Affordable on-site COVID-19 test using non-powered preconcentrator.

A simple, affordable point of care test (POCT) is necessary for on-site detection of coronavirus disease 2019 (COVID-19). The lateral flow assay (LFA) has great potential for use in POCT mainly because of factors such as low time consumption, low cost, and ease of use. However, it lacks sensitivity and limits of detection (LOD), which are essential for early diagnostics. In this study, we proposed a non-powered preconcentrator (NPP) based on nanoelectrokinetics for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Antigen (Ag) lateral flow assay. The non-powered preconcentrator is composed of glass fiber-based composite paper and ion permselective material, and it can be simply operated by force balancing gravitational, capillary, and depletion-induced forces. The proposed approach helps enrich the SARS-CoV-2 viral nucleocapsid (N) proteins based on a 10-min operation, and it improved the LOD by up to 10-fold. The corresponding virus enrichment, which was evaluated using the reverse-transcriptase polymerase chain reaction (RT-PCR), revealed an improvement in ΔCt values > 3. We successfully demonstrated the enhancement of the NPP-assisted LFA, we extended to applying it to clinical samples. Further, we demonstrated an affordable, easy-to-implement form of LFA by simply designing NPP directly on the LFA buffer tube.

Rapid and low-cost, and disposable electrical sensor using an extended gate field-effect transistor for cardiac troponin I detection.

Field effect transistor (FET) biosensor is based on metal oxide field effect transistor that is gated by changes in the surface charges induced the reaction of biomolecules. In most cases of FET biosensor, FET biosensor is not being reused after the reaction; therefore, it is an important concept of investigate the biosensor with simplicity, cheap and reusability. However, the conventional cardiac troponin I (cTnI) sensing technique is inadequate owing to its low sensitivity and high operational time and cost. In this study, we developed a rapid and low-cost, and disposable electrical sensor using an extended gate field-effect transistor (EGFET) to detect cTnI, as a key biomarker for myocardiac infarction. We first investigated pH sensing characteristics according to the pH level, which provided a logarithmically linear sensitivity in the pH sensing buffer solution of approximately 57.9 mV/pH. Subsequently, we prepared a cTnI sample and monitored the reaction between cTnI and cTnI antibodies through the changes in the drain current and transfer curves. Our results showed that the EGFET biosensor could successfully detect the cTnI levels as well as the pH with low-cost and rapid detection.

Nanoelectrokinetic-assisted lateral flow assay for COVID-19 antibody test.

A lateral flow assay (LFA) platform is a powerful tool for point-of-care testing (POCT), especially for self-testing. Although the LFA platform provides a simple and disposable tool for Coronavirus disease of 2019 (COVID-19) antigen (Ag) and antibody (Ab) screening tests, the lower sensitivity for low virus titers has been a bottleneck for practical applications. Herein, we report the combination of a microfluidic paper-based nanoelectrokinetic (NEK) preconcentrator and an LFA platform for enhancing the sensitivity and limit of detection (LOD). Biomarkers were electrokinetically preconcentrated onto a specific layer using the NEK preconcentrator, which was then coupled with LFA diagnostic devices for enhanced performance. Using this nanoelectrokinetic-assisted LFA (NEK-LFA) platform for self-testing, the severe acute respiratory syndrome coronavirus 2 Immunoglobulin G (SARS-CoV-2 IgG) sample was preconcentrated from serum samples. After preconcentration, the LOD of the LFA was enhanced by 32-fold, with an increase in analytical sensitivity (16.4%), which may offer a new opportunity for POCT and self-testing, especially in the COVID-19 pandemic and endemic global context.

Origami paper-based sample preconcentration using sequentially driven ion concentration polarization.

Ion concentration polarization (ICP) is one of the preconcentration techniques which can acquire a high preconcentration factor. Still, the main hurdles of ICP are its instability and low efficiency under physiological conditions with high ionic strength and abundant biomolecules. Here, we suggested a sequentially driven ICP process, which enhanced the electrokinetic force required for preconcentration, enabling enrichment of highly ionic raw samples without increasing the electric field. We acquired a 13-fold preconcentration factor (PF) in human serum using a paper-based origami structure consisting of multiple layers for three-dimensional sequential ICP (3D seq-ICP). Moreover, we demonstrated a paper-based enzyme-linked immunosorbent assay (ELISA) by 3D seq-ICP using tau protein, showing a 6-fold increase in ELISA signals.

