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.

Simultaneous Detection of Serum Glucose and Glycated Albumin on a Paper-Based Sensor for Acute Hyperglycemia and Diabetes Mellitus.

Diabetes mellitus is one of the most common chronic diseases worldwide. Generally, the levels of fasting or postprandial blood glucose and other biomarkers, such as glycated albumin, glycated hemoglobin, and 1,5-anhydroglucitol, are used to diagnose or monitor diabetes progression. In the present study, we developed a sensor to simultaneously detect the glucose levels and glycation ratios of human serum albumin using a lateral flow assay. Based on the specific enzymatic reactions and immunoassays, a spiked glucose solution, total human serum albumin, and glycated albumin were measured simultaneously. To test the performance of the developed sensor, clinical serum samples from healthy subjects and patients with diabetes were analyzed. The glucose level and glycation ratios of the clinical samples were determined with reasonable correlation. The R-squared values of glucose level and glycation ratio measurements were 0.932 and 0.930, respectively. The average detection recoveries of the sensor were 85.80% for glucose and 98.32% for the glycation ratio. The glucose level and glycation ratio in our results were crosschecked with reference diagnostic values of diabetes. Based on the outcomes of the present study, we propose that this novel platform can be utilized for the simultaneous detection of glucose and glycation ratios to diagnose and monitor diabetes mellitus.

Highly Sensitive Chemiluminescence-Based Lateral Flow Immunoassay for Cardiac Troponin I Detection in Human Serum.

Lateral flow assays (LFAs) have become the most common biosensing platforms for point-of-care testing due to their compliance with the ASSURED (affordable, sensitive, specific, user-friendly, rapid/robust, equipment-free, and deliverable to end-users) guidelines stipulated by the World Health Organization. However, the limited analytical sensitivity and low quantitative capability of conventional LFAs, which use gold nanoparticles (AuNPs) for colorimetric labeling, have prevented high-performance testing. Here, we report the development of a highly sensitive chemiluminescence (CL)-based LFA involving AuNPs conjugated with aldehyde-activated peroxidase and antibody molecules-i.e., AuNP-(ald)HRP-Ab-as a new conjugation scheme for high-performance testing in LFAs. When paired with the CL-based signal readout modality, the AuNP-(ald)HRP-Ab conjugate resulted in 110-fold enhanced sensitivity over the colorimetric response of a typical AuNP-Ab conjugate. To evaluate the performance of the CL-based LFA, we tested it with human cardiac troponin I (cTnI; a standard cardiac biomarker used to diagnose myocardial infarction) in standard and clinical serum samples. Testing the standard samples revealed a detection limit of 5.6 pg·mL-1 and acceptably reliable precision (with a coefficient of variation of 2.3%-8.4%), according to clinical guidelines. Moreover, testing the clinical samples revealed a high correlation (r = 0.97) with standard biochemical analyzers, demonstrating the potential clinical utility of the CL-based LFA for high-performance cTnI testing.

A Paper-Based Multiplexed Serological Test to Monitor Immunity against SARS-COV-2 Using Machine Learning.

The rapid spread of SARS-CoV-2 caused the COVID-19 pandemic and accelerated vaccine development to prevent the spread of the virus and control the disease. Given the sustained high infectivity and evolution of SARS-CoV-2, there is an ongoing interest in developing COVID-19 serology tests to monitor population-level immunity. To address this critical need, we designed a paper-based multiplexed vertical flow assay (xVFA) using five structural proteins of SARS-CoV-2, detecting IgG and IgM antibodies to monitor changes in COVID-19 immunity levels. Our platform not only tracked longitudinal immunity levels but also categorized COVID-19 immunity into three groups: protected, unprotected, and infected, based on the levels of IgG and IgM antibodies. We operated two xVFAs in parallel to detect IgG and IgM antibodies using a total of 40 µL of human serum sample in <20 min per test. After the assay, images of the paper-based sensor panel were captured using a mobile phone-based custom-designed optical reader and then processed by a neural network-based serodiagnostic algorithm. The serodiagnostic algorithm was trained with 120 measurements/tests and 30 serum samples from 7 randomly selected individuals and was blindly tested with 31 serum samples from 8 different individuals, collected before vaccination as well as after vaccination or infection, achieving an accuracy of 89.5%. The competitive performance of the xVFA, along with its portability, cost-effectiveness, and rapid operation, makes it a promising computational point-of-care (POC) serology test for monitoring COVID-19 immunity, aiding in timely decisions on the administration of booster vaccines and general public health policies to protect vulnerable populations.

