A microfluidic device for practical label-free CD4(+) T cell counting of HIV-infected subjects

Practical HIV diagnostics are urgently needed in resource-limited settings. While HIV infection can be diagnosed using simple, rapid, lateral flow immunoassays, HIV disease staging and treatment monitoring require accurate counting of a particular white blood cell subset, the CD4(+) T lymphocyte. To address the limitations of current expensive, technically demanding and/or time-consuming approaches, we have developed a simple CD4 counting microfluidic device. This device uses cell affinity chromatography operated under differential shear flow to specifically isolate CD4(+) T lymphocytes with high efficiency directly from 10 microliters of unprocessed, unlabeled whole blood. CD4 counts are obtained under an optical microscope in a rapid, simple and label-free fashion. CD4 counts determined in our device matched measurements by conventional flow cytometry among HIV-positive subjects over a wide range of absolute CD4 counts (R(2) = 0.93). This CD4 counting microdevice can be used for simple, rapid and affordable CD4 counting in point-of-care and resource-limited settings.     

Digital LAMP in a sample self-digitization (SD) chip

This paper describes the realization of digital loop-mediated DNA amplification (dLAMP) in a sample self-digitization (SD) chip. Digital DNA amplification has become an attractive technique to quantify absolute concentrations of DNA in a sample. While digital polymerase chain reaction is still the most widespread implementation, its use in resource-limited settings is impeded by the need for thermal cycling and robust temperature control. In such situations, isothermal protocols that can amplify DNA or RNA without thermal cycling are of great interest. Here, we accomplished the successful amplification of single DNA molecules in a stationary droplet array using isothermal digital loop-mediated DNA amplification. Unlike most (if not all) existing methods for sample discretization, our design allows for automated, loss-less digitization of sample volumes on-chip. We demonstrated accurate quantification of relative and absolute DNA concentrations with sample volumes of less than 2 μl. We assessed the homogeneity of droplet size during sample self-digitization in our device, and verified that the size variation was small enough such that straightforward counting of LAMP-active droplets sufficed for data analysis. We anticipate that the simplicity and robustness of our SD chip make it attractive as an inexpensive and easy-to-operate device for DNA amplification, for example in point-of-care settings.     

基于分子识别的免疫层析技术用于新冠肺炎感染的快速诊断

目前由新型冠状病毒(SARS-CoV-2)引发的新冠肺炎疫情仍在全球蔓延. 快速筛查并隔离感染者(包括无症状感染者)是遏制疫情传播的重要手段之一. 免疫层析技术是一种相对成熟的快速检测技术, 由于其操作简单、 反应时间短且结果稳定, 在生物标志物检测领域具有广阔的应用前景. 本文总结了目前免疫层析检测技术在新冠肺炎感染筛查领域的研究进展, 涵盖病毒抗体、 蛋白、 核酸等检测靶标, 并对不同检测方法的优势、 局限性进行了简要评述, 最后简单介绍了目前用于新冠肺炎感染筛查的免疫层析试纸的实际应用情况.     

Biofunctional paper via the covalent modification of cellulose

Paper-based analytical devices are the subject of growing interest for the development of low-cost point-of-care diagnostics, environmental monitoring technologies, and research tools for limited-resource settings. However, there are limited chemistries available for the conjugation of biomolecules to cellulose for use in biomedical applications. Herein, divinyl sulfone (DVS) chemistry was demonstrated to immobilize small molecules, proteins, and DNA covalently onto the hydroxyl groups of cellulose membranes through nucleophilic addition. Assays on modified cellulose using protein-carbohydrate and protein-glycoprotein interactions as well as oligonucleotide hybridization showed that the membrane's bioactivity was specific, dose-dependent, and stable over a long period of time. The use of an inkjet printer to form patterns of biomolecules on DVS-activated cellulose illustrates the adaptability of the DVS functionalization technique to pattern sophisticated designs, with potential applications in cellulose-based lateral flow devices.     

