Beads- and oil-free single molecule assay with immuno-rolling circle amplification for detection of SARS-CoV-2 from saliva.

Digital enzyme linked immunosorbent assays (ELISA) can be used to detect various antigens such as spike (S) or nucleocapsid (N) proteins of SARS-CoV-2, with much higher sensitivity compared to that achievable using conventional antigen tests. However, the use of microbeads and oil for compartmentalization in these assays limits their user-friendliness and causes loss of assay information due to the loss of beads during the process. To improve the sensitivity of antigen test, here, we developed an oil- and bead-free single molecule counting assay, with rolling circle amplification (RCA) on a substrate. With RCA, the signal is localized at the captured region of an antigen, and the signal from a single antigen molecule can be visualized using the same immune-reaction procedures as in the conventional ELISA. Substrate-based single molecule assay was theoretically evaluated for kd value, and the concentration of capture and detection antibodies. As a feasibility test, biotin-conjugated primer and mouse IgG conjugates were detected even at femto-molar concentrations with this digital immuno-RCA. Using this method, we detected the N protein of SARS-CoV-2 with a limit of detection less than 1 pg/mL more than 100-fold improvement compared to the detection using conventional ELISA. Furthermore, testing of saliva samples from COVID-19 patients and healthy controls (n = 50) indicated the applicability of the proposed method for detection of SARS-CoV-2 with 99.5% specificity and 90.9% sensitivity.

Photooxidation-induced fluorescence amplification system for an ultra-sensitive enzyme-linked immunosorbent assay (ELISA).

This report suggests a method of enhancing the sensitivity of chemifluorescence-based ELISA, using photooxidation-induced fluorescence amplification (PIFA). The PIFA utilized autocatalytic photooxidation of the chemifluorescent substrate, 10-acetyl 3,7-dihydroxyphenoxazine (ADHP, Amplex Red) to amplify the fluorescent product resorufin, initially oxidized by horse radish peroxidase (HRP). As the amplification rate is proportional to the initial level of resorufin, the level of antigen labeled by HRP is quantified by analyzing the profile of fluorescence intensity. The normalized profile was interpolated into an autocatalysis model, and the rate of increase at half-maximum time was quantified by the use of an amplification index (AI). The lower limit of detection, for resorufin or HRP, was less than one-tenth that of the plate reader. It requires only slight modification of the fluorescence reader and is fully compatible with conventional or commercial ELISA. When it is applied to a commercial ELISA kit for the detection of amyloid beta, it is verified that the PIFA assay enhanced the detection sensitivity by more than a factor of 10 and was compatible with a conventional 96-well ELISA assay kit. We anticipate this PIFA assay to be used in research for the detection of low levels of proteins and for the early diagnosis of various diseases with rare protein biomarkers, at ultra-low (pg/mL) concentrations.

Nanoelectrical characterization of individual exosomes secreted by Aß42-ingested cells using electrostatic force microscopy.

Quantifying the physical properties of individual exosomes containing amyloid-ß42 (Aß42) is crucial for a better understanding of an underpinning mechanism of Alzheimer's disease expression which is associated with the Aß42 transfer. Because of the lack of proper tools, however, there have been very few studies on how the amount of Aß42 affects the physical properties of exosomes. To answer the question, we investigated the physical properties of exosomes secreted by neuroblastoma by probing individual exosomes using electrostatic force microscopy. Interestingly, we observed that when the higher concentration of Aß42 oligomers was fed to cells, the higher surface charge of the exosomes appeared. This result indicates that the exosomes contain more Aß42 with the increase in Aß42 concentration in cell media, implying that they serve as transport vesicles for Aß42. Our approach could help to better understand how the neuronal exosomes are related to the propagation of neurodegenerative diseases and to seek how to make an early diagnosis of those diseases.

Isolation of extracellular vesicles from small volumes of plasma using a microfluidic aqueous two-phase system.

As conventional bulky methods for extracellular vesicle (EV) separation are unsuitable for small volumes of samples, microfluidic devices are thought to offer a solution for the integrated and automatic processing of EV separation. This study demonstrates a simple microfluidic aqueous two-phase system (ATPS) for EV separation with high recovery efficiency to overcome the limitation of previous devices, which require complex external equipment or high cost manufacturing. With polyethylene glycol and dextran in the microfluidic channel, the isolation mechanism of the microfluidic ATPS was analyzed by comparison between two-phase and one-phase systems. Our device could facilitate continuous EV isolation with 83.4% recovery efficiency and remove 65.4% of the proteins from the EV-protein mixture. EVs were also successfully isolated from human plasma at high recovery efficiency.

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.

Enhancing surface functionality of reduced graphene oxide biosensors by oxygen plasma treatment for Alzheimer's disease diagnosis.

We performed oxygen plasma treatment on reduced graphene oxide (rGO) to improve its surface reactivity with respect to biomolecular interactions. Oxygen-plasma-treated rGO surfaces were employed as reactive interfaces for the detection of amyloid-beta (Aß) peptides, the pathological hallmarks of Alzheimer's disease (AD), as the target analytes. By measuring the changes in electrical characteristics and confirmation through topographic analysis, the oxygen-plasma-treated rGO sensors had enhanced surface functionality for better antibody immobilization and sensing performance, with a 3.33-fold steeper slope for the electrical responses versus analyte concentration curve (logarithmic scale) compared to the untreated. The elicited biomolecular reactivity of the rGO surfaces with the oxygen plasma treatment remained at 46-51% of the initial value even after aging for 6h in ambient conditions. This phenomenon was also confirmed by pretreating the rGO surfaces with a blocking agent and subsequently subjecting them to antibody immobilization. Finally, the feasibility of the oxygen-plasma-treated rGO sensors as a diagnostic tool was evaluated with clinical samples of neural-derived exosomal Aß peptides extracted from apparent AD patients and normal controls (NC). In contrast to the untreated sensors (p=0.0460), the oxygen-plasma-treated rGO sensors showed a significant p-value in the identification of clinical samples of AD and NC subjects (p<0.001). These results suggest that oxygen plasma treatment improves sensor performance without complicated fabrication procedures and should aid in the development of novel diagnostic tools based on carbon nanomaterials.

Hydrodynamic filtration in microfluidic channels as size-selection process for giant unilamellar vesicles.

We have developed hydrodynamic filtration method in microfluidic device for the efficient size-selection of polydisperse lipid vesicles for giant unilamellar vesicles (GUVs), in which vesicles were formed by electroformation method. Combining pinched flow channel design before hydrodynamic filtration, GUVs were flowed and guided to filtration channels, in which small lipid vesicles were further filtered and GUV were remained in main flow channels. For increasing the selectivity of GUV in outlets, length of slit section, or relative flow rate were controlled and drain channels were introduced for avoiding back flow. At higher flow rate in a pinched flow, the fraction of recovered GUVs (>10 microm) were increased, in which most of small vesicles were filtered.

Electrical percolation networks of carbon nanotubes in a shear flow.

The effect of shear on the electrical percolation network of carbon nanotube (CNT)-polymer composites is investigated using computer simulations. Configurations of CNTs in a simple shear, obtained by using Monte Carlo simulations, are used to locate the electrical percolation network of CNTs and calculate the electric conductivity. When exposed to the shear, CNTs align parallel to the shear direction and the electrical percolation threshold CNT concentration decreases. Meanwhile, after a certain period of the shear imposition above a critical shear rate, CNTs begin to form an aggregate and the percolating network of CNTs is broken, thus decreasing the electric conductivity significantly. We also construct quasiphase diagrams for the aggregate formation and the electrical percolation network formation to investigate the effect of the shear rate and CNT concentration.