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

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

Homogeneous Immunosensor Based on Luminescence Resonance Energy Transfer for Glycated Hemoglobin Detection Using Upconversion Nanoparticles

We report an immunosensor based on luminescence resonance energy transfer (LRET) to detect homogeneous glycated hemoglobin (HbA1c). This system uses near-infrared (NIR)-to-visible rare-earth upconversion nanoparticles (UCNPs), such as NaYF₄:Yb³⁺, Er³⁺, as the donor and HbA1c as the acceptor. The HbA1c used as target molecules showed absorption at 541 nm, which corresponded with the emission of the UCNPs. When HbA1c was added, LRET occurred between the donor and acceptor under laser irradiation of 980 nm because of the specific recognition between the anti-HbA1c monocolonal antibody-functionalized UCNPs and HbA1c. In the absence of HbA1c, there was strong upconversion luminescence intensity; however, in its presence, the distance between the donor and acceptor decreased to enable energy transfer, consequently quenching the luminescence of the UCNPs. The proposed method was successfully applied to HbA1c detection in blood samples. Our results indicate that the LRET-based immunosensor allows for specific and sensitive detection of HbA1c in a homogeneous manner

Selective Assembly and Guiding of Actomyosin Using Carbon Nanotube Network Monolayer Patterns

We report a new method for the selective assembly and guiding of actomyosin using carbon nanotube patterns. In this method, monolayer patterns of the single-walled carbon nanotube (swCNT) network were prepared via the self-limiting mechanism during the directed assembly process, and they were used to block the adsorption of both myosin and actin filaments on specific substrate regions. The swCNT network patterns were also used as an efficient barrier for the guiding experiments of actomyosin. This is the first result showing that inorganic nanostructures such as carbon nanotubes can be used to control the adsorption and activity of actomyosin. This strategy is advantageous over previous methods because it does not require complicated biomolecular linking processes and nonbiological nanostructures are usually more stable than biomolecular linkers

Absorption-Modulated SiO₂@Au Core–Satellite Nanoparticles for Highly Sensitive Detection of SARS-CoV‑2 Nucleocapsid Protein in Lateral Flow Immunosensors

The worldwide spread of coronavirus disease 2019 (COVID-19) highlights the need for rapid, simple, and accurate tests to detect various variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The antigen test, based on the lateral flow immunoassay (LFI), is a suitable “first line of defense” test that enables early identification and timely isolation of patients to minimize viral transmission among communities. However, it is generally less accurate than nucleic acid testing, and its sensitivity needs improvement. Here, a novel rapid detection method is designed to sensitively detect SARS-CoV-2 using isolated gold nanoparticle (AuNP)-assembled SiO2 core–satellite nanoparticles (SiO2@Au CSNPs). Well-grown AuNP satellites in the synthesis of SiO2@Au CSNPs significantly enhanced their light absorption, increased the detection sensitivity, and lowered the detection limit by 2 orders of magnitude relative to conventional gold colloids. The proposed system enabled highly sensitive detection of the SARS-CoV-2 nucleocapsid protein with a detection limit of 0.24 pg mL–1 within 20 min. This is the first study to develop a highly sensitive antigen test using the absorption-modulated SiO2@Au CSNPs. Our findings demonstrate the capacity of this platform to serve as an effective sensing strategy for managing pandemic conditions and preventing the spread of viral infections

Solid-Phase Photocatalysts: Physical Vapor Deposition of Au Nanoislands on Porous TiO₂ Films for Millimolar H₂O₂ Production within a Few Minutes

The production of hydrogen peroxide (H2O2) using photocatalytic nanoparticles is an emerging field with applications in organic synthesis, biosensors, and fuel cells. Colloidal photocatalysts are, however, limited in their applications because of poor efficiency and stability. In this study, millimolar production of H2O2 was achieved within 5 min using solid-phase photocatalystsAu nanoislands (NIs) on porous TiO2 filmswithout supplying O2 or stirring the solution. Au/TiO2 showed an almost 80-fold greater productivity than TiO2, which can be explained by considering two factors. First, the physical-vapor-deposited Au resulted in the formation of Au NIs of various sizes on TiO2, whose work functions were size-dependent. Thus, the combination of small Au NIs, TiO2, and large Au NIs allowed the introduction of potential gradients through TiO2, and also the reduced potential barriers at the small Au NI/TiO2 junctions; thereby minimizing the recombination of electron–hole pairs, and effectively extracting them. Second, the porous TiO2 films may effectively scatter UV light, leading to enhanced electron–hole pair generation. The inherent properties of the solid-phase photocatalysts could also circumvent stability issues caused by aggregation. These solid-phase photocatalysts should facilitate the development of high-efficiency H2O2 generation and promote technology based on H2O2-mediated processes

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

Selective Assembly and Guiding of Actomyosin Using Carbon Nanotube Network Monolayer Patterns

We report a new method for the selective assembly and guiding of actomyosin using carbon nanotube patterns. In this method, monolayer patterns of the single-walled carbon nanotube (swCNT) network were prepared via the self-limiting mechanism during the directed assembly process, and they were used to block the adsorption of both myosin and actin filaments on specific substrate regions. The swCNT network patterns were also used as an efficient barrier for the guiding experiments of actomyosin. This is the first result showing that inorganic nanostructures such as carbon nanotubes can be used to control the adsorption and activity of actomyosin. This strategy is advantageous over previous methods because it does not require complicated biomolecular linking processes and nonbiological nanostructures are usually more stable than biomolecular linkers

Multichannel Surface Plasmon Resonance Imaging and Analysis of Micropatterned Self-Assembled Monolayers and Protein Affinity Interactions

Multichannel images of 11-mercaptoundecanoic acid and 11-mercapto-1-undecanol self-assembled monolayers together with a biospecific interferon-γ (IFN-γ)/anti-IFN-γ antibody immunoaffinity interaction were observed by the two-dimensional surface plasmon resonance (2D-SPR) imaging system. With the fabricated 2D-SPR imaging system, adopting a white light source in combination with a narrow band-pass filter, sharp images were resolved, minimizing the diffraction patterns on the resulting images. Micropatterning of self-assembled monolayers was acheived by exploiting the UV photolysis of thiol bonding, instead of conventional photolithography. The line profile calibration of the image contrast with ellipsometric analysis enabled us to discriminate the change in monolayer thickness within a sub-nanometer scale. For the protein interactions on the surface, the biospecific affinity recognition reaction of IFN-γ antigen with surface-immobilized antibody was analyzed. Through the signal amplification strategy based on the enzyme-catalyzed precipitation reaction in a sandwich-type immunoassay, biospecific antigen binding was found detectable down to a concentration of 1 ng/mL