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.

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.

High sensitive and broad-range detection of cortisol in human saliva using a trap lateral flow immunoassay (trapLFI) sensor.

Cortisol, a steroid hormone, is a main biomarker of psychological stress. Early diagnosis and proper treatment of such stress is crucial to prevent the excessive secretion of cortisol. However, cortisol has a low molecular weight and cannot provide sufficient recognition sites for sandwich immunoreaction; it has previously been measured using a competitive immunoassay instead of a general sandwich immunoassay. The disadvantage of this approach is that quantitative measurements are limited because of the narrow measurable range that is key for biosensors. To overcome this limitation, we propose a new detection platform that enables small molecules such as cortisol to be quantified with high detection sensitivity. A trap lateral flow immunoassay (trapLFI) sensor has deletion and detection zones instead of the test and control zones in general lateral flow immunoassay (LFI) sensors. The conjugates used to minimize possible detection targets at low concentration are gold nanoparticles that include an antibody against cortisol and an enzyme for signal generation. Target-bound conjugates are captured in the detection zone, whereas conjugates not binding with targets are trapped in the deletion zone. Using this platform, enzyme-catalyzed color signals increase in the detection zone and decrease in the deletion zone with the concentration of cortisol. The ratio of signal from deletion zone and detection zone supplied a wide analytical range (0.01-100 ng mL-1) with high detection sensitivity (9.9 pg mL-1). Analysis of 15 human saliva samples showed a good correlation with conventional ELISA results (R2 = 0.9432).

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.

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.

A rapid assay provides on-site quantification of tetrahydrocannabinol in oral fluid.

Tetrahydrocannabinol (THC), the primary psychoactive ingredient of cannabis, impairs cognitive and motor function in a concentration-dependent fashion. Drug testing is commonly performed for employment and law enforcement purposes; however, available tests produce low-sensitive binary results (lateral flow assays) or have long turnaround (gas chromatography­mass spectrometry). To enable on-site THC quantification in minutes, we developed a rapid assay for oral THC analysis called EPOCH (express probe for on-site cannabis inhalation). EPOCH features distinctive sensor design such as a radial membrane and transmission optics, all contained in a compact cartridge. This integrated approach permitted assay completion within 5 min with a detection limit of 0.17 ng/ml THC, which is below the regulatory guideline (1 ng/ml). As a proof of concept for field testing, we applied EPOCH to assess oral fluid samples from cannabis users (n = 43) and controls (n = 43). EPOCH detected oral THC in all specimens from cannabis smokers (median concentration, 478 ng/ml) and THC-infused food consumers. Longitudinal monitoring showed a fast drop in THC concentrations within the first 6 hours of cannabis smoking (half-life, 1.4 hours).

Addressable micropatterning of multiple proteins and cells by microscope projection photolithography based on a protein friendly photoresist.

We report a new method for the micropatterning of multiple proteins and cells with micrometer-scale precision. Microscope projection photolithography based on a new protein-friendly photoresist, poly(2,2-dimethoxy nitrobenzyl methacrylate-r-methyl methacrylate-r-poly(ethylene glycol) methacrylate) (PDMP), was used for the fabrication of multicomponent protein/cell arrays. Microscope projection lithography allows precise registration between multiple patterns as well as facile fabrication of microscale features. Thin films of PDMP became soluble in near-neutral physiological buffer solutions upon UV exposure and exhibited excellent resistance to protein adsorption and cell adhesion. By harnessing advantages in microscope projection photolithography and properties of PDMP thin films, we could successfully fabricate protein arrays composed of multiple proteins. Furthermore, we could extend this method for the patterning of two different types of immune cells for the potential study of immune cell interactions. This technique will in general be useful for protein chip fabrication and high-throughput cell-cell communication study.

Enhanced biomolecular detection based on localized surface plasmon resonance (LSPR) using enzyme-precipitation reaction.

An enzyme-catalyzed precipitation reaction was employed as a means to increase the change in the LSPR signal after intermolecular bindings between antigens and antibodies occurred on gold nanodot surfaces. The gold nanodot array with an diameter of 175 nm and a thickness of 20 nm was fabricated on a glass wafer using thermal nanoimprint lithography. The human interleukin (hIL) 5 antibody was immobilized on the gold nanodot, followed by binding of hIL 5 to the anti-hIL 5. Subsequently, a biotinylated anti-hIL 5 and a alkaline phosphatase conjugated with streptavidin were simultaneously introduced. A mixture of 5-bromo-4-chloro-3-indolyl phosphate p-toluidine (BCIP) and nitro blue tetrazolium (NBT) was then used for precipitation, which resulted from the biocatalytic reaction of the alkaline phosphatase on gold nanodot. The LSPR spectra were obtained after each binding process. Using this analysis, the enzyme-catalyzed precipitation reaction on gold nanodots was found to be effective in amplifying the change in the peak wavelength of LSPR after molecular bindings.

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.

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.

Improved near infrared-mediated hydrogel formation using diacrylated Pluronic F127-coated upconversion nanoparticles.

