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.

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.

Paper-based 1,5-anhydroglucitol quantification using enzyme-based glucose elimination.

The monosaccharide 1,5-anhydroglucitol (1,5-AG) is a known indicator of glucose levels. Conventional 1,5-AG quantification methods with enzyme-based sensors using pyranose oxidase (PROD) require elimination of interference from the sample (a laborious and time-consuming process), as PROD cannot distinguish 1,5-AG from other sugars. We developed a one-step paper-based sensor for detecting 1,5-AG using glucose oxidase, catalase, and mutarotase that eliminates excess glucose, which interferes with 1,5-AG detection. This sensor consists of two compartments for the quantification of glucose and 1,5-AG and reflects the concentration of these targets after reaction with water or spiked human urine. The limit of detection of the sensor was 0.9 mg dL-1 for glucose and 3.2 µg mL-1 for 1,5-AG.

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.

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.

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.

Chemically Driven, Water-Soluble Composites of Carbon Nanotubes and Silver Nanoparticles as Stretchable Conductors.

In the past decade, hybrid materials for highly stretchable, conductive electrodes have received tremendous attention in the fields of emerging wearable electronic, optoelectronic, and sensing devices. Here, we present a previously unrecognized aqueous route to producing stretchable conductors composed of silver nanoparticles (AgNPs) and single-walled carbon nanotubes (SWNTs) embedded in a polyurethane (PU) matrix, in contrast to ones dispersed in toxic organic solvents reported to date. The intact chemical interaction between one-dimensional SWNTs, for endowing the capability of establishing conductive pathways even in stretching conditions, and AgNPs, for enabling high conductivity of the composites, is achieved in an aqueous medium with an anionic polyelectrolyte, poly(acrylic acid), that undergoes pH-dependent conformational evolution. With this aqueous approach, we demonstrate that AgNP-SWNT-PU composites supported on PDMS substrates have the conductivities of 620 and 120 S cm-1 in unstrained and 90% elongated conditions, respectively, and display repeatable reversibility at a strain of 60%.