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.

Advanced trap lateral flow immunoassay sensor for the detection of cortisol in human bodily fluids.

Paper-based biosensors based on lateral flow immunoassay (LFI) are promising candidates for POC diagnosis because of their ease of use and rapid target detection. However, the low sensitivity of LFI limits its application, and signal amplification has been used in numerous studies to increase its sensitivity. We developed an advanced trap LFI (α-trapLFI), a simple-to-use sensor, with an additional step for signal amplification. Here, signal amplification is automatically implemented following delayed release of enhancement solution induced by water-soluble polyvinyl alcohol tape. As the polyvinyl alcohol tape is exposed to water, its polymer structure is perturbed (within 5 min), allowing ions to pass through. This new sensor was designed to have a short time delay between the flow of solutions used for the immunoassay and signal amplification. The α-trapLFI was subsequently used to detect cortisol with high sensitivity (9.1 pg∙mL-1) over a broad detection range (0.01-1000 ng∙mL-1) in bodily fluids. Furthermore, an excellent correlation was obtained by analyzing 20 human real saliva samples using this sensor and a conventional ELISA (R2 = 0.90). The new sensor will be helpful in detecting various small molecules for simple, rapid, and portable POC diagnosis of stress disorders.

Ultrasensitive Detection Platform of Disease Biomarkers Based on Recombinase Polymerase Amplification with H-Sandwich Aptamers.

The detection of trace protein biomarkers is essential in the diagnostic field. Protein detection systems ranging from widely used enzyme-linked immunosorbent assays to simple, inexpensive approaches, such as lateral flow immunoassays, play critical roles in medical and drug research. Despite continuous progress, current systems are insufficient for the diagnosis of diseases that require high sensitivity. In this study, we developed a heterogeneous sandwich-type sensing platform based on recombinase polymerase amplification using DNA aptamers specific to the target biomarker. Only the DNA bound to the target in the form of a heterogeneous sandwich was selectively amplified, and the fluorescence signal of an intercalating dye added before the amplification reaction was detected, thereby enabling high specificity and sensitivity. We applied this method for the detection of protein biomarkers for various infectious diseases including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and observed attomolar-level detection of biomarkers and low cross-reactivity between different viruses. We also confirmed detection efficiency of the proposed method using clinical samples. These results demonstrate that the proposed sensing platform can be used to diagnose various diseases requiring high sensitivity, specificity, and accuracy.

Reactant/polymer hybrid films on p-n junction photodetectors for self-powered, non-invasive glucose biosensors.

The portability of electronic-based biosensors is limited because of the use of batteries and/or solutions containing reactants such as enzymes for assay, which limits the utility of such biosensors in point-of-care (POC) testing. In this study, we report on the development of a self-powered biosensor composed of only portable components: a reactant-containing poly (ethylene glycol) (PEG) film for the colorimetric assay, and a self-powered n-InGaZnO/p-Si photodetector. The PEG film containing enzymes and color-developing agents was formed on a glass slide by spin coating. The self-powered biosensor was fabricated by placing the hybrid film on the p-n junction photodetector, and applied in non-invasive glucose detection (salivary glucose). Injection of the target-containing solution dissolved the PEG that led to the release of enzymes and color-developing agents, resulting in a colorimetric assay. The colorimetric assay could attenuate the light reaching the photodetector, thus facilitating target concentration verification by measuring the photocurrent. Our self-powered biosensor has two main advantages: (i) all components of the biosensor are portable and (ii) dilution of target concentration is avoided as the reagents are in the PEG film. Therefore, the self-powered biosensor, without solution-phase components, could be highly beneficial for creating portable, sensitive biosensors for POC testing.

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.

Highly sensitive and universal detection strategy based on a colorimetric assay using target-specific heterogeneous sandwich DNA aptamer.