Rapid and sensitive lateral flow immunoassay method for determining alpha fetoprotein in serum using europium (III) chelate microparticles-based lateral flow test strips

Alpha-fetoprotein (AFP), a primary marker for many diseases including various cancers, is important in clinical tumor diagnosis and antenatal screening. Most immunoassays provide high sensitivity and accuracy for determining AFP, but they are expensive, often complex, time-consuming procedures. A simple and rapid point-of-care system that integrates Eu (III) chelate microparticles with lateral flow immunoassay (LFIA) has been developed to determine AFP in serum with an assay time of 15 min. The approach is based on a sandwich immunoassay performed on lateral flow test strips. A fluorescence strip reader was used to measure the fluorescence peak heights of the test line (H_T) and the control line (H_C); the H_T/H_C ratio was used for quantitation. The Eu (III) chelate microparticles-based LFIA assay exhibited a wide linear range (1.0–1000 IU mL⁻¹) for AFP with a low limit of detection (0.1 IU mL⁻¹) based on 5ul of serum. Satisfactory specificity and accuracy were demonstrated and the intra- and inter-assay coefficients of variation (CV) for AFP were both <10%. Furthermore, in the analysis of human serum samples, excellent correlation (n = 284, r = 0.9860, p < 0.0001) was obtained between the proposed method and a commercially available CLIA kit. Results indicated that the Eu (III) chelate microparticles-based LFIA system provided a rapid, sensitive and reliable method for determining AFP in serum, indicating that it would be suitable for development in point-of-care testing.     

A simple and efficient method for potential point-of-care diagnosis of human papillomavirus genotypes: combination of isothermal recombinase polymerase amplification with lateral flow dipstick and reverse dot blot

Cervical cancer is the second most common cancer in the world’s woman population with a high incidence in developing countries where diagnostic conditions for the cancer are poor. The main culprit causing the cancer is the human papillomavirus (HPV). HPV is divided into three major groups, i.e., high-risk (HR) group, probable high-risk (pHR) group, and low-risk (LR) group according to their potential of causing cervical cancer. Therefore, developing a sensitive, reliable, and cost-effective point-of-care diagnostic method for the virus genotypes in developing countries even worldwide is of high importance for the cancer prevention and control strategies. Here we present a combined method of isothermal recombinase polymerase amplification (RPA), lateral flow dipstick (LFD), and reverse dot blot (RDB), in quick point-of-care identification of HPV genotypes. The combined method is highly specific to HPV when the conserved L1 genes are used as targeted genes for amplification. The method can be used in identification of HPV genotypes at point-of-care within 1 h with a sensitivity of low to 100 fg of the virus genomic DNA. We have demonstrated that it is an excellent diagnostic point-of-care assay in monitoring the disease without time-consuming and expensive procedures and devices.

     

Development of multiplex loop mediated isothermal amplification (m-LAMP) label-based gold nanoparticles lateral flow dipstick biosensor for detection of pathogenic Leptospira

In recent years extensive numbers of molecular diagnostic methods have been developed to meet the need of point-of-care devices. Efforts have been made towards producing rapid, simple and inexpensive DNA tests, especially in the diagnostics field. We report on the development of a label-based lateral flow dipstick for the rapid and simple detection of multiplex loop-mediated isothermal amplification (m-LAMP) amplicons. A label-based m-LAMP lateral flow dipstick assay was developed for the simultaneous detection of target DNA template and a LAMP internal control. This biosensor operates through a label based system, in which probe-hybridization and the additional incubation step are eliminated. We demonstrated this m-LAMP assay by detecting pathogenic Leptospira, which causes the re-emerging disease Leptospirosis. The lateral flow dipstick was developed to detect of three targets, the LAMP target amplicon, the LAMP internal control amplicon and a chromatography control. Three lines appeared on the dipstick, indicating positive results for all representative pathogenic Leptospira species, whereas two lines appeared, indicating negative results, for other bacterial species. The specificity of this biosensor assay was 100% when it was tested with 13 representative pathogenic Leptospira species, 2 intermediate Leptospira species, 1 non-pathogenic Leptospira species and 28 other bacteria species. This study found that this DNA biosensor was able to detect DNA at concentrations as low as 3.95 × 10⁻¹ genomic equivalent ml⁻¹. An integrated m-LAMP and label-based lateral flow dipstick was successfully developed, promising simple and rapid visual detection in clinical diagnostics and serving as a point-of-care device.     

