Single-tier point-of-care serodiagnosis of Lyme disease.

Point-of-care (POC) serological testing provides actionable information for several difficult to diagnose illnesses, empowering distributed health systems. Accessible and adaptable diagnostic platforms that can assay the repertoire of antibodies formed against pathogens are essential to drive early detection and improve patient outcomes. Here, we report a POC serologic test for Lyme disease (LD), leveraging synthetic peptides tuned to be highly specific to the LD antibody repertoire across patients and compatible with a paper-based platform for rapid, reliable, and cost-effective diagnosis. A subset of antigenic epitopes conserved across Borrelia burgdorferi genospecies and targeted by IgG and IgM antibodies, were selected based on their seroreactivity to develop a multiplexed panel for a single-step measurement of combined IgM and IgG antibodies from LD patient sera. Multiple peptide epitopes, when combined synergistically using a machine learning-based diagnostic model, yielded a high sensitivity without any loss in specificity. We blindly tested the platform with samples from the U.S. Centers for Disease Control & Prevention (CDC) LD repository and achieved a sensitivity and specificity matching the lab-based two-tier results with a single POC test, correctly discriminating cross-reactive look-alike diseases. This computational LD diagnostic test can potentially replace the cumbersome two-tier testing paradigm, improving diagnosis and enabling earlier effective treatment of LD patients while also facilitating immune monitoring and surveillance of the disease in the community.

Deep Learning-Enabled Multiplexed Point-of-Care Sensor using a Paper-Based Fluorescence Vertical Flow Assay.

Multiplexed computational sensing with a point-of-care serodiagnosis assay to simultaneously quantify three biomarkers of acute cardiac injury is demonstrated. This point-of-care sensor includes a paper-based fluorescence vertical flow assay (fxVFA) processed by a low-cost mobile reader, which quantifies the target biomarkers through trained neural networks, all within <15 min of test time using 50 µL of serum sample per patient. This fxVFA platform is validated using human serum samples to quantify three cardiac biomarkers, i.e., myoglobin, creatine kinase-MB, and heart-type fatty acid binding protein, achieving less than 0.52 ng mL-1 limit-of-detection for all three biomarkers with minimal cross-reactivity. Biomarker concentration quantification using the fxVFA that is coupled to neural network-based inference is blindly tested using 46 individually activated cartridges, which shows a high correlation with the ground truth concentrations for all three biomarkers achieving >0.9 linearity and <15% coefficient of variation. The competitive performance of this multiplexed computational fxVFA along with its inexpensive paper-based design and handheld footprint makes it a promising point-of-care sensor platform that can expand access to diagnostics in resource-limited settings.

Recent Progress in Lyme Disease and Remaining Challenges.

Lyme disease (also known as Lyme borreliosis) is the most common vector-borne disease in the United States with an estimated 476,000 cases per year. While historically, the long-term impact of Lyme disease on patients has been controversial, mounting evidence supports the idea that a substantial number of patients experience persistent symptoms following treatment. The research community has largely lacked the necessary funding to properly advance the scientific and clinical understanding of the disease, or to develop and evaluate innovative approaches for prevention, diagnosis, and treatment. Given the many outstanding questions raised into the diagnosis, clinical presentation and treatment of Lyme disease, and the underlying molecular mechanisms that trigger persistent disease, there is an urgent need for more support. This review article summarizes progress over the past 5 years in our understanding of Lyme and tick-borne diseases in the United States and highlights remaining challenges.

Quantitative particle agglutination assay for point-of-care testing using mobile holographic imaging and deep learning.