A simple, universal, and sensitive colorimetric biosensor for detecting of various biomarkers was devised using a target-specific DNA aptamer, as the recognition element, and engineered with streptavidin-fusion replication protein A 70 kDa (RPA70A) linked to biotin-horseradish peroxidase, as the colorimetric element. To improve sensitivity and stability compared to other colorimetric sensing platforms, we developed a novel detection strategy by integrating a newly selected heterogeneous sandwich DNA aptamer and protein engineering in this study. The proposed method is based on a change in color from colorless to blue due to the interaction of the aptamer with RPA70A in the presence of the target; this color change could be observed by the naked eye or measured with a UV-vis spectrometer. We confirmed its high sensitivity and specificity for two model targets using their aptamers under optimal experimental conditions. In addition, the feasibility of the assay was investigated in clinical samples containing NPs of influenza A or B virus. These results suggest that our detection system developed herein can be universally applied to the diagnosis of various diseases owing to its stability, sensitivity, and specificity.

Development of DNA Aptamers against the Nucleocapsid Protein of Severe Fever with Thrombocytopenia Syndrome Virus for Diagnostic Application: Catalytic Signal Amplification using Replication Protein A-Conjugated Liposomes.

Most prevalent infectious diseases worldwide are caused by mediators such as insects and characterized by high mortality and morbidity, thereby creating a global public health concern. Therefore, a sensitive, selective detection platform for diagnosing diseases in the early stages of infection is needed to prevent disease spread and to protect public health. Here, we developed novel DNA aptamers specific to the nucleocapsid protein (NP) of the severe fever with thrombocytopenia syndrome (SFTS) virus and synthesized ssDNA-binding protein-conjugated liposomes encapsulated with horseradish peroxidase (HRP) for application in a simple and universal platform. This platform achieved highly sensitive detection of the NP by measuring the colorimetric signal following lysis of the HRP encapsulated liposomes, mediated by a mixture of 3,3',5,5'-tetramethylbenzidine and H2O2 solution. The limit of detection was 0.009 ng·mL-1, and NP was successfully detected in diluted human serum with a high recovery rate. Moreover, this method was specific and did not exhibit cross-reactivity among NPs of other virus types. These results demonstrated the efficacy of the proposed method as a highly sensitive, specific, and universal diagnostic tool for potential application in monitoring of the early stages of infectious diseases.

Development of Replication Protein A-Conjugated Gold Nanoparticles for Highly Sensitive Detection of Disease Biomarkers.

Paper-based lateral flow immunoassays (LFIAs) using conventional sandwich-type immunoassays are one of the most commonly used point-of-care (PoC) tests. However, the application of gold nanoparticles (AuNPs) in LFIAs does not meet sensitivity requirements for the detection of infectious diseases or biomarkers present at low concentrations in body fluids because of the limited number of AuNPs that can bind to the target. To overcome this problem, we first developed a single-stranded DNA binding protein (RPA70A, DNA binding domain A of human Replication Protein A 70 kDa) conjugated to AuNPs for a sandwich assay using a capture antibody immobilized in the LFIA and an aptamer as a detection probe, thus, enabling signal intensity enhancement by attaching several AuNPs per aptamer. We applied this method to detect the influenza nucleoprotein (NP) and cardiac troponin I (cTnI). We visually detected spiked targets at a low femtomolar range, with limits of detection for NP in human nasal fluid and for cTnI in serum of 0.26 and 0.23 pg·mL-1, respectively. This technique showed significantly higher sensitivity than conventional methods that are widely used in LFIAs involving antibody-conjugated AuNPs. These results suggest that the proposed method can be universally applied to the detection of substances requiring high sensitivity and can be used in the field of PoC testing for early disease diagnosis.

Advanced Colorimetric Paper Sensors Using Color Focusing Effect Based on Asymmetric Flow of Fluid.

Although paper-based colorimetric sensors utilizing enzymatic reactions are well suited for real-field diagnosis, their widespread use is hindered by signal blurring at the detection spot due to the action of capillary forces on the liquid and the corresponding membrane. In this study, we eliminated signal losses commonly observed during enzyme-mediated colorimetric sensing and achieved pattern-free quantitative analysis of glucose and uric acid by mixing enzymes and color-forming reagents with chitosan oligosaccharide lactate (COL), which resulted in perfectly focused colorimetric signals at the detection spot, using asymmetric flow induced by changing the flow rate of the COL-treated paper. The targets were calibrated with 0-500 mg/dL of glucose and 0-200 mg/dL of uric acid, and the limit of detection was calculated to be 0.6 and 0.03 mg/dL, respectively. In human urine, the correlation has a high response between the measured and spiked concentrations, and the stability of the enzyme mixture including COL increased by 41% for glucose oxidase mixture and 29% for uricase mixture, compared to the corresponding mixtures without COL. Thus, the color focusing and pattern-free sensor, which have the advantages of easy fabrication, easy handling, and high stability, should be applied to real-field diagnosis.

