Capillary Microfluidic Chips for Point-of-Care Testing

We might be at the turning point where research in microfluidics undertaken in academia and industrial research laboratories, and substantially sponsored by public grants, may provide a range of portable and networked diagnostic devices. In this Progress Report, an overview on microfluidic devices that may become the next generation of point-of-care (POC) diagnostics is provided. First, we describe gaps and opportunities in medical diagnostics and how microfluidics can address these gaps using the example of immunodiagnostics. Next, we conceptualize how different technologies are converging into working microfluidic POC diagnostics devices. Technologies are explained from the perspective of sample interaction with components of a device. Specifically, we detail materials, surface treatment, sample processing, microfluidic elements (such as valves, pumps, and mixers), receptors, and analytes in the light of various biosensing concepts. Finally, we discuss the integration of components into accurate and reliable devices.

ABSTRACTCryptococcal disease (CD) is a leading cause of mortality among individuals with advanced HIV disease (AHD). Screening with serum cryptococcal antigen (sCrAg) lateral flow assay (LFA) enables early detection of subclinical disease but requires venipuncture and laboratory processing. Clinic-based point of care (POC) CrAg screening tests using urine or fingerprick whole blood could facilitate early diagnosis of CD. We evaluated the diagnostic performance of POC clinic-based fingerprick whole blood and urine CrAg compared to the gold standard laboratory sCrAg LFA in screening for CD among asymptomatic individuals with CD4 counts of <200 cells/μL in Harare, Zimbabwe. sCrAg positive participants who consented to a lumbar puncture also had cerebrospinal fluid (CSF) CrAg testing and titers for CSF-positive specimens. A total of 1,333 individuals were screened, and over half (56.6%) were males. The median (interquartile range) CD4 count was 27.5 (11–46) cells/μL. We found a sensitivity of 63.8% (95% CI: 54.8–72.1) and specificity of 84.0% (95% CI: 81.7–86.0) for urine CrAg, and a sensitivity of 48.0% (95% CI: 39.1–57.1) and specificity of 99.5% (95% CI: 98.9–99.8) was found for fingerprick whole blood. The sensitivity of both POC CrAg tests increased in individuals with sCrAg titers of ≥1:160, CD4 count of <50 cells/μL and disseminated central nervous system (CNS) disease. Clinic-based POC urine and fingerprick whole blood CrAg testing performed better in screening for CD among AHD patients with CNS disease. More sensitive assays to identify AHD patients with asymptomatic CD are needed.IMPORTANCE Cryptococcal disease (CD) remains a leading cause of morbidity and mortality among individuals with advanced HIV disease (AHD). Identifying point of care (POC) approaches to screening for CD in asymptomatic individuals is important to guide therapeutic management. We evaluated the use of POC fingerprick whole blood and urine testing for cryptococcal disease in patients with AHD as compared with laboratory-based serum antigen testing. POC fingerprick whole blood and urine testing had low sensitivity and specificity in asymptomatic individuals with AHD. Most analysis has focused on evaluating test performance in symptomatic individuals. Here we show that POC testing with whole blood and urine samples should not be used to screen for asymptomatic CD in AHD.

Life Sciences Discovery and Technology Highlights

Point of care (POC) testing in communities, home settings, and primary healthcare centers is believed to have tremendous potential in reducing delays in diagnosing and initiating treatment for diseases such as HIV, tuberculosis, syphilis, and malaria. Quick diagnosis and further management decisions completed in the same clinical encounter or at least the same day, while the patient waits, promise to overcome delays associated with conventional laboratory-based testing. However, the availability of cheap, simple, and rapid tests that can be conducted outside laboratories does not automatically ensure successful POC testing. In order to understand the new roles and challenges medical devices such as these encounter, we need to study how diagnostics are used at the POC and integrated into workflow and patient pathways. This chapter reviews selected results from a qualitative research project on barriers to POC testing in India and South Africa and discusses them comparatively. The project used semi-structured interviews and focus group discussions to examine diagnostic practices across major diseases and actors in homes, clinics, communities, hospitals, and laboratories in South Africa and India. In comparing selected results, it becomes clear that both countries have very different diagnostic eco-systems that provide very different conditions for POC testing. The chapter concludes by reflecting on how to take such insights into account when designing POC testing programs.