Particle agglutination assays are widely adopted immunological tests that are based on antigen-antibody interactions. Antibody-coated microscopic particles are mixed with a test sample that potentially contains the target antigen, as a result of which the particles form clusters, with a size that is a function of the antigen concentration and the reaction time. Here, we present a quantitative particle agglutination assay that combines mobile lens-free microscopy and deep learning for rapidly measuring the concentration of a target analyte; as its proof-of-concept, we demonstrate high-sensitivity C-reactive protein (hs-CRP) testing using human serum samples. A dual-channel capillary lateral flow device is designed to host the agglutination reaction using 4 µL of serum sample with a material cost of 1.79 cents per test. A mobile lens-free microscope records time-lapsed inline holograms of the lateral flow device, monitoring the agglutination process over 3 min. These captured holograms are processed, and at each frame the number and area of the particle clusters are automatically extracted and fed into shallow neural networks to predict the CRP concentration. 189 measurements using 88 unique patient serum samples were utilized to train, validate and blindly test our platform, which matched the corresponding ground truth concentrations in the hs-CRP range (0-10 µg mL-1) with an R2 value of 0.912. This computational sensing platform was also able to successfully differentiate very high CRP concentrations (e.g., >10-500 µg mL-1) from the hs-CRP range. This mobile, cost-effective and quantitative particle agglutination assay can be useful for various point-of-care sensing needs and global health related applications.

Deep learning-enabled point-of-care sensing using multiplexed paper-based sensors.

We present a deep learning-based framework to design and quantify point-of-care sensors. As a use-case, we demonstrated a low-cost and rapid paper-based vertical flow assay (VFA) for high sensitivity C-Reactive Protein (hsCRP) testing, commonly used for assessing risk of cardio-vascular disease (CVD). A machine learning-based framework was developed to (1) determine an optimal configuration of immunoreaction spots and conditions, spatially-multiplexed on a sensing membrane, and (2) to accurately infer target analyte concentration. Using a custom-designed handheld VFA reader, a clinical study with 85 human samples showed a competitive coefficient-of-variation of 11.2% and linearity of R 2 = 0.95 among blindly-tested VFAs in the hsCRP range (i.e., 0-10 mg/L). We also demonstrated a mitigation of the hook-effect due to the multiplexed immunoreactions on the sensing membrane. This paper-based computational VFA could expand access to CVD testing, and the presented framework can be broadly used to design cost-effective and mobile point-of-care sensors.

Measurement of serum phosphate levels using a mobile sensor.

The measurement of serum phosphate concentration is crucial for patients with advanced chronic kidney disease (CKD) and those on maintenance dialysis, as abnormal phosphate levels may be associated with severe health risks. It is important to monitor serum phosphate levels on a regular basis in these patients; however, such measurements are generally limited to every 0.5-3 months, depending on the severity of CKD. This is due to the fact that serum phosphate measurements can only be performed at regular clinic visits, in addition to cost considerations. Here we present a portable and cost-effective point-of-care device capable of measuring serum phosphate levels using a single drop of blood (<60 µl). This is achieved by integrating a paper-based microfluidic platform with a custom-designed smartphone reader. This mobile sensor was tested on patients undergoing dialysis, where whole blood samples were acquired before starting the hemodialysis and during the three-hour treatment. This sampling during the hemodialysis, under patient consent, allowed us to test blood samples with a wide range of phosphate concentrations, and our results showed a strong correlation with the ground truth laboratory tests performed on the same patient samples (Pearson coefficient r = 0.95 and p < 0.001). Our 3D-printed smartphone attachment weighs about 400 g and costs less than 80 USD, whereas the material cost for the disposable test is <3.5 USD (under low volume manufacturing). This low-cost and easy-to-operate system can be used to measure serum phosphate levels at the point-of-care in about 45 min and can potentially be used on a daily basis by patients at home.

Point-of-Care Serodiagnostic Test for Early-Stage Lyme Disease Using a Multiplexed Paper-Based Immunoassay and Machine Learning.

Caused by the tick-borne spirochete Borrelia burgdorferi, Lyme disease (LD) is the most common vector-borne infectious disease in North America and Europe. Though timely diagnosis and treatment are effective in preventing disease progression, current tests are insensitive in early stage LD, with a sensitivity of <50%. Additionally, the serological testing currently recommended by the U.S. Center for Disease Control has high costs (>$400/test) and extended sample-to-answer timelines (>24 h). To address these challenges, we created a cost-effective and rapid point-of-care (POC) test for early-stage LD that assays for antibodies specific to seven Borrelia antigens and a synthetic peptide in a paper-based multiplexed vertical flow assay (xVFA). We trained a deep-learning-based diagnostic algorithm to select an optimal subset of antigen/peptide targets and then blindly tested our xVFA using human samples (N(+) = 42, N(-) = 54), achieving an area-under-the-curve (AUC), sensitivity, and specificity of 0.950, 90.5%, and 87.0%, respectively, outperforming previous LD POC tests. With batch-specific standardization and threshold tuning, the specificity of our blind-testing performance improved to 96.3%, with an AUC and sensitivity of 0.963 and 85.7%, respectively.