In situ hydrogel synthesis based on photopolymerization has been recognized as a promising strategy that can be used for tissue augmentation. In this study, we developed an efficient in situ gelation method to prepare bulk hydrogels via near infrared (NIR)-mediated photopolymerization using acrylated polyethylene glycol and diacrylated Pluronic F127-coated upconversion nanoparticles (UCNPs). In our system, upon 980-nm laser irradiation, UCNPs transmit visible light, which triggers the activation of eosin Y to initiate polymerization. We found that the UCNPs coated with diacrylated Pluronic F127 can enhance the photopolymerization efficiency and thus enable the production of bulk hydrogel with requirement of a lower NIR light power compared to that required with the bare UCNPs. This photopolymerization approach will be beneficial to achieve in situ polymerization in vivo for various biomedical applications such as cell/drug delivery and construction of tissue augments.

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.

Label-free analysis of the characteristics of a single cell trapped by acoustic tweezers.

Single-cell analysis is essential to understand the physical and functional characteristics of cells. The basic knowledge of these characteristics is important to elucidate the unique features of various cells and causative factors of diseases and determine the most effective treatments for diseases. Recently, acoustic tweezers based on tightly focused ultrasound microbeam have attracted considerable attention owing to their capability to grab and separate a single cell from a heterogeneous cell sample and to measure its physical cell properties. However, the measurement cannot be performed while trapping the target cell, because the current method uses long ultrasound pulses for grabbing one cell and short pulses for interrogating the target cell. In this paper, we demonstrate that short ultrasound pulses can be used for generating acoustic trapping force comparable to that with long pulses by adjusting the pulse repetition frequency (PRF). This enables us to capture a single cell and measure its physical properties simultaneously. Furthermore, it is shown that short ultrasound pulses at a PRF of 167 kHz can trap and separate either one red blood cell or one prostate cancer cell and facilitate the simultaneous measurement of its integrated backscattering coefficient related to the cell size and mechanical properties.

Electrically conductive graphene/polyacrylamide hydrogels produced by mild chemical reduction for enhanced myoblast growth and differentiation.

Graphene and graphene derivatives, such as graphene oxide (GO) and reduced GO (rGO), have been extensively employed as novel components of biomaterials because of their unique electrical and mechanical properties. These materials have also been used to fabricate electrically conductive biomaterials that can effectively deliver electrical signals to biological systems. Recently, increasing attention has been paid to electrically conductive hydrogels that have both electrical activity and a tissue-like softness. In this study, we synthesized conductive graphene hydrogels by mild chemical reduction of graphene oxide/polyacrylamide (GO/PAAm) composite hydrogels to obtain conductive hydrogels. The reduced hydrogel, r(GO/PAAm), exhibited muscle tissue-like stiffness with a Young's modulus of approximately 50kPa. The electrochemical impedance of r(GO/PAAm) could be decreased by more than ten times compared to that of PAAm and unreduced GO/PAAm. In vitro studies with C2C12 myoblasts revealed that r(GO/PAAm) significantly enhanced proliferation and myogenic differentiation compared with unreduced GO/PAAm and PAAm. Moreover, electrical stimulation of myoblasts growing on r(GO/PAAm) graphene hydrogels for 7days significantly enhanced the myogenic gene expression compared to unstimulated controls. As results, our graphene-based conductive and soft hydrogels will be useful as skeletal muscle tissue scaffolds and can serve as a multifunctional platform that can simultaneously deliver electrical and mechanical cues to biological systems. STATEMENT OF SIGNIFICANCE: Graphene-based conductive hydrogels presenting electrical conductance and a soft tissue-like modulus were successfully fabricated via mild reduction of graphene oxide/polyacrylamide composite hydrogels to study their potential to skeletal tissue scaffold applications. Significantly promoted myoblast proliferation and differentiation were obtained on our hydrogels. Additionally, electrical stimulation of myoblasts via the graphene hydrogels could further upregulate myogenic gene expressions. Our graphene-incorporated conductive hydrogels will impact on the development of new materials for skeletal muscle tissue engineering scaffolds and bioelectronics devices, and also serve as novel platforms to study cellular interactions with electrical and mechanical signals.

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.

Near-Infrared-Light-Assisted Photothermal Polymerization for Transdermal Hydrogelation and Cell Delivery.

Photopolymerization of hydrogels has been widely used to encapsulate cells and support their growth in 3D environments. However, common light sources (i.e., ultraviolet and visible light) strongly interact with biological systems and are therefore inappropriate for in vivo applications, such as transdermal polymerization. Using near infrared (NIR) light that minimally interacts with living tissues, this study investigates NIR light-assisted photothermal polymerization (NAPP) of diacrylated polyethylene glycol (PEGDA), in which interactions between NIR light and gold nanorods activate a thermal initiator (i.e., AIPH), resulting in generation of radicals for polymerization of PEGDA. Gelation parameters, including precursor concentrations and NIR power, are investigated to minimize the use of initiator and temperature increases (<43 °C) during NAPP. Cell viability is as high as 80% after NAPP-based encapsulation. Incorporation of polyethylene glycol (PEG) modified with a cell-adhesive peptide moiety (Arg-Gly-Asp) into the gel system further enables prolongation of cell viability during incubation up to 7 d. NAPP results in successful transdermal gelation and good viability of the transplanted cells. Thus, this new cell encapsulation approach, demonstrated for the first time in this study, will benefit various applications, including cell delivery and remote control over cellular environments.

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.

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.