Optical trapping allows manipulating very small objects – varying from Angstoms to micron size particles – without physical contact by taking advantage of laser light. This technique, relying on light momentum transfer, allows manipulating biological matter (such as cells, organelles, vesicles or chemically functionalized artificial particles, etc.) and offers promising potentialities for research in biotechnology and biochemistry. Individually confining a large number of microscopic objects opens new ways for the downscaling of analysis tools for drug screening, particles sorting and the assessment of statistical data. In particular, the combination of optical trapping with microfluidics greatly enlarges the possibilities offered by both techniques. This PhD work takes place in a research aiming at developing novel bio-analytical instrumentation relying on large arrays of optical traps compatible with microfluidic systems. The dissertation focuses on the use of micro-optics for generating extended matrices of three-dimensional optical traps capable of capturing a large number of particles within microfluidic environments. The stability of optical traps is first studied with special emphasis on the conditions present in microfluidic flows, both from a theoretical and an experimental point of view, and trapping forces achievable on biological cells are investigated. A multiple optical trapping system relying on microlens arrays, adapted to work on commercial microscopes, is shown to be capable of generating arrays of more than 500 three-dimensional traps. Simultaneously trapping in multiple planes is also evidenced using matrices of microlenses, thanks to a self-imaging effect of the array of traps. Furthermore, possibilities offered by multiple optical trapping within microfluidic environments are explored. Polystyrene spheres as well as biological particles, such as native vesicles, can be trapped in arrays, manipulated inside microfluidic devices and analyzed in parallel through fluorescence microscopy while extremely small quantities of chemical reagents are flown past the array. Finally, for the sake of system miniaturization and further extension of the number of traps, multiple three-dimensional optical trapping based on arrays of miniaturized high numerical aperture parabolic mirrors is proposed, allowing the optics necessary for optical trapping, fluorescence excitation and collection to be integrated at the level of a microfluidic chip. Such miniaturized mirror matrices did not even exist before this thesis; their fabrication and characterization are detailed in this dissertation.

Background: In sub-Saharan Africa, gonococcal and chlamydial infections are usually managed using the syndromic approach. However, many infections are asymptomatic in women, and the syndromic algorithm has poor sensitivity and...

BackgroundHeightened pressures on hospitals and Emergency Medical Services (EMS) due to growing demand and staff shortages have led to prolonged ambulance response times and delays in handing over the care...

Structure optimization method of microfluidic paper chip based on image grey-level statistics for chromogenic reaction

Silver nanoparticles (AgNP) can help prevent infection of virus and bacteria. The size and morphology of AgNP can be crucial to function, with smaller nanoparticles (< 20 nm) able to penetrate the cell wall. This is significant as oxidative stress and genotoxicity are associated with some sizes and coatings of AgNP, contraindicating the use of AgNP to reduce infection. We present evidence that a microfluidic chip can synthesize larger sizes and distributions of AgNP from the nano-to-micro size range. We show results from a microfluidic mixing chip that can produce a wide range of nano-to-micro size (~ 24–400 nm) AgNP. Synthesis is based on a modified Turkevich method, using a single-step AgNP synthesis on the microfluidic chip using two chemical components, trisodium citrate (NaCit) and AgNO3. To make AgNP more accessible, we describe the microfluidic chip and conditions capable of synthesis. We also describe how modification of flow rate and chemical reagent concentration change particle diameter during production. In our experiments, we found that AgNP production created a visible adsorption line in the microfluidic device, possibly owing to AgNP surface interaction at the polydimethylsiloxane (PDMS) interface. We characterize these particles with dynamic light scattering (DLS), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). Based on optical light microscopy, we hypothesize that AgNP formation primarily occurs at the interface between the two chemical reagent streams. We also conclude that AgNP size increases could be due to interaction with the PDMS surface, which is known to be porous. Future work will help to understand how surface interaction may influence the formation of larger particles.

Lateral flow assay (LFA) is an important point-of-care (POC) test platform due to the associated portability, on-site testing, and low cost for diagnosis of pathogen infections and disease biomarkers. However, compared to high-end analyzers in hospitals, LFA devices, in particular, paper- based LFA tests, fall short in accuracy. This study focuses on two ways to improve LFAs: (1) using cellulose fibers, rather than glass fibers for a sample pad, and (2) incorporating a one-step simple, facile, and low cost PVA dam into the LFA. Both strategies (cellulose fiber as a sample pad and water dissolvable PVA dam) contributed to delaying the controlled biomolecule’s flow through the nitrocellulose membrane’s capillary channels resulting in increased bio-recognition time, thus contributing to the enhancement of LFA sensitivity. PVA modified cellulose fiber-based LFA demonstrated 10 times higher sensitivity than the cellulose fiber-based unmodified LFA, whereas 2 times enhancement was obtained in the cellulose fiber-based sample pad LFA compared to the glass fiber-based sample pad LFA. Ultimately, 20 times increase in sensitivity was achieved in the modified LFA device. This study shows that PVA and eco-friendly cellulose fibers could be incorporated into other paper based POC testing devices for future development.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10570-021-04083-3.