Rapid and Simple Detection of Ochratoxin A using Fluorescence Resonance Energy Transfer on Lateral Flow Immunoassay (FRET-LFI).

The detection of mycotoxins is crucial because of their toxicity in plants, animals, and humans. It is very important to determine whether food products are contaminated with mycotoxins such as ochratoxin A (OTA), as mycotoxins can survive heat treatments and hydrolysis. In this study, we designed a fluorescence resonance energy transfer (FRET)-based system that exploits antibody-antigen binding to detect mycotoxins more rapidly and easily than other currently available methods. In addition, we were able to effectively counteract the matrix effect in the sample by using a nitrocellulose membrane that enabled fluorescence measurement in coffee samples. The developed FRET on lateral flow immunoassay (FRET-LFI) system was used to detect OTA at a limit of detection (LOD) of 0.64 ng∙mL-1, and the test can be completed in only 30 min. Moreover, OTA in coffee samples was successfully detected at a LOD of 0.88 ng∙mL-1, overcoming the matrix effect, owing to the chromatographic properties of the capillary force of the membrane. We believe that the developed system can be used as a powerful tool for the sensitive diagnosis of harmful substances such as mycotoxins and pesticides for environmental and food quality control monitoring.

Paper-based multiplexed vertical flow assay for point-of-care testing.

We developed a multiplexed point-of-care immunodiagnostic assay for antibody detection in human sera made through the vertical stacking of functional paper layers. In this multiplexed vertical flow immunodiagnostic assay (xVFA), a colorimetric signal is generated by gold nanoparticles captured on a spatially-multiplexed sensing membrane containing specific antigens. The assay is completed in 20 minutes, following which the sensing membrane is imaged by a cost-effective mobile-phone reader. The images are sent to a server, where the results are rapidly analyzed and relayed back to the user. The performance of the assay was evaluated by measuring Lyme-specific antibodies in human sera as model target antibodies. The presented platform is rapid, simple, inexpensive, and allows for simultaneous and quantitative measurement of multiple antibodies and/or antigens making it a suitable point-of-care platform for disease diagnostics.

A hook effect-free immunochromatographic assay (HEF-ICA) for measuring the C-reactive protein concentration in one drop of human serum.

The immunochromatographic (ICA) assay is a highly promising platform for rapid and simple detection of C-reactive protein (CRP) which is an indicator of the different phases of various diseases, as well as of inflammation and infection. However, the hook effect in the ICA assay limits the quantification of CRP levels at high CRP concentrations. Methods: In this study, we developed a hook effect-free immunochromatographic assay (HEF-ICA) to detect CRP over a wide concentration range. The hook effect results from the simultaneous reaction of an excess target antigens with both immobilized and labeled antibodies respectively. To reduce the potential occurrence of this simultaneous reaction, we separated the migration of the target antigen and gold nanoparticle (AuNP)-labeled antibodies on a nitrocellulose membrane and analyzed the time profiles by modifying the ICA structure. Results: The signal intensity of HEF-ICA was saturated at high CRP concentrations, without decreasing. The titration curve of HEF-ICA was adjusted with the Hill equation, and HEF-ICA was performed with the following parameters: limit of detection, 43 ng mL-1; dynamic range, 119 ng mL-1 to 100 µg mL-1. The accuracy of the newly developed assay was evaluated using 33 clinical samples via comparison with a clinical chemistry analyzer. Conclusion: HEF-ICA enabled the measurement of a wide range of CRP concentrations without the hook effect, and was suitable for point-of-care testing with fingertip blood sampling, as only a minute sample volume (2.5 µL) was required.