One-Step Nucleic Acid Purification and Noise-Resistant Polymerase Chain Reaction by Electrokinetic Concentration for Ultralow-Abundance Nucleic Acid Detection

Nucleic acid amplification tests (NAATs)integrated on a chip hold great promise for point-of-care diagnostics. Currently, nucleic acid (NA) purification remains time-consuming and labor-intensive, and it takes extensive efforts to optimize the amplification chemistry. Using selective electrokinetic concentration, we report one-step, liquid-phase NA purification that is simpler and faster than conventional solid-phase extraction. By further re-concentrating NAs and performing polymerase chain reaction (PCR) in a microfluidic chamber, our platform suppresses non-specific amplification caused by non-optimal PCR designs. We achieved the detection of 5 copies of M. tuberculosis genomic DNA (equaling 0.3 cell) in real biofluids using both optimized and non-optimal PCR designs, which is 10- and 1000-fold fewer than those of the standard bench-top method, respectively. By simplifying the workflow and shortening the development cycle of NAATs, our platform may find use in point-of-care diagnosis.     

Lateral flow dipstick test for genotyping of 15 beta-globin gene (HBB) mutations with naked-eye detection

For definitive diagnosis of thalassemia carriers and patients, as well as for prenatal diagnosis, genotype analysis is of fundamental importance. We report a dry-reagent, lateral flow dipstick test that enables visual genotyping (detection by naked eye) of 15 mutations common in Mediterranean populations in the beta-globin gene (HBB). The method comprises 3 simple steps: (i) PCR amplification of a single 1896 bp segment of the beta globin gene flanking all 15 mutations; (ii) a multiplex (10-plex and/or 30-plex) primer extension reaction of the unpurified amplification product using allele-specific primers. Biotin is incorporated in the extended product; (iii) a dry-reagent multi-allele (10-plex) dipstick assay for visual detection of the primer extension reaction products within minutes. The total time required for PCR, primer extension reaction and the dipstick assay is ∼2 h. The method was evaluated by genotyping 45 DNA samples of known genotypes and 54 blind samples. The results were fully concordant with reference methods. The method is simple, rapid, and cost-effective. Detection by the dipstick assay does not require specialized instrumentation or highly qualified personnel. The proposed method could be a particularly useful tool in laboratories with limited resources and a basis for point-of-care diagnostics especially in combination with PCR amplification from whole blood.     

A sensitive lateral flow biosensor for Escherichia coli O157:H7 detection based on aptamer mediated strand displacement amplification

Foodborne diseases caused by pathogens are one of the major problems in food safety. Convenient and sensitive point-of-care rapid diagnostic tests for food-borne pathogens have been a long-felt need of clinicians. Commonly used methods for pathogen detection rely on conventional culture-based tests, antibody-based assays and polymerase chain reaction (PCR)-based techniques. These methods are costly, laborious and time-consuming. Herein, we present a simple and sensitive aptamer based biosensor for rapid detection of Escherichia coli O157:H7 (E. coli O157:H7). In this assay, two different aptamers specific for the outmembrane of E. coli O157:H7 were used. One of the aptamers was used for magnetic bead enrichment, and the other was used as a signal reporter for this pathogen, which was amplified by isothermal strand displacement amplification (SDA) and further detected by a lateral flow biosensor. Only the captured aptamers on cell membrane were amplified, limitations of conventional DNA amplification based method such as false-positive can be largely reduced. The generated signals (red bands on the test zone of a lateral flow strip) can be unambiguously read out by the naked eye. As low as 10 colony forming units (CFU) of E. coli O157:H7 were detected in this study. Without DNA extraction, the reduced handling and simpler equipment requirement render this assay a simple and rapid alternative to conventional methods.     

Isothermal nucleic acid amplification technologies for point-of-care diagnostics: a critical review

Nucleic Acid Testing (NAT) promises rapid, sensitive and specific diagnosis of infectious, inherited and genetic disease. The next generation of diagnostic devices will interrogate the genetic determinants of such conditions at the point-of-care, affording clinicians prompt reliable diagnosis from which to guide more effective treatment. The complex biochemical nature of clinical samples, the low abundance of nucleic acid targets in the majority of clinical samples and existing biosensor technology indicate that some form of nucleic acid amplification will be required to obtain clinically relevant sensitivities from the small samples used in point-of-care testing (POCT). This publication provides an overview and thorough review of existing technologies for nucleic acid amplification. The different methods are compared and their suitability for POCT adaptation are discussed. Current commercial products employing isothermal amplification strategies are also investigated. In conclusion we identify the factors impeding the integration of the methods discussed in fully automated, sample-to-answer POCT devices.     