Self-Powered Biosensors Using Various Light Sources in Daily Life Environments: Integration of p-n Heterojunction Photodetectors and Colorimetric Reactions for Biomolecule Detection.

Electronic biosensors operating without power supply are high in demand owing to increasing interest in point-of-care (POC) coupled with portable and wearable electronic devices for smart healthcare services. Although self-powered electronic sensors have emerged with the promise of resolving the energy supply problems, achieving sufficient sensitivity to targets in real samples is highly challenging because of the matrix effect caused by electroactive species. In this study, we developed a self-powered biosensor platform by combining n-indium gallium zinc oxide (IGZO)/p-Si heterojunction photodetectors and physically separated colorimetric reactions. The self-powered biosensors were applied to glucose detection in real human samples using light sources from daily life environments such as fluorescent light and sunlight. The sensors showed high sensitivity and stability from 0.01 to 10 mg mL-1 of glucose in human saliva and urine without matrix effect from the electroactive species in real samples. In addition, a small change in glucose concentration in human serum was distinguishable with a resolution of 0.01 mg mL-1. Notably, these results were obtained using well-developed and widely used materials like Si and IGZO with simple deposition techniques. Moreover, this self-powered biosensing platform can be universally applied for the detection of all biomolecules being detected by colorimetric assays. To the best of our knowledge, this is the first report on such self-powered biosensors, which could be a promising candidate for future POC biosensors integrated with portable and wearable electronic devices.

Fabrication of Glass Microchannel via Glass Imprinting using a Vitreous Carbon Stamp for Flow Focusing Droplet Generator.

This study reports a cost-effective method of replicating glass microfluidic chips using a vitreous carbon (VC) stamp. A glass replica with the required microfluidic microstructures was synthesized without etching. The replication method uses a VC stamp fabricated by combining thermal replication using a furan-based, thermally-curable polymer with carbonization. To test the feasibility of this method, a flow focusing droplet generator with flow-focusing and channel widths of 50 µm and 100 µm, respectively, was successfully fabricated in a soda-lime glass substrate. Deviation between the geometries of the initial shape and the vitreous carbon mold occurred because of shrinkage during the carbonization process, however this effect could be predicted and compensated for. Finally, the monodispersity of the droplets generated by the fabricated microfluidic device was evaluated.

A highly sensitive and widely adaptable plasmonic aptasensor using berberine for small-molecule detection.

Localized surface plasmon resonance (LSPR) biosensors allow label-free detection of small molecules in molecular binding events; however, they are limited by a relatively low sensitivity and narrow dynamic range. Here, we report highly sensitive small-molecule detection by LSPR peak shift exploiting the G-quadruplex (GQx) structure-binding characteristic of known GQx binders to enhance the LSPR signal of a plasmonic aptasensor. Six known GQx binders (thiazole orange, malachite green, crystal violet, zinc protoporphyrin IX, thioflavin T, and berberine) were tested for their ability to enhance the LSPR signal. Among these, berberine (BER) induced the largest LSPR peak shift by interacting with the GQx structure formed by the aptamer/target binding event on a gold nanorod surface. This specific binding performance was confirmed by the fluorescence signal of BER and through repeated cycles of BER addition and washing on the plasmonic sensing chip. The proposed plasmonic aptasensor respectively showed limit of detection (LOD) of 0.56, 0.63, 0.87 and 1.05 pM for ochratoxin A, aflatoxin B1, adenosine triphosphate and potassium ions, which was 1000-fold higher than that in BER-free condition, and a wide dynamic range from 10 pM to 10µM. In addition, the proposed LSPR aptasensor could effectively be used to quantitatively analyze small molecules in real samples.