Surface Reaction of Electroosmotic Flow-Driven Free Antigens With Immobilized Magnetic-Microbeads-Tagged-Antibodies in Microchannels.

Immunoassays based on reactions between target pathogen (antigen; Ag) and antibody (Ab) are frequently used for Ag detection. An external magnetic field was used to immobilize magnetic microbeads-tagged-antibodies (mMB-Ab) on the surface of a microchannel in the capture zone. The mMB-Ab was subsequently used for Ag detection. The objective of this numerical study, with experimental validation, is to assess the surface reaction between mMB-Ab and Ag in the presence of electro-osmotic flow (EOF). First, immobilization of mMB-Ab complex in the wall of the capture zone was achieved. Subsequently, the Ag was transported by EOF toward the capture zone to bind with the immobilized mMB-Ab. Lastly, mMB-Ab:Ag complex was formed and immobilized in the capture zone. A finite volume solver was used to implement the above steps. The surface reaction between the mMB-Ab and Ag was investigated in the presence of electric fields (E): 150 V/cm-450 V/cm and Ag concentrations: 0.001 M-1000 M. The depletion of mMB-Ab increases with time as the E decreases. Furthermore, as the concentration of Ag decreases, the depletion of mMB-Ab increases with time. These results quantify the detection of Ag using the EOF device; thus, signifying its potential for rapid throughput screening of Ag. This platform technology can lead to the development of portable devices for the detection of target cells, pathogens, and biomolecules for testing water systems, biological fluids, and biochemicals.

Label-free electrochemical biosensor for direct detection of Oncostatin M (OSM) inflammatory bowel diseases (IBD) biomarker in human serum.

Oncostatin M (OSM) is an interleukin-6 (IL-6) member family cytokine implicated in the pathogenesis of chronic diseases including inflammatory bowel disease (IBD). OSM is a novel diagnostic biomarker over-expressed in the serum of IBD patients. This paper reports on the first electrochemical OSM immunosensor, developed using a multistep fabrication process aimed at covalently immobilizing OSM antibodies on a mixed self-assembled monolayer coated gold working electrode. Cyclic voltammetry, atomic force microscopy (AFM), IR spectroscopy and optical characterizations were used to validate the sensor functionalization protocol. Electrochemical impedance spectroscopy (EIS) measurements were performed to assess the reliability of the immunosensor preparation and to verify the antibody-antigen complexes formation. The label-free immunosensor showed high sensitivity identifying OSM at clinically relevant concentrations (37-1000 pg mL-1) with low detection limit of 2.86 pg mL-1. Both sensitivity and selectivity of the proposed immunosensor were also demonstrated in human serum in the presence of interfering biomarkers, making it an innovative potential platform for the OSM biomarker detection in IBD patients' serum.

Influence of different conjugation methods for activating antibodies on polymeric nanoparticles: Effects for polyclonal expansion of human CD8+ T cells.

Particle-based systems have become a state-of-the-art method for in vitro expanding cytotoxic T cells by tailoring their surface with activating molecules. However, commonly used methods utilize facile carbodiimide chemistry leading to uncontrolled orientation of the immobilized antibodies on the particle surface that can lead to poor binding to target cells. To address this, selective coupling strategies utilizing regioselective chemical groups such as disulfide bridges offer a simple approach. In this work we present a set of methods to investigate the effect of polymeric nanoparticles, conjugated with either regioselective- or randomly-immobilized antiCD3 and antiCD28 antibodies, on the activation potential, expansion and expression of activation markers in T cells. We show that nanoparticles with well-oriented monovalent antibodies conjugated via maleimide require fewer ligands on the surface to efficiently expand T cells compared to bivalent antibodies randomly-immobilized via carbodiimide conjugation. Analysis of the T cell expression markers reveal that the T cell phenotype can be fine-tuned by adjusting the surface density of well-oriented antibodies, while randomly immobilized antibodies showed no differences despite their ligand density. Both conjugation techniques induced cytotoxic T cells, evidenced by analyzing their Granzyme B secretion. Furthermore, antibody orientation affects the immunological synapse and T cell activation by changing the calcium influx profile upon activation. Nanoparticles with well-oriented antibodies showed lower calcium influx compared to their bivalent randomly-immobilized counterparts. These results highlight the importance of controlling the antibody density and orientation on the nanoparticle surface via controlled coupling chemistries, helping to develop improved particle-based expansion protocols to enhance T cell therapies.

Au/PANI@PtCu-based electrochemical immunosensor for ultrasensitive determination of pro-gastrin-releasing peptide.

An ultrasensitive sandwich-type electrochemical immunosensor for pro-gastrin-releasing peptide (ProGRP) detection was constructed based on PtCu nanodendrites functionalized Au/polyaniline nanospheres (Au/PANI@PtCu). The prepared Au/PANI@PtCu nanocomposites not only possessed excellent electro-catalytic activity of H2O2 reduction due to the synergistic effect between the Au/PANI and PtCu NDs but also provided large specific surface area for detection of antibodies (Ab2) immobilization. In addition, Au nanoparticles encapsulated multi-wall carbon nanotubes (AuNPs@MWCNTs) were also applied to modify the glassy carbon electrode interface for loading numerous capture antibodies (Ab1). In the presence of target ProGRP, a sandwich-type electrochemical immunosensor showed a strong current response from the electro-catalysis of Au/PANI@PtCu toward H2O2 reduction. Benefiting from the exceptional electro-catalytic performance of Au/PANI@PtCu and the high conductivity of AuNPs@MWCNTs, the sandwich-type immunoassay exhibited remarkable sensitivity in detection. The linear range extended from 100 fg/mL to 10 ng/mL, while achieving an impressively low limit of detection of 77.62 fg/mL.

