D static cultures at day 6, showing the added value of 3D perfused culture for the induction of recall responses. Interestingly, the perfusion of mRNA-based SARS-CoV-2 vaccines also led to strong B cell maturation and specific B cell amplification, indicating that mRNA-derived spike could be expressed and efficiently presented in the LO chip. Conclusion(s): We developed a versatile Lymphoid Organ-Chip model suitable for the rapid evaluation of B cell recall responses. The model is responsive to protein and mRNA-encoded antigens, highlighting its potential in the evaluation of SARS-CoV-2 vaccine boosting strategies.

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 handheld plug-and-play microfluidic liquid handling automation platform for immunoassays.

Lab-on-a-chip technologies and microfluidics have pushed miniaturized liquid handling to unprecedented precision, integration, and automation, which improved the reaction efficiency of immunoassays. However, most microfluidic immunoassay systems still require bulky infrastructures, such as external pressure sources, pneumatic systems, and complex manual tubing and interface connections. Such requirements prevent plug-and-play operation at the point-of-care (POC) settings. Here we present a fully automated handheld general microfluidic liquid handling automation platform with a plug-and-play 'clamshell-style' cartridge socket, a miniature electro-pneumatic controller, and injection-moldable plastic cartridges. The system achieved multi-reagent switching, metering, and timing control on the valveless cartridge using electro-pneumatic pressure control. As a demonstration, a SARS-CoV-2 spike antibody sandwich fluorescent immunoassay (FIA) liquid handling was performed on an acrylic cartridge without human intervention after sample introduction. A fluorescence microscope was used to analyze the result. The assay showed a limit of detection at 31.1 ng/mL, comparable to some previously reported enzyme-linked immunosorbent assays (ELISA). In addition to automated liquid handling on the cartridge, the system can operate as a 6-port pressure source for external microfluidic chips. A rechargeable battery with a 12 V 3000 mAh capacity can power the system for 42 h. The footprint of the system is 16.5 × 10.5 × 7 cm, and the weight is 801 g, including the battery. The system can find many other POC and research applications requiring complex liquid manipulation, such as molecular diagnostics, cell analysis, and on-demand biomanufacturing.

Comparison of Illumination Methods for Flow-Through Optofluidic Biosensors.

Optofluidic biosensors have become an important medical diagnostic tool because they allow for rapid, high-sensitivity testing of small samples compared to standard lab testing. For these devices, the practicality of use in a medical setting depends heavily on both the sensitivity of the device and the ease of alignment of passive chips to a light source. This paper uses a model previously validated by comparison to physical devices to compare alignment, power loss, and signal quality for windowed, laser line, and laser spot methods of top-down illumination.

Integrated smart analytics of nucleic acid amplification tests via paper microfluidics and deep learning in cloud computing

Pandemics such as COVID-19 have exposed global inequalities in essential health care. Here, we proposed a novel analytics of nucleic acid amplification tests (NAATs) by combining paper microfluidics with deep learning and cloud computing. Real-time amplifications of synthesized SARS-CoV-2 RNA templates were performed in paper devices. Information pertained to on-chip reactions in time-series format were transmitted to cloud server on which deep learning (DL) models were preloaded for data analysis. DL models enable prediction of NAAT results using partly gathered real-time fluorescence data. Using information provided by the G-channel, accurate prediction can be made as early as 9 min, a 78% reduction from the conventional 40 min mark. Reaction dynamics hidden in amplification curves were effectively leveraged. Positive and negative samples can be unbiasedly and automatically distinguished. Practical utility of the approach was validated by cross-platform study using clinical datasets. Predicted clinical accuracy, sensitivity and specificity were 98.6%, 97.6% and 99.1%. Not only the approach reduced the need for the use of bulky apparatus, but also provided intelligent, distributable and robotic insights for NAAT analysis. It set a novel paradigm for analyzing NAATs, and can be combined with the most cutting-edge technologies in fields of biosensor, artificial intelligence and cloud computing to facilitate fundamental and clinical research.Copyright © 2023 Elsevier Ltd

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.

On-site bioaerosol sampling and detection in microfluidic platforms.

