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A paper-based electrochemical immunosensor was developed for the detection of cancer antigen 125 (CA125) by screen-printing technique. The reduced graphene oxide/thionine/gold nanoparticles (rGO/Thi/AuNPs) nanocomposites were compounded and coated onto the working electrode of immunosensor for CA125 antibody (anti-CA125) immobilization and detection signal amplification. The detection principle was based on the fact that the immunocomplex formed by specify binding of CA125 antibody and antigen could reduce the current response of thionine, which was proportional to the corresponding concentration of CA125 antigen. The immunoassay results showed that the linear range of CA125 was from 0.1 U mL−1 to 200 U mL−1 with the limit of detection (LOD) of 0.01 U mL−1 at signal to noise of 3. Quality control serum samples measured by our proposed immunosensor showed acceptable agreement with traditional ELISA method with the relative error less than 8.05%. The immunosensor exhibited good electrochemical performance with high reproducibility, reliability, stability and accuracy. The proposed immunosensor could be used for the determination of CA125 and had the potential for point-of-care testing (POCT) of other tumor marker. • A novel electrochemical immunosensor based on rGO/Thi/AuNPs nanocomposites was developed for the determination of CA125. • The rGO/Thi/AuNPs nanocomposites were synthesized to improve the sensitivity of immunosensor. • The immunosensor showed acceptable agreement with traditional ELISA method in clinical diagnosis. • It could be used for point-of-care testing of tumor markers and had the potential for disease screening in remote region. [ABSTRACT FROM AUTHOR]

In this work, we report the development of a general strategy for enhancing the efficiency of target capture in immunoassays, using a bifunctional fusion protein construct which incorporates a substrate-anchoring moiety for the high-abundance immobilization of an antigen-binding domain. This approach was informed by the development of a pseudo first-order rate constant model, and tested in a paper-based assay format using a fusion construct consisting of an rcSso7d binding module and a cellulose-binding domain. These rcSso7d-CBD fusion proteins were solubly expressed and purified from bacteria in high molar yields, and enable oriented, high-density adsorption of the rcSso7d binding species to unmodified cellulose within a 30-second incubation period. These findings were validated using two distinct, antigen-specific rcSso7d variants, which were isolated from a yeast surface display library via flow cytometry. Up to 1.6 micromoles of rcSso7d-CBD was found to adsorb per gram of cellulose, yielding a volume-averaged binder concentration of up to 760 μM within the resulting active material. At this molar abundance, the target antigen is captured from solution with nearly 100% efficiency, maximizing the attainable sensitivity for any given diagnostic system. [ABSTRACT FROM AUTHOR]

Abstract: Paper based colorimetric biosensing platform utilizing cross-linked siloxane 3-aminopropyltriethoxysilane (APTMS) as probe was developed for the detection of a broad range of targets including H2O2, glucose and protein biomarker. APTMS was extensively used for the modification of filter papers to develop paper based analytical devices. We discovered when APTMS was cross-linked with glutaraldehyde (GA), the resulting complex (APTMS–GA) displays brick-red color, and a visual color change was observed when the complex reacted with H2O2. By integrating the APTMS–GA complex with filter paper, the modified paper enables quantitative detection of H2O2 through the monitoring of the color intensity change of the paper via software Image J. Then, with the immobilization of glucose oxidase (GOx) onto the modified paper, glucose can be detected through the detection of enzymatically generated H2O2. For protein biomarker prostate specific antigen (PSA) assay, we immobilized capture, not captured anti-PSA antibody (Ab1) onto the paper surface and using GOx modified gold nanorod (GNR) as detection anti-PSA antibody (Ab2) label. The detection of PSA was also achieved via the liberated H2O2 when the GOx label reacted with glucose. The results demonstrated the possibility of this paper based sensor for the detection of different analytes with wide linear range. The low cost and simplicity of this paper based sensor could be developed for “point-of-care” analysis and find wide application in different areas. [Copyright &y& Elsevier]

Electrochemical paper-based analytical devices (ePADs) are useful analytical devices that serve as point-of-care testing (POCT) devices for various clinical biomarkers in view of their simplicity, portability, and low-cost format. However, multistep reagent manipulation usually restricts the performance of the device for end users. Herein, we developed a sequential ePAD for sequential immunosensing fluid delivery by integrating dual flow behaviors (fast-flow/delayed) within a single paper platform for the simultaneous detection of hepatitis B surface antigen (HBsAg) and hepatitis C core antigen (HCVcAg). In the present work, a fast-flow channel was used for the automated washing of unbound antigens, while a delayed channel was created to store a redox reagent for further electrochemical analysis with a single buffer loading (the analysis time can be completed within 500 s). Hence, the undesirable complex procedure of multi-step reagent manipulation is scarcely needed by the user. The detection limit of the proposed ePAD was as low as 18.2 pg mL−1 for HBsAg and 1.19 pg mL−1 for HCVcAg. In addition, this proposed ePAD was also proven to be effective in real clinical sera from patients to verify its biological applicability. The ePAD sensor shows high promise as an easy-to-use, portable, and extendable sensor for other multiplex biological assays. [Display omitted] • ePAD for simultaneous HBsAg and HCVcAg was created. • First time employment of dual flow behaviors on ePAD for hepatitis markers sensing. • ePAD shows an easy to use, portability, and broad sensing applicability. • The sensor was applied to detect hepatitis markers in real clinical sera. [ABSTRACT FROM AUTHOR]