A Paper-Based Device for Performing Loop-Mediated Isothermal Amplification with Real-Time Simultaneous Detection of Multiple DNA Targets.

Paper-based diagnostic devices have many advantages as a one of the multiple diagnostic test platforms for point-of-care (POC) testing because they have simplicity, portability, and cost-effectiveness. However, despite high sensitivity and specificity of nucleic acid testing (NAT), the development of NAT based on a paper platform has not progressed as much as the others because various specific conditions for nucleic acid amplification reactions such as pH, buffer components, and temperature, inhibitions from technical differences of paper-based device. Here, we propose a paper-based device for performing loop-mediated isothermal amplification (LAMP) with real-time simultaneous detection of multiple DNA targets. We determined the optimal chemical components to enable dry conditions for the LAMP reaction without lyophilization or other techniques. We also devised the simple paper device structure by sequentially stacking functional layers, and employed a newly discovered property of hydroxynaphthol blue fluorescence to analyze real-time LAMP signals in the paper device. This proposed platform allowed analysis of three different meningitis DNA samples in a single device with single-step operation. This LAMP-based multiple diagnostic device has potential for real-time analysis with quantitative detection of 102-105 copies of genomic DNA. Furthermore, we propose the transformation of DNA amplification devices to a simple and affordable paper system approach with great potential for realizing a paper-based NAT system for POC testing.

An immunochromatographic biosensor combined with a water-swellable polymer for automatic signal generation or amplification.

An immunochromatographic assay (ICA) strip is one of the most widely used platforms in the field of point-of-care biosensors for the detection of various analytes in a simple, fast, and inexpensive manner. Currently, several approaches for sequential reactions in ICA platforms have improved their usability, sensitivity, and versatility. In this study, a new, simple, and low-cost approach using automatic sequential-reaction ICA strip is described. The automatic switching of a reagent pad from separation to attachment to the test membrane was achieved using a water-swellable polymer. The reagent pad was dried with an enzyme substrate for signal generation or with signal-enhancing materials. The strip design and system operation were confirmed by the characterization of the raw materials and flow analysis. We demonstrated the operation of the proposed sensor by using various chemical reaction-based assays, including metal-ion amplification, enzyme-colorimetric reaction, and enzyme-catalyzed chemiluminescence. Furthermore, by employing C-reactive protein as a model, we successfully demonstrated that the new water-swellable polymer-based ICA sensor can be utilized to detect biologically relevant analytes in human serum.

One-touch-activated blood multidiagnostic system using a minimally invasive hollow microneedle integrated with a paper-based sensor.

The development of real-time innocuous blood diagnosis has been a long-standing goal in healthcare; an improved, miniature, all-in-one point-of-care testing (POCT) system with low cost and simplified operation is highly desired. Here, we present a one-touch-activated blood multidiagnostic system (OBMS) involving the synergistic integration of a hollow microneedle and paper-based sensor, providing a number of unique characteristics for simplifying the design of microsystems and enhancing user performance. In this OBMS, all functions of blood collection, serum separation, and detection were sequentially automated in one single device that only required one-touch activation by finger-power without additional operations. For the first time, we successfully demonstrated the operation of this system in vivo in glucose and cholesterol diagnosis, showing a great possibility for human clinical application and commercialization. Additionally, this novel system offers a new approach for the use of microneedles and paper sensors as promising intelligent elements in future real-time healthcare monitoring devices.

Tear-off patterning: a simple method for patterning nitrocellulose membranes to improve the performance of point-of-care diagnostic biosensors.