As the recent coronavirus disease (COVID-19) pandemic and several severe illnesses such as Middle East respiratory syndrome coronavirus (MERS-CoV), Influenza A virus (IAV) flu, and severe acute respiratory syndrome (SARS) have been found to be airborne, the importance of monitoring bioaerosols for the control and prevention of airborne epidemic diseases outbreaks is increasing. However, current aerosol collection and detection technologies may be limited to on-field use for real-time monitoring because of the relatively low concentrations of targeted bioaerosols in air samples. Microfluidic devices have been used as lab-on-a-chip platforms and exhibit outstanding capabilities in airborne particulate collection, sample processing, and target molecule analysis, thereby highlighting their potential for on-site bioaerosol monitoring. This review discusses the measurement of airborne microorganisms from air samples, including sources and transmission of bioaerosols, sampling strategies, and analytical methodologies. Recent advancements in microfluidic platforms have focused on bioaerosol sample preparation strategies, such as sorting, concentrating, and extracting, as well as rapid and field-deployable detection methods for analytes on microfluidic chips. Furthermore, we discuss an integrated platform for on-site bioaerosol analyses. We believe that our review significantly contributes to the literature as it assists in bridging the knowledge gaps in bioaerosol monitoring using microfluidic platforms.

Present status of microfluidic PCR chip in nucleic acid detection and future perspective

Polymerase chain reaction (PCR) amplifies specific fragment of DNA molecules and has been extensively applied in fields of pathogens and gene mutation detection, food safety and clinical diagnosis which on the other hand, holds the drawbacks of large size instrument, high heat dissipation etc. It has been demonstrated that microfluidics technique coupling with PCR reaction exhibits characteristics of integration, automatization, miniaturization, and portability. Meanwhile, various designed fabrication of microchip could contribute to diverse applications. In this review, we summarized major works about a variety of microfluidic chips equipped with several kinds of PCR techniques (PCR, RT-PCR, mPCR, dPCR) and detection methods like fluorescence, electrochemistry, and electrophoresis detection. The development and application of PCR-based microfluidic chip in pathogen and gene mutation detection, diseases prevention and diagnosis, DNA hybridization and low-volume sample treatment were also discussed. Copyright © 2022 Elsevier B.V.

Molecular detection of SARS-COV-2 in exhaled breath at the point-of-need.

The SARS-CoV-2 pandemic has highlighted the need for improved technologies to help control the spread of contagious pathogens. While rapid point-of-need testing plays a key role in strategies to rapidly identify and isolate infectious patients, current test approaches have significant shortcomings related to assay limitations and sample type. Direct quantification of viral shedding in exhaled particles may offer a better rapid testing approach, since SARS-CoV-2 is believed to spread mainly by aerosols. It assesses contagiousness directly, the sample is easy and comfortable to obtain, sampling can be standardized, and the limited sample volume lends itself to a fast and sensitive analysis. In view of these benefits, we developed and tested an approach where exhaled particles are efficiently sampled using inertial impaction in a micromachined silicon chip, followed by an RT-qPCR molecular assay to detect SARS-CoV-2 shedding. Our portable, silicon impactor allowed for the efficient capture (>85%) of respiratory particles down to 300 nm without the need for additional equipment. We demonstrate using both conventional off-chip and in-situ PCR directly on the silicon chip that sampling subjects' breath in less than a minute yields sufficient viral RNA to detect infections as early as standard sampling methods. A longitudinal study revealed clear differences in the temporal dynamics of viral load for nasopharyngeal swab, saliva, breath, and antigen tests. Overall, after an infection, the breath-based test remains positive during the first week but is the first to consistently report a negative result, putatively signalling the end of contagiousness and further emphasizing the potential of this tool to help manage the spread of airborne respiratory infections.

Lab-on-a-Chip Devices for Point-of-Care Medical Diagnostics

The recent coronavirus (COVID-19) pandemic has underscored the need to move from traditional lab-centralized diagnostics to point-of-care (PoC) settings. Lab-on-a-chip (LoC) platforms facilitate the translation to PoC settings via the miniaturization, portability, integration, and automation of multiple assay functions onto a single chip. For this purpose, paper-based assays and microfluidic platforms are currently being extensively studied, and much focus is being directed towards simplifying their design while simultaneously improving multiplexing and automation capabilities. Signal amplification strategies are being applied to improve the performance of assays with respect to both sensitivity and selectivity, while smartphones are being integrated to expand the analytical power of the technology and promote its accessibility. In this chapter, we review the main technologies in the field of LoC platforms for PoC medical diagnostics and survey recent approaches for improving these assays. [GRAPHICS] .