Reagent Filming for Universal Point-of-Care Diagnostics.

Simplifying assays while maintaining the robustness of reagents is a challenge in diagnostics. This problem is exacerbated when translating quality diagnostic assays to developing countries that lack resources and infrastructure such as trained health workers, high-end equipment, and cold-chain systems. To solve this problem, in this study, a simple solution that films assay reagents to simplify the operation of diagnostic assays and preserve the stability of diagnostic reagents without using cold chains is presented. A polyvinyl-alcohol-based water-soluble film is used to encapsulate premeasured and premixed reagents. The reagent film, produced through a simple and scalable cast-drying process, provides a glassy inner matrix with abundant hydroxyl groups that can stabilize various reagents (ranging from chemicals to biological materials) by restricting molecular mobility and generating hydrogen bonds. The reagent film is applied to an enzymatic glucose assay, a high-sensitivity immunoassay for cardiac troponin, and a molecular assay for viral RNA detection, to test its practicability and universal applicability. The film-based assays result in excellent analytical/diagnostic performance and stable long-term reagent storage at elevated temperatures (at 25 or 37 °C, for six months), demonstrating clinical readiness. This technology advances the development and distribution of affordable high-quality diagnostics to resource-limited regions.

Automated, Universal, and Mass-Producible Paper-Based Lateral Flow Biosensing Platform for High-Performance Point-of-Care Testing.

Paper-based lateral flow assays (LFAs) are among the most widely used biosensing platforms for point-of-care testing (POCT). However, the conventional colloidal gold label of LFAs show low sensitivity and limited quantitative capacity. Alternatively, the use of enzyme/chemical reaction-based signal amplification with structural modifications has enhanced analytical capacity but requires multiple user interventions as a trade-off, increasing complexity, test imprecision, and time. These platforms are also difficult to manufacture, limiting their practical applications. In this study, within the current LFA production framework, we developed a highly sensitive, automated, universal, and manufacturable LFA biosensing platform by (i) incorporating gold nanoparticles into a polymer-networked peroxidase with an antibody as a new scheme for enhanced enzyme conjugation and (ii) integrating a mass-producible and time-programmable amplification part based on a water-swellable polymer for automating the sequential reactions in the immunoassay and signal amplification, without compromising performance, simplicity, and production feasibility. We applied this platform to evaluate cardiac troponin I (cTnI), a gold-standard biomarker for myocardial infarction diagnosis. Quantitative analysis of cTnI in clinical setting remains limited to the laboratory-based high-end and costly standard equipment. Coupled with an enzyme-catalyzed chemiluminescence method, this platform enables automated, cost-effective (0.66 USD per test), and high-performance testing of human cTnI in serum samples within 20 min with a detection range of 6 orders of magnitude, detection limit of 0.84 pg mL-1 (595-fold higher than conventional cTnI-LFA), and a coefficient of variation of 2.9-8.5%, which are comparable to the standard equipment and acceptable for clinical use. Moreover, cTnI analysis results using clinical serum/plasma samples revealed a strong correlation (R2 = 0.991) with contemporary standard equipment, demonstrating the practical application of this platform for high-performance POCT.

Glycation ratio determination through simultaneous detection of human serum albumin and glycated albumin on an advanced lateral flow immunoassay sensor.