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is emerging as a global pandemic outbreak. To date, approximately one million deaths and over 32 million cases have been reported. This ongoing pandemic urgently requires an accurate testing device that can be used in the field in a fast manner. Serological assays to detect antibodies have been proven to be a great complement to the standard method of reverse transcription-polymerase chain reaction (RT-PCR), particularly after the second week of infection. We have developed a specific and sensitive immunosensor for immunoglobulin detection produced against SARS-CoV-2. Unlike other lateral flow-based assays (LFAs) involving the utilization of multiple antibodies, we have reported a label-free paper-based electrochemical platform targeting SARS-CoV-2 antibodies without the specific requirement of an antibody. The presence of SARS-CoV-2 antibodies will interrupt the redox conversion of the redox indicator, resulting in a decreased current response. This electrochemical sensor was proven effective in real clinical sera from patients with satisfactory results. In addition, the proposed format was also extended to antigen detection (the spike protein of SARS-CoV-2), which presents new possibilities for diagnosing COVID-19. • Electrochemical sensor for COVID-19 diagnostic. • Serological assay for SARS-CoV-2 antibodies was demonstrated. • Sensitivity enhancement compared to the traditional LFA devices. • Applicable to real clinical sera with satisfactory results. [ABSTRACT FROM AUTHOR]

COVID-19 has evolved into a global pandemic. Early and rapid detection is crucial to control of the SARS-CoV-2 transmission. While representing the gold standard for early diagnosis, nucleic acid tests for SARS-CoV-2 are often complicated and time-consuming. Serological rapid antibody tests are characterized by high rates of false-negative diagnoses, especially during early infection. Here, we developed a novel nanozyme-based chemiluminescence paper assay for rapid and sensitive detection of SARS-CoV-2 spike antigen, which integrates nanozyme and enzymatic chemiluminescence immunoassay with the lateral flow strip. The core of our paper test is a robust Co–Fe@hemin-peroxidase nanozyme that catalyzes chemiluminescence comparable with natural peroxidase HRP and thus amplifies immune reaction signal. The detection limit for recombinant spike antigen of SARS-CoV-2 was 0.1 ng/mL, with a linear range of 0.2-100 ng/mL. Moreover, the sensitivity of test for pseudovirus could reach 360 TCID 50 /mL, which was comparable with ELISA method. The strip recognized SARS-CoV-2 antigen specifically, and there was no cross reaction with other coronaviruses or influenza A subtypes. This testing can be completed within 16 min, much shorter compared to the usual 1-2 h required for currently used nucleic acid tests. Furthermore, signal detection is feasible using the camera of a standard smartphone. Ingredients for nanozyme synthesis are simple and readily available, considerably lowering the overall cost. In conclusion, our paper test provides a high-sensitive point-of-care testing (POCT) approach for SARS-CoV-2 antigen detection, which should greatly facilitate early screening of SARS-CoV-2 infections, and considerably lower the financial burden on national healthcare resources. • Point-of-care antigen tests for screening of SARS-CoV-2 infection were established. • Chemiluminescence improves sensitivity of paper test and benefits early screening. • Nanozyme with high peroxidase activity and stability catalyzes chemiluminescence. • Nanozyme chemiluminescence paper test brings high efficiency and low cost. [ABSTRACT FROM AUTHOR]

Lateral flow assays (LFAs) have currently attracted broad interest for point-of-care (POC) diagnostics, but their application has been restricted by poor quantification and limited sensitivity. While the former has been currently solved to some extent by the development of handheld or smartphone-based readers, the latter has not been addressed fully, particularly the potential influences of environmental conditions (e.g., temperature and relative humidity (RH)), which have not yet received serious attention. The present study reports the use of a portable temperature–humidity control device to provide an optimum environmental requirement for sensitivity improvement in LFAs, followed by quantification by using a smartphone. We found that a RH beyond 60% with temperatures of 55–60 °C and 37–40 °C produced optimum nucleic acid hybridization and antigen–antibody interaction in LFAs, respectively representing a 10-fold and 3-fold signal enhancement over ambient conditions (25 °C, 60% RH). We envision that in the future the portable device could be coupled with a fully integrated paper-based sample-to-answer biosensor for sensitive detection of various target analytes in POC settings. [ABSTRACT FROM AUTHOR]