Cell detection and counting through cell lysate impedance spectroscopy in microfluidic devices

Cell-based microfluidic devices have attracted interest for a wide range of applications. While optical cell counting and flow cytometry-type devices have been reported extensively, sensitive and efficient non-optical methods to detect and quantify cells attached over large surface areas within microdevices are generally lacking. We describe an electrical method for counting cells based on the measurement of changes in conductivity of the surrounding medium due to ions released from surface-immobilized cells within a microfluidic channel. Immobilized cells are lysed using a low conductivity, hypotonic media and the resulting change in impedance is measured using surface patterned electrodes to detect and quantify the number of cells. We found that the bulk solution conductance increases linearly with the number of isolated cells contributing to solution ion concentration. The method of cell lysate impedance spectroscopy is sensitive enough to detect 20 cells microL(-1), and offers a simple and efficient method for detecting and enumerating cells within microfluidic devices for many applications including measurement of CD4 cell counts in HIV patients in resource-limited settings. To our knowledge, this is the most sensitive approach using non-optical setups to enumerate immobilized cells. The microfluidic device, capable of isolating specific cell types from a complex bio-fluidic and quantifying cell number, can serve as a single use cartridge for a hand-held instrument to provide simple, fast and affordable cell counting in point-of-care settings.     

Carbon nanoparticles in lateral flow methods to detect genes encoding virulence factors of Shiga toxin-producing <Emphasis Type="Italic">Escherichia coli</Emphasis>

The use of carbon nanoparticles is shown for the detection and identification of different Shiga toxin-producing Escherichia coli virulence factors (vt1, vt2, eae and ehxA) and a 16S control (specific for E. coli) based on the use of lateral flow strips (nucleic acid lateral flow immunoassay, NALFIA). Prior to the detection with NALFIA, a rapid amplification method with tagged primers was applied. In the evaluation of the optimised NALFIA strips, no cross-reactivity was found for any of the antibodies used. The limit of detection was higher than for quantitative PCR (q-PCR), in most cases between 10⁴ and 10⁵ colony forming units/mL or 0.1–0.9 ng/μL DNA. NALFIA strips were applied to 48 isolates from cattle faeces, and results were compared to those achieved by q-PCR. E. coli virulence factors identified by NALFIA were in very good agreement with those observed in q-PCR, showing in most cases sensitivity and specificity values of 1.0 and an almost perfect agreement between both methods (kappa coefficient larger than 0.9). The results demonstrate that the screening method developed is reliable, cost-effective and user-friendly, and that the procedure is fast as the total time required is <1 h, which includes amplification.     

A self-powered, one-step chip for rapid, quantitative and multiplexed detection of proteins from pinpricks of whole blood

We describe an automated, self-powered chip based on lateral flow immunoassay for rapid, quantitative, and multiplex protein detection from pinpricks of whole blood. The device incorporates on-chip purification of blood plasma by employing inertial forces to focus blood cells away from the assay surface, where plasma proteins are captured and detected on antibody "barcode" arrays. Power is supplied from the capillary action of a piece of adsorbent paper, and sequentially drives, over a 40 minute period, the four steps required to capture serum proteins and then develop a multiplex immunoassay. An 11 protein panel is assayed from whole blood, with high sensitivity and high reproducibility. This inexpensive, self-contained, and easy to operate chip provides a useful platform for point-of-care diagnoses, particularly in resource-limited settings.     

Quantitative evaluation of oligonucleotide surface concentrations using polymerization-based amplification

Quantitative evaluation of minimal polynucleotide concentrations has become a critical analysis among a myriad of applications found in molecular diagnostic technology. Development of high-throughput, nonenzymatic assays that are sensitive, quantitative and yet feasible for point-of-care testing are thus beneficial for routine implementation. Here, we develop a nonenzymatic method for quantifying surface concentrations of labeled DNA targets by coupling regulated amounts of polymer growth to complementary biomolecular binding on array-based biochips. Polymer film thickness measurements in the 20-220 nm range vary logarithmically with labeled DNA surface concentrations over two orders of magnitude with a lower limit of quantitation at 60 molecules/microm(2) (approximately 10(6) target molecules). In an effort to develop this amplification method towards compatibility with fluorescence-based methods of characterization, incorporation of fluorescent nanoparticles into the polymer films is also evaluated. The resulting gains in fluorescent signal enable quantification using detection instrumentation amenable to point-of-care settings.     