These data supplement a publication describing the auto-fluorescence of materials commonly used in paper-based microfluidics. The data presented include excitation-emission matrices of nitrocellulose, cellulose, and glass fiber manufactured by GE and Millipore, among others, and fluorescence lifetime data for Millipore HF120 nitrocellulose. The abstract of the publication follows: ------------------------------------------------------------- Porous media made of nitrocellulose and glass fiber are common “paper” substrates for lateral flow assays, microfluidic paper analytical devices, and other point-of-care diagnostic assays. Such assays commonly use optical labels such as gold nanoparticles, latex beads, or fluorescent nanoparticles to visualize the presence of analytes. Fluorescent labels are commonly used in bioassays to enhance sensitivity, but paper autoluminescence worsens signal-to-noise ratios of paper-based fluorescence assays. To date, there exists no systematic investigation of autoluminescence wavelengths or lifetimes of paper membranes. In response, we quantified the autoluminescence of commonly-used papers across the visible spectrum via excitation-emission spectroscopy and time-resolved fluorescence spectroscopy, and demonstrate that autoluminescence is solely due to autofluorescence with lifetimes of about 5 ns in the visible spectrum. Counterintuitively, we found that spectroscopy alone does not provide sufficient information to select candidate paper substrates for fluorophore-labeled assays. Therefore, we developed a simple quantitative framework to select a low-fluorescence substrate that minimizes both the overlap of paper and fluorophore emission spectra and the fluorescence intensity on an imaging system of interest (such as a gel imager). Use of this framework was shown to improve the limit of detection of an influenza A nucleoprotein immunoassay by over 50%. The tools developed in this manuscript may similarly enable other assay developers to screen appropriate, low-fluorescence paper substrates and enhance the sensitivity of paper-based fluorescence assays.