Glycated albumin synthesized in a non-enzymatic reaction with high glucose levels in human plasma is a long-term biomarker for understanding average glucose levels over the past few weeks. Glycated albumin level determination requires an enzymatic assay involving an expensive, complicated, and laborious process, including the specific hydrolysis of albumin and the oxidation of glycated amino acids. In this study, we developed two advanced lateral flow immunoassays (LFIAs) for the simultaneous determination of total human serum albumin and glycated albumin concentrations using a colorimetric signal. Additionally, through a sequential reaction on our advanced LFIA, the selectivity of glycated albumin was improved. We quantified both HSA and GA with wide detection ranges of 1 ng mL-1-1 mg mL-1 and 0.5 µg mL-1-3.6 mg mL-1, respectively. Various serum samples with different glycation ratios were analyzed using this sensor and demonstrated a reasonable recovery range. This indicated that our platform could directly determinate the glycation ratio of various samples, and therefore, be applicable in point-of-care glucose status monitoring.

Paper/Soluble Polymer Hybrid-Based Lateral Flow Biosensing Platform for High-Performance Point-of-Care Testing.

As a global shift continues to occur in high burden diseases toward developing countries, the importance of medical diagnostics based on point-of-care testing (POCT) is rapidly increasing. However, most diagnostic tests that meet clinical standards rely on high-end analyzers in central hospitals. Here, we report the development of a simple, low-cost, mass-producible, highly sensitive/quantitative, automated, and robust paper/soluble polymer hybrid-based lateral flow biosensing platform, paired with a smartphone-based reader, for high-performance POCT. The testing architecture incorporates a polymeric barrier that programs/automates sequential reactions via a polymer dissolving mechanism. The smartphone-based reader with simple opto-mechanical parts offers a stable framework for accurate quantification. Analytical performance of this platform was evaluated by testing human cardiac troponin I (cTnI), a preferred biomarker for the diagnosis of myocardial infarction, in serum/plasma samples. Coupled with catalytic/colorimetric gold-ion amplification, this platform produced results within 20 min with a detection limit of 0.92 pg mL-1 and a coefficient of variation <10%, which is equivalent to the performance of a high-sensitivity standard analyzer, and operated within acceptable levels stipulated by clinical guidelines. Moreover, cTnI clinical sample tests indicate a high correlation (r = 0.981) with the contemporary analyzers, demonstrating the clinical utility of this platform in high-performance POCT.

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.

Optical birefringence of liquid crystals for label-free optical biosensing diagnosis.

PURPOSE: We present a polarization-sensitive optical detection platform for label-free quantitative optical biosensing diagnosis using liquid crystals (LCs). This is capable of determining quantitatively the optical birefringence of optical cells containing LCs, whose orientation depends on the immobilized biomolecules. PATIENTS AND METHODS: This technique uses a polarization-dependent double-port detection without any polarizer at a single wavelength and removes the need of aligning optical cells of LCs in the azimuthal direction, with respect to the light path through the optical cell. Thus, this technique enables a stand-alone detection in a relatively compact format without an additional optical instrument, such as a retardation compensator, a Michael-Levy chart, and a spectrophotometer, in order to determine the optical birefringence quantitatively. RESULTS: We demonstrate that bovine serum albumin immobilized on the gold surface of the cell hybrid interfaces that support both homeotropic and planar anchoring of LCs causes optical phase retardation change which can be determined quantitatively. We also provide estimation of the zenithal orientation of LCs near the gold surface of the hybrid interfaces, based on the phase retardation determined. The estimated limit of bovine serum albumin detection is approximately 2.1 µM. CONCLUSION: This optical technique with LCs can serve an optical platform for label-free quantitative diagnosis of proteins in a real time manner.

Detection of heavy-metal ions using liquid crystal droplet patterns modulated by interaction between negatively charged carboxylate and heavy-metal cations.