Highly efficient and scalable biomarker preconcentrator based on nanoelectrokinetics.

Micro/nanofluidics are excellent candidates for biological sample preparation. However, the limited process volume in micro/nanofluidics is the main hurdle limiting their practical applications. To date, most micro/nanofluidics have processed sample volumes of several microliters and have rarely been used to handle large-volume samples. Herein, we propose a microfluidic paper-based large-volume preconcentrator (u-LVP) for enrichment and purification of biomarkers (e.g., miRNA) using ion concentration polarization. A Nafion (ion-selective nanoporous membrane)-functionalized multilayer cellulose paper enables microscale division of milliliter-scale samples, thus electrokinetically separating and preconcentrating the biomarker in different locations within the u-LVP. By inserting collecting discs at optimal positions in the u-LVP, the enriched biomarker is simply recovered with high efficiency. With this approach, as an exemplary biomarker, miRNA-21 in human serum was separated from proteins and preconcentrated with an effective preconcentration factor exceeding 6.63 and a recovery rate above 84%. Thus, our platform offers new opportunities and benefits for biomarker, diagnostic, prognostic, and therapeutic research.

Enhanced paper-based ELISA for simultaneous EVs/exosome isolation and detection using streptavidin agarose-based immobilization.

EVs/exosomes are considered as the next generation of biomarkers, including for liquid biopsies. Consequently, the quantification of EVs/exosomes is crucial for facilitating EV/exosome research and applications. Paper-based enzyme-linked immunosorbent assay (p-ELISA) is a portable diagnostic system with low cost that is simple and easy to use; however, it shows low sensitivity and linearity. In this study, we develop p-ELISA for targeting EVs/exosomes by using streptavidin agarose resin-based immobilization (SARBI). This method reduces assay preparation times, provides strong binding, and retains good sensitivity and linearity. The time required for the total assay, including preparation steps and surface immobilization, was shortened to ∼2 h. We evaluated SARBI p-ELISA systems with/without CD63 capture Ab and then with fetal bovine serum (FBS) and EVs/exosome-depleted fetal bovine serum (dFBS). The results provide evidence supporting the selective capture ability of SARBI p-ELISA. We obtain semiquantitative p-ELISA results using an exosome standard (ES) and human serum (HS), with R2 values of 0.95 and 0.92, respectively.

Electrokinetic Size-Based Spatial Separation of Micro/Nanospheres Using Paper-Based 3D Origami Preconcentrator.

Sample preparation steps (e.g., preconcentration and separation) are key to enhancing sensitivity and reliability in biomedical and analytical chemistry. However, conventional methods (e.g., ultracentrifugation) cause significant loss of sample as well as their contamination. In this study, we developed a paper-based three-dimensional (3D) origami ion concentration polarization preconcentrator (POP) for highly efficient and facile sample preparation. The unique design of POP enables simultaneous preconcentration and spatial separation of target analytes rapidly and economically. The POP comprises accordion-like multifolded layers with convergent wicking areas that can separate analytes based on their sizes in different layers, which can then be easily isolated by unfolding the POP. We first demonstrated 100-fold preconcentration of albumin and its isolation on the specific layers. Then, we demonstrated the simultaneous preconcentration and spatial separation of microspheres of three different sizes (with diameters of 0.02, 0.2, and 2 µm) on the different layers.

Battery operated preconcentration-assisted lateral flow assay.

Paper-based analytical devices (e.g. lateral flow assays) are highly advantageous as portable diagnostic systems owing to their low costs and ease of use. Because of their low sensitivity and detection limits for biomolecules, these devices have several limitations in applications for real-field diagnosis. Here, we demonstrate a paper-based preconcentration enhanced lateral flow assay using a commercial ß-hCG-based test. Utilizing a simple 9 V battery operation with a low power consumption of approximately 81 µW, we acquire a 25-fold preconcentration factor, demonstrating a clear sensitivity enhancement in the colorimetric lateral flow assay; consequently, clear colors are observed in a rapid kit test line, which cannot be monitored without preconcentration. This device can also facilitate a semi-quantitative platform using the saturation value and/or color intensity in both paper-based colorimetric assays and smartphone-based diagnostics.