Limit of detection (LOD), speed and cost for some of the most important diagnostics tools for COVID-19 i.e., lateral flow assays (LFA), enzyme-linked immunosorbent assay (ELISA) and Polymerase Chain Reaction (PCR) all benefitted from both the financial and regulatory support brought about by the pandemic. Before COVID-19, LFA was the fastest and least expensive but came with the poorest limit of detection (LOD). The slowest, most expensive but with the best (lowest) LOD was PCR. In between those two extremes, in terms of cost, LOD and speed, was an ELISA . Today, through the “COVID-19 accelerator effect” there are point of care (POC) reverse transcriptase quantitative PCR (ie., RT-qPCR) COVID-19 tests on the market that provide sample to answer in under 20 minutes, while retaining the same very low LOD but at a lower cost [1]. There are even POC PCR tests under development that come in at less than 10 minutes: beating both LFA and ELISAs in terms of time and LOD [2]. Although RT-qPCR has gained the most in overall performance, signal-amplified POC LFAs are possible now with an LOD that rivals that of an ELISA.However, implementing the next generation PCR equipment in point-of-care settings, remains very challenging because of difficulties in reaching a 10 minute or below sample to answer time , instrument complexity and cost. In this work, we discuss the engineering challenges involved in the development of high-speed microfluidic PCR equipment. To guide this review/comparison of PCR advances we introduce three parameters. The first one is the overall sample to answer time (t), the second is called lambda (λ) and is a measure of the degree to which stochastic effects interfere with obtaining analytical results and thereby setting the minimal number of copies required per volume. The third parameter gamma (γ), introduced first by Dong et al [3], is the ratio of thermal cycling time (in min) to the heat cycled sample volume (in mL). It describes the trade-off between the speed of the PCR system and the desired level of sensitivity. It can be interpreted as a system efficiency: the most efficient system heat cycles the largest amount of sample in the smallest amount of time.With these three criteria in mind we set out to suggest designs for an optimized POC reverse transcriptase (RT) quantitative qPCR or RT-qPCR system. Applications of RT-qPCR include quantifying gene expression levels, validating RNA interference (RNAi), and detecting pathogens such as viruses. The aim of this specific POC RT-qPCR system is to provide sample to answer in ten minutes or less for a respiratory pathogens panel, with an LOD of 500 copies/mL and the capability to multiplex (8 chambers) while remaining portable and low cost.Finally we introduce two of the most daunting, remaining, microfluidics challenges that both need to be tackled to implement an ideal microfluidic platform like the POC RT-qPCR system described above. One is liquid storage and the other is proportional valves on a disposable microfluidic platform. Liquid storage is essential when the assay requires repeated washes, if these washes can come from one and the same reservoir via a proportional valve, real estate is gained and system complexity is reduced. The storage issue is further complicated when the design also calls for storing lyophilized or dried reagents on the same disposable. We compare some of the most recent approaches to liquid storage and proportional valving that we believe will enable the fast POC diagnostic platforms of the future. 1. Tsang, Hin Fung, Wai Ming Stanley Leung, Lawrence Wing Chi Chan, William Chi Shing Cho, and Sze Chuen Cesar Wong. “Performance Comparison of the Cobas® Liat® and Cepheid® GeneXpert® Systems on SARS-CoV-2 Detection in Nasopharyngeal Swab and Posterior Oropharyngeal Saliva.” Expert Review of Molecular Diagnostics 21, no. 5 (May 4, 2021): 515–18. 2. BUCKLAND, Justin, Tom JELLICOE, Alex STOKOE, and Amaru ARAYA-WILLIAMS. “VARIABLE TEMPERATURE REACTOR, HEATER AND CONTROL CIRCUIT FOR THE SAME.” Patent Application, January 30, 2020. 3. Dong, Xiaobin, Luyao Liu, Yunping Tu, Jing Zhang, Guijun Miao, Lulu Zhang, Shengxiang Ge, Ningshao Xia, Duli Yu, and Xianbo Qiu. “Rapid PCR Powered by Microfluidics: A Quick Review under the Background of COVID-19 Pandemic.” Trends in Analytical Chemistry 143 (October 2021): 116377.

Prostate cancer is a life-threatening malignant disease, one the most common cancer types in men and leading cause of death among men because of cancer. Considering the state of the art for the diagnosis of prostate cancer a man overall has to suffer uncomfortable and invasive medical tests, usually in the environment of a hospital or medical clinic, long waiting times for the results of the medical assessment, and, nonetheless still risking the possibility to obtain an uncertain medical outcome. In this case the results of the diagnosis tests do not clearly and certainly indicate the actual existence or absence of cancer. Therefore, the medical test should be repeated and/or further inconvenient tests should be performed increasing the time and financial costs, as well as, the emotional stress for the patient. Meanwhile, reliable, low-cost, and accessible therapeutic compound monitoring systems for individualized healthcare, and especially for treatment of malignant diseases, such as prostate cancer and AIDS are a very important aspect in medical practice. This requirements are even more highlighted for drugs demonstrating very narrow therapeutic window, that is also depicted at low concentrations. Moreover, point-of-care biosensors for therapeutic compounds monitoring bring the treatment to the patient0s bedside, minimizing the interfering factors in medical treatments, providing a flexible and robust cut-of-technology for the monitoring of treatment, with higher efficacy and lower side effects for patients. The realization of novel ultrasensitive nano-bio-sensors for the direct and label-free detection of chemical and biological species, that present high reliability, robustness and the advantage of a quick data acquisition is therefore, the main objective of the thesis. The work presented in this thesis aims to achieve optimum sensing output in both diagnostics and therapeutics fields, for detecting a malignant disease at early stages, and for efficient screening of therapeutic drugs used in chemotherapy, allowing the possibility for continuous monitoring. Silicon nanowires with their unique properties such as the high surface-to-volume ratio and the size comparable to biomolecules, and combined with the specificity of immune-sensing techniques, are natural candidates for the fabrication of nanosensors. Moreover, memristive properties, exhibited by nanofabricated devices coupled with biological processes, introduce innovating solutions to the biosensing filed. In addition, graphene-based nanostructures, only recently suggested in literature, are hereby demonstrated as novel structures in electrochemistry for effective drug monitoring. Finally, complete biosensing platforms are suggested through the design and realization of a printed circuit electronic board for automatized sensing readout, along with the fabrication of a microfluidic circuit for straightforward bio-functionalization and bio-detection procedures. These aspects pave the way for the actual development of point-of-care devices, a significant aspect for treatment in hospitals and bench to bedside applications. New possibilities for multi-panel detection are also presented by the selective functionalization of individual nanodevices on a singe chip. This new aspect shows high potential for Lab-on-a Chip applications and multiplex detection of different biomarkers on the same chip, or for both biomarkers and therapeutic compounds paving the way for theranostics.