Herein, we demonstrated a simple, sensitive, and rapid label-free detection method for heavy-metal (HM) ions using liquid crystal (LC) droplet patterns on a solid surface. Stearic-acid-doped LC droplet patterns were spontaneously generated on an n-octyltrichlorosilane (OTS)-treated glass substrate by evaporating a solution of the nematic LC, 4-cyano-4'-pentylbiphenyl (5CB), dissolved in heptane. The optical appearance of the droplet patterns was a dark crossed texture when in contact with air, which represents the homeotropic orientation of the LC. This was caused by the steric interaction between the LC molecules and the alkyl chains of the OTS-treated surface. The dark crossed appearance of the acid-doped LC patterns was maintained after the addition of phosphate buffered saline (PBS) solution (pH 8.1 at 25°C). The deprotonated stearic-acid molecules self-assembled through the LC/aqueous interface, thereby supporting the homeotropic anchoring of 5CB. However, the optical image of the acid-doped LC droplet patterns incubated with PBS containing HM ions appeared bright, indicating a planar orientation of 5CB at the aqueous/LC droplet interface. This dark to bright transition of the LC patterns was caused by HM ions attached to the deprotonated carboxylate moiety, followed by the sequential interruption of the self-assembly of the stearic acid at the LC/aqueous interface. The results showed that the acid-doped LC pattern system not only enabled the highly sensitive detection of HM ions at a sub-nanomolar concentration but it also facilitated rapid detection (<10 min) with simple procedures.

Label-free detection of viruses on a polymeric surface using liquid crystals.

In this study, we demonstrated a label-free detection of viruses using liquid crystals (LCs) on a polymeric surface with periodic nanostructures. The polymeric nanostructures, which hold sinusoidal anisotropic patterns, were created by a sequential process of poly-(dimethylsiloxane) buckling and replication of the patterns on a poly-(urethane acrylate) surface containing a film of gold. After immobilization of human cytomegalovirus- and adenovirus-antibodies onto the polymeric surface treated with a mixed self-assembled monolayer, a uniform appearance reflecting the uniform orientation of 4-cyano-4'-pentylbiphenyl (5CB) was observed. Conversely, binding of viruses to their antibody decorated surface induced a random appearance of 5CB from the random orientation of 5CB. The uniform to random orientational transition of 5CB indicates that the anisotropic topography of the polymeric surface was masked after specific binding of viruses to the antibody decorated surface. We evaluated the specificity of the binding events by confirming topographical changes and optical thickness using atomic force microscopy and ellipsometry, respectively. These results demonstrate that polymeric surfaces with continuous anisotropic patterns can be used to amplify the presence of nanoscopic virions into an optical response of LC, as well as expand the scope of LC-based biological detection on polymeric solid surfaces.

Measuring ligand-receptor binding events on polymeric surfaces with periodic wave patterns using liquid crystals.

In this study, we measured ligand-receptor binding events on polymeric substrates with periodic nanostructures using liquid crystals (LCs). Periodic sinusoidal wave patterns were generated through the buckling of the poly-(dimethylsiloxane) (PDMS) substrate on a cylindrical surface followed by replicating the associated relief structures on a poly-(urethane acrylate) (PUA) surface, where a film of gold was deposited. Avidin was then covalently immobilized onto a gold surface decorated with carboxylic acid-terminated self-assembled monolayer via NHS/EDC chemistry. The optical response of the device showed that the orientation of nematic 4-cyano-4'-pentylbiphenyl (5CB) was parallel to the plane of the avidin surface. However, the formation of the avidin-biotin complexes disturbed the sinusoidal topographies of the surface, and induced a random orientation of LCs, which produced a distinctive change in the optical response. We also confirmed the specific ligand-receptor interactions using ellipsometry and atomic force microscopy (AFM). These results suggest that polymeric surfaces with continuous wavy features could be used to develop novel LC-based sensors for the detection of ligand-receptor binding events.