Engineering innovative interfaces for point-of-care diagnostics

The ongoing Coronavirus disease 2019 (COVID-19) pandemic illustrates the need for sensitive and reliable tools to diagnose and monitor diseases. Traditional diagnostic approaches rely on centralized laboratory tests that result in long wait times to results and reduce the number of tests that can be given. Point-of-care tests (POCTs) are a group of technologies that miniaturize clinical assays into portable form factors that can be run both in clinical areas —in place of traditional tests— and outside of traditional clinical settings —to enable new testing paradigms. Hallmark examples of POCTs are the pregnancy test lateral flow assay and the blood glucose meter. Other uses for POCTs include diagnostic assays for diseases like COVID-19, HIV, and malaria but despite some successes, there are still unsolved challenges for fully translating these lower cost and more versatile solutions. To overcome these challenges, researchers have exploited innovations in colloid and interface science to develop various designs of POCTs for clinical applications. Herein, we provide a review of recent advancements in lateral flow assays, other paper based POCTs, protein microarray assays, microbead flow assays, and nucleic acid amplification assays. Features that are desirable to integrate into future POCTs, including simplified sample collection, end-to-end connectivity, and machine learning, are also discussed in this review.     

On-chip sample preparation by controlled release of antibodies for simple CD4 counting

We present a simple system for CD4 and CD8 counting for point-of-care HIV staging in low-resource settings. Automatic sample preparation is achieved through a dried reagent coating inside a thin (26 μm) counting chamber, allowing the delayed release of fluorochrome conjugated monoclonal antibodies after the filling of the chamber with whole blood by capillary flow. A custom-built image cytometer is used to capture fluorescence images representing more than 1 μl of blood. The thin layer of blood in combination with the large image area allows the use of whole blood from a finger prick without the need for dilution, lysis or cell enrichment. Automatic cell counting of CD4(+) and CD8(+) T-lymphocytes correlates well with results obtained by flow cytometry.     

Efficient on-chip isolation of HIV subtypes

HIV has caused a global pandemic over the last three decades. There is an unmet need to develop point-of-care (POC) viral load diagnostics to initiate and monitor antiretroviral treatment in resource-constrained settings. Particularly, geographical distribution of HIV subtypes poses significant challenges for POC immunoassays. Here, we demonstrated a microfluidic device that can effectively capture various subtypes of HIV particles through anti-gp120 antibodies, which were immobilized on the microchannel surface. We first optimized an antibody immobilization process using fluorescent antibodies, quantum dot staining and AFM studies. The results showed that anti-gp120 antibodies were immobilized on the microchannel surface with an elevated antibody density and uniform antibody orientation using a Protein G-based surface chemistry. Further, RT-qPCR analysis showed that HIV particles of subtypes A, B and C were captured repeatably with high efficiencies of 77.2 ± 13.2%, 82.1 ± 18.8, and 80.9 ± 14.0% from culture supernatant, and 73.2 ± 13.6, 74.4 ± 14.6 and 78.3 ± 13.3% from spiked whole blood at a viral load of 1000 copies per mL, respectively. HIV particles of subtypes A, B and C were captured with high efficiencies of 81.8 ± 9.4%, 72.5 ± 18.7, and 87.8 ± 3.2% from culture supernatant, and 74.6 ± 12.9, 75.5 ± 6.7 and 69.7 ± 9.5% from spiked whole blood at a viral load of 10,000 copies per mL, respectively. The presented immuno-sensing device enables the development of POC on-chip technologies to monitor viral load and guide antiretroviral treatment (ART) in resource-constrained settings.     

Electro-kinetically driven route for highly sensitive blood pathology on a paper-based device

Enhancing the sensitivity of colorimetric detection in paper-devices is a quintessential step in achieving frugal diagnosis. Here, we demonstrate an effective way of improving the detection sensitivity of paper-based devices, as mediated by electro-kinetic mechanisms. By directly employing blood plasma, we investigate the electro-kinetic clustering of glucose, a neutral molecule in paper devices. Under the influence of uniform electric field, dispersed glucose gets accumulated in the paper strips. Due to the combination of EOF and electrophoretic migration, we achieve twofold increase in the colour intensity for both normal and diabetic samples. This approach is robust and possesses better sensitivity than conventional colorimetric assays and can be easily extended to other body fluid based diagnosis. These results may turn out to be of profound importance in improving the quality of pathological diagnosis in low-cost paper-based point-of-care devices deployed in resource-limited settings.