BackgroundUganda Ministry of Health (MOH) recommends a first HIV DNA-PCR test at 4–6 weeks for early infant diagnosis (EID) of HIV-exposed infants (HEI) and immediate return of results. WHO recommends initiating antiretroviral therapy (ART) ≤ 7 days from HIV diagnosis. In 2019, MOH introduced point-of-care (POC) whole-blood EID testing in 33 health facilities and scaled up to 130 facilities in 2020. We assessed results turnaround time and ART linkage pre-POC and during POC testing.MethodsWe evaluated EID register data for HEI at 10 health facilities with POC and EID testing volume of ≥ 12 infants/month from 2018 to 2021. We abstracted data for 12 months before and after POC testing rollout and compared time to sample collection, results receipt, and ART initiation between periods using medians, Wilcoxon, and log-rank tests.ResultsData for 4.004 HEI were abstracted, of which 1.685 (42%) were from the pre-POC period and 2.319 (58%) were from the period during POC; 3.773 (94%) had a first EID test (pre-POC: 1.649 [44%]; during POC: 2.124 [56%]). Median age at sample collection was 44 (IQR 38–51) days pre-POC and 42 (IQR 33–50) days during POC (p < 0.001). Among 3.773 HEI tested, 3.678 (97%) had test results. HIV-positive infants’ (n = 69) median age at sample collection was 94 (IQR 43–124) days pre-POC and 125 (IQR 74–206) days during POC (p = 0.04). HIV positivity rate was 1.6% (27/1.617) pre-POC and 2.0% (42/2.061) during POC (p = 0.43). For all infants, median days from sample collection to results receipt by infants’ caregivers was 28 (IQR 14–52) pre-POC and 1 (IQR 0–25) during POC (p < 0.001); among HIV-positive infants, median days were 23 (IQR 7–30) pre-POC and 0 (0–3) during POC (p < 0.001). Pre-POC, 4% (1/23) HIV-positive infants started ART on the sample collection day compared to 33% (12/37) during POC (p < 0.001); ART linkage ≤ 7 days from HIV diagnosis was 74% (17/23) pre-POC and 95% (35/37) during POC (p < 0.001).ConclusionPOC testing improved EID results turnaround time and ART initiation for HIV-positive infants. While POC testing expansion could further improve ART linkage and loss to follow-up, there is need to explore barriers around same-day ART initiation for infants receiving POC testing.

The COVID-19 pandemic greatly impacted the in vitro diagnostic market, leading to the development of new technologies such as point-of-care testing (POCT), multiplex testing, and digital health platforms. In this study, we present a self-contained microfluidic chip integrated with an internet-of-things (IoT)-based point-of-care (POC) device for rapid and sensitive diagnosis of respiratory viruses. Our platform enables sample-to-answer diagnostics within 70 min by automating RNA extraction, reverse transcription-loop-mediated isothermal amplification (RT-LAMP), and fluorescence detection. The microfluidic chip is designed to store all the necessary reagents for the entire diagnostic assay, including a lysis buffer, a washing buffer, an elution buffer, and a lyophilized RT-LAMP cocktail. It can perform nucleic acid extraction, aliquoting, and gene amplification in multiple reaction chambers without cross-contamination. The IoT-based POC device consists of a Raspberry Pi 4 for device control and data processing, a CMOS sensor for measuring fluorescence signals, a resistive heater panel for temperature control, and solenoid valves for controlling the movement of on-chip reagent solutions. The proposed device is portable and features a touchscreen for user control and result display. We evaluated the performance of the platform using 11 clinical respiratory virus samples, including 5 SARS-CoV-2 samples, 2 influenza A samples, and 4 influenza B samples. All tested clinical samples were accurately identified with high specificity and fidelity, demonstrating the ability to simultaneously detect multiple respiratory viruses. The combination of the integrated microfluidic chip with the POC device offers a simple, cost-effective, and scalable solution for rapid molecular diagnosis of respiratory viruses in resource-limited settings.