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.

Smart Diagnostics: Combining Artificial Intelligence and In Vitro Diagnostics.

We are beginning a new era of Smart Diagnostics-integrated biosensors powered by recent innovations in embedded electronics, cloud computing, and artificial intelligence (AI). Universal and AI-based in vitro diagnostics (IVDs) have the potential to exponentially improve healthcare decision making in the coming years. This perspective covers current trends and challenges in translating Smart Diagnostics. We identify essential elements of Smart Diagnostics platforms through the lens of a clinically validated platform for digitizing biology and its ability to learn disease signatures. This platform for biochemical analyses uses a compact instrument to perform multiclass and multiplex measurements using fully integrated microfluidic cartridges compatible with the point of care. Image analysis digitizes biology by transforming fluorescence signals into inputs for learning disease/health signatures. The result is an intuitive Score reported to the patients and/or providers. This AI-linked universal diagnostic system has been validated through a series of large clinical studies and used to identify signatures for early disease detection and disease severity in several applications, including cardiovascular diseases, COVID-19, and oral cancer. The utility of this Smart Diagnostics platform may extend to multiple cell-based oncology tests via cross-reactive biomarkers spanning oral, colorectal, lung, bladder, esophageal, and cervical cancers, and is well-positioned to improve patient care, management, and outcomes through deployment of this resilient and scalable technology. Lastly, we provide a future perspective on the direction and trajectory of Smart Diagnostics and the transformative effects they will have on health care.

Microfluidics: Simulating the gut microbiome for early cancer detection

Background: Cancer is the second leading cause of death globally and ∼39.5% of people will be diagnosed with cancer at some point during their lifetimes. Thus, there is an unmet need to identify novel strategies for early cancer detection and prevention. The emerging evidence suggests that the gut microbiome has a role in promoting cancer. This microbiome including bacteria plays a vital role in maintaining homeostasis in the body. An imbalance in bacterial composition may cause diseases including cancer. Here we developed a microfluidic chip that can accurately simulate the gut microbiome to test the effects of bacteria and therapies on cancer cells. Methods and Results: To test the causal effect of bacteria on cancer, we developed a new highthroughput microfluidic device for simulating the environment of the gut. Initially, we used the photolithography technique where we designed the chip in AutoCAD and fabricated using photoresist resins and Polydimethylsiloxane (PDMS). Next, we tested the effect of bacteria on the growth of colorectal cancer cells. For this, we cultured colorectal cancer cells (HCT-116) with lipopolysaccharide (LPS), which is found in the outer membrane of bacteria, as well as the Bacillus bacteria in our microfluidics. Our data show that both LPS and Bacillus significantly accelerate the growth of cancer cells 2.02 times (p value = 0.012) and 1.58 times (p value = 0.011), respectively, over a 4 day culture period. These results show that the increased presence of certain bacteria can promote cancer cell growth and that our chip can be used to test the specific correlation between bacteria and cancer cell growth. The previously described method was inefficient and time-consuming. To overcome this limitation, we designed a new chip that allows running 16 samples at once with improved efficiency and accuracy. The template of the device that had 16 microfluidic channels was printed by a 3D printer and used for PDMS replica molding. The PDMS device was attached to the modified multiwell plate to feed media to and collect waste from each channel in a high-throughput manner. In the initial design, the bacteria grew faster than cancer cells taking over the chips. Our new design has dual layered chambers to keep bacteria and cancer cells separated by a membrane, allowing only bacterial secretions to pass through the membrane to cancer cells, mimicking the human gut. The new design also allowed the chip to maintain continuous microfluidic flow and a hypoxic environment. Conclusion: Our research demonstrates that the new microfluidic device has broader implications including simulating other body organs such as the lung and liver, and testing the impact of viruses such as influenza and COVID-19 on human cells. This device can be used to test both the effect of bacteria and new treatment on clinical samples for the identification of personalized therapy, thus reducing the need for mouse model testing, which is a lengthy and expensive process.