This article describes a new method, referred to as "tear-off patterning," for patterning nitrocellulose (NC) membranes in order to fabricate NC-based point-of-care (POC) diagnostic devices. Paper-based microfluidic sensors usually employ hydrophobic barrier coatings such as paraffin wax on either paper or membranes. Herein, complex patterns were fabricated by stamping the target area with dimethyl sulfoxide before tearing off the stamped area. Fluid flow and morphological analyses were performed in order to characterize the patterned membranes. Furthermore, the myoglobin and creatine kinase-MB levels in human serum were measured simultaneously using a dual-fluidic-channel-patterned NC membrane in order to confirm the usefulness of the patterning method for fabricating POC biosensors. The proposed method for patterning NC membranes offers clear advantages, such as the ability to fabricate complex designs and patterns without a hydrophobic barrier after protein immobilization in a laboratory and in a simple, low-cost manner. We believe that this method can be used to develop various POC diagnostic biosensors at the research and development stage and can help improve the performance and features of POC diagnostic devices.

A three-line lateral flow assay strip for the measurement of C-reactive protein covering a broad physiological concentration range in human sera.

The lateral flow assay (LFA) strip sensor possesses many advantages as a diagnostic device, including the capabilities of rapid, one-step assay performance, and high throughput production. A major limitation of the sensor, however, is its difficulty in measuring a broad concentration range of target proteins, including C-reactive protein (CRP), due to the "hook effect." In this study, we report the use of a three-line LFA strip sensor, adding an antigen line to the conventional two-line LFA sensor, for detecting CRP within a broad concentration range in human sera. We introduced an antigen line between test and control lines in the LFA sensor. The antigen line was formed by dispensing a CRP antibody solution followed by a CRP solution in nitrocellulose membrane. All other conditions were identical to those applied to the conventional LFA strip sensor. The CRP level in test samples was generated by data processing from the intensities of three lines. The strip sensor measured a linear detection range of CRP concentration from 1 ng/mL to 500 µg/mL within 10 min, with a calculated detection range of 0.69 ng/mL-1.02 mg/mL. Using the developed three-line LFA sensor, 50 clinical samples were measured at a detection range of 0.4-84.7 µg/mL. This novel and easy-to-use CRP sensor can be a useful tool for rapid, sensitive, and cost-effective detection of a broad physiological concentration range of CRP capabilities that are vital for various diagnostic applications.

Homogeneous assay of target molecules based on chemiluminescence resonance energy transfer (CRET) using DNAzyme-linked aptamers.

We have designed a single-stranded DNAzyme-aptamer sensor for homogeneous target molecular detection based on chemiluminescence resonance energy transfer (CRET). The structure of the engineered single-stranded DNA (ssDNA) includes the horseradish peroxidase (HRP)-like DNAzyme, optimum-length linker (10-mer-length DNA), and target-specific aptamer sequences. A quencher dye was modified at the 3' end of the aptamer sequence. The incorporation of hemin into the G-quadruplex structure of DNAzyme yields an active HRP-like activity that catalyzes luminol to generate a chemiluminescence (CL) signal. In the presence of target molecules, such as ochratoxin A (OTA), adenosine triphosphate (ATP), or thrombin, the aptamer sequence was folded due to the formation of the aptamer/analyte complex, which induced the quencher dye close to the DNAzyme structure. Consequently, the CRET occurred between a DNAzyme-catalyzed chemiluminescence reaction and the quencher dye. Our results showed that CRET-based DNAzyme-aptamer biosensing enabled specific OTA analysis with a limit of detection of 0.27ng/mL. The CRET platform needs no external light source and avoids autofluorescence and photobleaching, and target molecules can be detected specifically and sensitively in a homogeneous manner.

An interference-free and rapid electrochemical lateral-flow immunoassay for one-step ultrasensitive detection with serum.