Abstract: This paper reports a method of highly sensitive detection of thrombin using a surface-enhanced Raman scattering (SERS)-based magnetic aptasensor. Magnetic beads and gold nanoparticles (Au NPs) were used as supporting substrates and sensing probes, respectively. For this purpose, 15-mer thrombin-binding aptamers (TBA15) were immobilized onto the surface of magnetic beads, and then thrombin antigens and 29-mer thrombin-binding aptamer (TBA29)-conjugated Au NPs were sequentially added for the formation of sandwich aptamer complexes. Quantitative analysis was performed by monitoring the intensity variation of a characteristic SERS signal of Raman reporter molecules. Because all of the reactions occur in solution, this SERS-based immunoassay technique can solve the diffusion-limited kinetic problems on a solid substrate. The limit of detection (LOD) of thrombin, determined by the SERS-based aptasensor, was estimated to be 0.27pM. The proposed method is expected to be a good clinical tool for the diagnosis of a thrombotic disease. [Copyright &y& Elsevier]

Abstract: This paper describes a method for the effective and self-oriented immobilization of antibodies on magnetic silica-nanoparticles using a multimeric protein G. Cysteine-tagged recombinant dimers and trimers of protein G were produced in Escherichia coli BL21 by repeated linking of protein G monomers with a flexible (GGGGS)3 linker. Amino-functionalized silica-coated magnetic nanoparticles (SiO2-MNPs, Fe3O4@SiO2) were prepared and coupled to the protein G multimers, giving the final magnetic immunosensor. The optimal conditions for the reaction between the protein Gs and the SiO2-MNPs was a time of 60min and a concentration of 100μg/mL, resulting in coupling efficiencies of 77%, 67% and 55% for the monomeric, dimeric and trimeric protein Gs, respectively. Subsequently, anti-hepatitis B surface antigen (HBsAg) was immobilized onto protein G-coupled SiO2-MNPs. The quantitative efficiency of antibody immobilization found the trimeric protein G to be the best, followed by the dimeric and monomeric proteins, which differs from the coupling efficiencies. Using all three protein constructs in an HBsAg fluoroimmunoassay, the lowest detectable concentrations were 500, 250 and 50ng/mL for the monomeric, dimeric and trimeric protein G-coupled SiO2-MNPs, respectively. Therefore, multimeric protein Gs, particularly the trimeric form, can be employed to improve antibody immobilization and, ultimately, enhance the sensitivity of immunoassays. In addition, the multimeric protein Gs devised in this study can be utilized in other immunosensors to bind the antibodies at a high efficiency and in the proper orientation. [Copyright &y& Elsevier]

Abstract: This paper describes the development of an immunosensor coupled to glassy carbon electrode (GCE) modified with multiwall carbon nanotubes (MWCNT) (CNT-GCE) integrated with microfluidic systems for rapid and sensitive quantification of prostate specific antigen (PSA) in human serum samples. Mouse monoclonal (5G6) to PSA antibodies were immobilized on a rotating disk. PSA in the serum sample are allowed to react immunologically with the immobilized anti-tPSA and horseradish peroxidase (HRP) enzyme-labeled second antibodies specific to PSA. HRP, in the presence of hydrogen peroxide (H2O2) catalyzes the oxidation of 4-tert-butylcatechol (4-TBC), whose back electrochemical reduction was detected on CNT-GCE at −0.15V. The electrochemical detection can be done within 1min and total assay time was 30min. The calculated detection limits for electrochemical detection and the ELISA procedure are 0.08 and 0.5μgL−1, respectively and the intra- and inter-assay coefficients of variation were below 4.5%. The electrochemical immunosensor showed higher sensitivity and lower time consumed than the standard spectrophotometric detection ELISA method, which shows potential for detecting PSA in clinical diagnosis. [Copyright &y& Elsevier]

Abstract: An integrated microfluidic device with injecting, flushing, and sensing functions was realized using valves that operate based on direct electrowetting. The device consisted of two substrates: a glass substrate with driving and sensing electrodes and a poly(dimethylsiloxane) (PDMS) substrate. Microfluidic transport was achieved using the spontaneous movement of solutions in hydrophilic flow channels formed with a dry-film photoresist layer. The injection and flushing of solutions were controlled by gold working electrodes, which functioned as valves. The valves were formed either in the channels or in a through-hole in the glass substrate. To demonstrate the system''s applicability to an immunoassay, the detection of immobilized antigens was performed as a partial simulation of a sandwich immunoassay. Human α-fetoprotein (AFP) or an anti-human AFP antibody was immobilized on a platinum working electrode in the chamber using a plasma-polymerized film (PPF). By applying a potential to the injection valves, necessary solutions were injected one by one through the channels into a reaction chamber at the center of the chip and incubated for reasonable periods of time. The solutions were then flushed through the flushing valve and absorbed in a filter paper placed under the device. After incubation with the corresponding antibodies labeled with glucose oxidase (GOD), electrochemical detection was conducted. In both cases, the obtained current depended on the amount of immobilized antigen. The calibration curves were sigmoidal, and the detection limit was 0.1ng. The developed microfluidic system could potentially be a fundamental component for a micro immunoassay of the next generation. [Copyright &y& Elsevier]