Strategies for sensitivity enhancement of point-of-care devices

Point-of-care (POC) technology reduces the time required for diagnosis at a reduced cost to facilitate early treatment, continuous monitoring, and prevention of fatal outcomes. Biosensors are the key to the development of reliable and accurate POC devices as they are capable of detecting clinical biomarkers based on bio-recognition events. Paper-based microfluidics and lateral flow assays (LFAs) are the most commonly used techniques for the development of POC devices. Electrochemical biosensors provide high sensitivity and reproducibility in comparison to optical biosensors. Sensitivity enhancement of POC devices is imperative to lower their detection limit for improved analysis of target biomarkers at low concentrations. In this review, we have discussed the need for sensitivity enhancement in POC devices. Various sensitivity enhancement strategies such as physical, chemical, electrochemical, nanomaterial, nucleic acid, enzymatic, label-based, etc. are discussed along with numerous examples. The role of biosensors in the sensitivity enhancement of POC devices is also described herein. We have illustrated the relationship between sensitivity and the limit of detection of POC devices. Several sensitivity enhancement strategies that have been either adopted or have the potential to be realized for POC devices have been summarized in tabular form. In terms of future perspectives, the sensitivity enhancement of POC devices for the detection of important biomarkers is yet to be comprehended copiously amid the rising market for POC devices.

Lab-on-a-Chip Immunoassay for Prediction of Severe COVID-19 Disease.

Most people infected by the SARS-CoV-2 virus which causes COVID-19 disease experience mild or no symptoms. Severe forms of the disease are often marked by a hyper-inflammatory response known as a cytokine storm. Thus, biomarker tests which can identify these patients and place them on the appropriate treatment regime at the earliest possible phase would help to improve outcomes. Here we describe an automated microarray-based immunoassay using the Fraunhofer lab-on-a-chip platform for analysis of C-reactive protein due to its role in the hyper-inflammatory response.

A Rapid User-Friendly Lab-on-a-Chip Microarray Platform for Detection of SARS-CoV-2 Variants.

Since the original SARS-CoV-2 virus emerged from Wuhan, China, in late December 2019, a number of variants have arisen with enhanced infectivity, and some may even be capable of escaping the existing vaccines. Here we describe a rapid automated nucleic acid microarray hybridization and readout in less than 15 min using the Fraunhofer lab-on-a-chip platform for identification of bacterial species and antibiotic resistance. This platform allows a fast adaptation of new biomarkers enabling identification of different genes and gene mutations, such as those seen in the case the SARS-CoV-2 variants.

A Supported Lipid Bilayer-Based Lab-on-a-Chip Biosensor for the Rapid Electrical Screening of Coronavirus Drugs.

With the rapid spread and multigeneration variation of coronavirus, rapid drug development has become imperative. A major obstacle to addressing this issue is adequately constructing the cell membrane at the molecular level, which enables in vitro observation of the cell response to virus and drug molecules quantitatively, shortening the drug experiment cycle. Herein, we propose a rapid and label-free supported lipid bilayer-based lab-on-a-chip biosensor for the screening of effective inhibition drugs. An extended gate electrode was prepared and functionalized by an angiotensin-converting enzyme II (ACE2) receptor-incorporated supported lipid bilayer (SLB). Such an integrated system can convert the interactions of targets and membrane receptors into real-time charge signals. The platform can simulate the cell membrane microenvironment in vitro and accurately capture the interaction signal between the target and the cell membrane with minimized interference, thus observing the drug action pathway quantitatively and realizing drug screening effectively. Due to these label-free, low-cost, convenient, and integrated advantages, it is a suitable candidate method for the rapid drug screening for the early treatment and prevention of worldwide spread of coronavirus.

Microfluidic Platforms for the Isolation and Detection of Exosomes: A Brief Review.

Extracellular vesicles (EVs) are a group of communication organelles enclosed by a phospholipid bilayer, secreted by all types of cells. The size of these vesicles ranges from 30 to 1000 nm, and they contain a myriad of compounds such as RNA, DNA, proteins, and lipids from their origin cells, offering a good source of biomarkers. Exosomes (30 to 100 nm) are a subset of EVs, and their importance in future medicine is beyond any doubt. However, the lack of efficient isolation and detection techniques hinders their practical applications as biomarkers. Versatile and cutting-edge platforms are required to detect and isolate exosomes selectively for further clinical analysis. This review paper focuses on lab-on-chip devices for capturing, detecting, and isolating extracellular vesicles. The first part of the paper discusses the main characteristics of different cell-derived vesicles, EV functions, and their clinical applications. In the second part, various microfluidic platforms suitable for the isolation and detection of exosomes are described, and their performance in terms of yield, sensitivity, and time of analysis is discussed.