Point-of-care testing (POCT) of biomarkers in clinical samples is of great importance for rapid and cost-effective diagnosis. However, it is extremely challenging to develop an electrochemical POCT technique retaining both ultrasensitivity and simplicity. We report an interference-free electrochemical lateral-flow immunoassay that enables one-step ultrasensitive detection with serum. The electrochemical-chemical-chemical (ECC) redox cycling combined with an enzymatic reaction of an enzyme label is used to obtain high signal amplification. The ECC redox cycling involving Ru(NH3)6(3+), enzyme product, and tris(3-carboxyethyl)phosphine (TCEP) depends on pH, because the formal potentials of an enzyme product and TCEP increase with decreasing pH although that of Ru(NH3)6(3+) is pH-independent. With consideration of the pH dependence of ECC redox cycling, a noble combination of enzyme label, substrate, and product [ß-galactosidase, 4-amino-1-naphthyl ß-D-galactopyranoside, and 4-amino-1-naphthol, respectively] is introduced to ensure fast and selective ECC redox cycling of the enzyme product along with a low background level. The selective ECC redox cycling at a low applied potential (0.05 V vs. Ag/AgCl) minimizes the interference effect of electroactive species (L-ascorbic acid, acetaminophen, and uric acid) in serum. A detection limit of 0.1 pg mL(-1) for troponin I is obtained only 11 min after serum dropping without the use of an additional solution. Moreover, the lateral-flow immunoassay is applicable to the analysis of real clinical samples.

An automatic enzyme immunoassay based on a chemiluminescent lateral flow immunosensor.

Microfluidic integrated enzyme immunosorbent assay (EIA) sensors are efficient systems for point-of-care testing (POCT). However, such systems are not only relatively expensive but also require a complicated manufacturing process. Therefore, additional fluidic control systems are required for the implementation of EIAs in a lateral flow immunosensor (LFI) strip sensor. In this study, we describe a novel LFI for EIA, the use of which does not require additional steps such as mechanical fluidic control, washing, or injecting. The key concept relies on a delayed-release effect of chemiluminescence substrates (luminol enhancer and hydrogen peroxide generator) by an asymmetric polysulfone membrane (ASPM). When the ASPM was placed between the nitrocellulose (NC) membrane and the substrate pad, substrates encapsulated in the substrate pad were released after 5.3 ± 0.3 min. Using this delayed-release effect, we designed and implemented the chemiluminescent LFI-based automatic EIA system, which sequentially performed the immunoreaction, pH change, substrate release, hydrogen peroxide generation, and chemiluminescent reaction with only 1 sample injection. In a model study, implementation of the sensor was validated by measuring the high sensitivity C-reactive protein (hs-CRP) level in human serum.

Vertical flow immunoassay (VFA) biosensor for a rapid one-step immunoassay.

A highly rapid, one-step immunoassay of high sensitivity C-reactive protein (hsCRP) using a biosensor with a vertical flow immunoassay (VFA) was developed. The VFA biosensor was primarily composed of a sample pad, conjugate pad, FTH film and nitrocellulose (NC) membrane, which were all vertically stacked upon one another. Anti-hsCRP and secondary antibodies were consecutively immobilized on the NC membrane at the position below the holes. Gold nanoparticles (AuNPs) conjugated with another anti-hsCRP antibody were encapsulated in the conjugation pad. Various assay conditions, including the size of the hole and the sample volume, were optimized. Under optimized conditions, hsCRP concentrations from 0.01 to 10 µg mL(-1) were detected within 2 min. In comparison with a lateral flow assay (LFA) system, the VFA sensor showed a gradual increase of signal in a concentration-dependent manner without a hook effect in the tested range.

Graphene-based chemiluminescence resonance energy transfer for homogeneous immunoassay.

We report on chemiluminescence resonance energy transfer (CRET) between graphene nanosheets and chemiluminescent donors. In contrast to fluorescence resonance energy transfer, CRET occurs via nonradiative dipole-dipole transfer of energy from a chemiluminescent donor to a suitable acceptor molecule without an external excitation source. We designed a graphene-based CRET platform for homogeneous immunoassay of C-reactive protein (CRP), a key marker for human inflammation and cardiovascular diseases, using a luminol/hydrogen peroxide chemiluminescence (CL) reaction catalyzed by horseradish peroxidase. According to our results, anti-CRP antibody conjugated to graphene nanosheets enabled the capture of CRP at the concentration above 1.6 ng mL(-1). In the CRET platform, graphene played a key role as an energy acceptor, which was more efficient than graphene oxide, while luminol served as a donor to graphene, triggering the CRET phenomenon between luminol and graphene. The graphene-based CRET platform was successfully applied to the detection of CRP in human serum samples in the range observed during acute inflammatory stress.