A sensitive sandwich-type electrochemical immunosensor using nitrogen-doped graphene/metal-organic framework-derived CuMnCoOx and Au/MXene for the detection of breast cancer biomarker.

In terms of cancer-related deaths among women, breast cancer (BC) is the most common. Clinically, human epidermal growth receptor 2 (HER2) is one of the most commonly used diagnostic biomarkers for facilitating BC cell proliferation and malignant growth. In this study, a disposable gold electrode (DGE) modified with gold nanoparticle-decorated Ti3C2Tx (Au/MXene) was utilized as a sensing platform to immobilize the capturing antibody (Ab1/Au/MXene). Subsequently, nitrogen-doped graphene (NG) with a metal-organic framework (MOF)-derived copper-manganese-cobalt oxide, tagged as NG/CuMnCoOx, was used as a probe to label the detection antibody (Ab2). A sandwich-type immunosensor (NG/CuMnCoOx/Ab2/HER2-ECD /Ab1/Au/MXene/DGE) was developed to quantify HER2-ECD. NG/CuMnCoOx enhances the conductivity, electrocatalytic active sites, and surface area to immobilize Ab2. In addition, Au/MXene facilitates electron transport and captures more Ab1 on its surface. Under optimal conditions, the resultant immunosensor displayed an excellent linear range of 0.0001 to 50.0 ng. mL-1. The detection limit was 0.757 pg·mL-1 with excellent selectivity, appreciable reproducibility, and high stability. Moreover, the applicability for determining HER2-ECD in human serum samples indicates its ability to monitor tumor markers clinically.

Ultrasensitive and Rapid Detection of Procalcitonin via Waveguide-Enhanced Nanogold-Linked Immunosorbent Assay for Early Sepsis Diagnosis.

Sepsis, a life-threatening inflammatory response, demands economical, accurate, and rapid detection of biomarkers during the critical "golden hour" to reduce the patient mortality rate. Here, we demonstrate a cost-effective waveguide-enhanced nanogold-linked immunosorbent assay (WENLISA) based on nanoplasmonic waveguide biosensors for the rapid and sensitive detection of procalcitonin (PCT), a sepsis-related inflammatory biomarker. To enhance the limit of detection (LOD), we employed sandwich assays using immobilized capture antibodies and detection antibodies conjugated to gold nanoparticles to bind the target analyte, leading to a significant evanescent wave redistribution and strong nanoplasmonic absorption near the waveguide surface. Experimentally, we detected PCT for a wide linear response range of 0.1 pg/mL to 1 ng/mL with a record-low LOD of 48.7 fg/mL (3.74 fM) in 8 min. Furthermore, WENLISA has successfully identified PCT levels in the blood plasma of patients with sepsis and healthy individuals, offering a promising technology for early sepsis diagnosis.

Sandwich-type immunosensor based on aminated 3D-rGOF-NH2 and CMK-3-Fc-MgAl-LDH multilayer nanocomposites for detection of CA125.

Cancer antigen 125 (CA125)1 is the most important biological screening indicator used to monitor epithelial ovarian cancers, and it plays a vital role in distinguishing ovarian cancers from benign diseases. Biosensors show great potential in the analysis and detection of disease markers. In this study, we designed electrochemical sensors based on three-dimensional amino-functionalized reduced graphene oxide (3D-rGOF-NH2),2 MgAl layered double hydroxide nanocomposites containing ordered mesoporous carbon (CMK-3),3 and ferrocene carboxylic acids（Fc-COOH）4for the detection of CA125. 3D-rGOF-NH2 possesses good conductivity, a large surface area, and high porosity, enabling more immobilized nanoparticles to be deposited on its surface with excellent stability. CMK-3@Fc@MgAl-LDH nanocomposite was used as a carrier to enhance the immobilization of antibodies and the loading of Fc, conductors to enhance conductivity, and enhancers to gradually amplify the signal of Fc. The surface morphology, elemental composition, and surface groups of the materials were characterized using scanning electron microscopy (SEM),5 transmission electron microscopy (TEM),6 and X-ray diffraction (XRD)7 techniques. The response signal of the electrochemical sensor was measured by DPV. Under the optimal conditions, the electrochemical sensor obtained a linear detection range of 0.01 U/mL-100 U/mL with a detection limit of 0.00417 U/mL.

Significance of the antibody orientation for the lateral flow immunoassays: A mini-review.

Lateral flow immunoassays (LFIAs) are well-established and broadly commercialized tools in the field of point-of-care testing due to their simplicity, rapidity, cost-effectiveness, and low requirements for users and equipment. However, the insensitivity and the possibility of producing inaccurate results associated with conventional LFIAs have impeded their wide-ranging implementation, especially for monitoring ultra-trace level of analytes. Moreover, the heterogeneous distribution of amino acids on the surface of antibody (Ab) results in a lack of precise control over their orientation, which ultimately leads to unsatisfactory detection performance. To address those concerns, herein we provide an overview of the emerging efforts to prepare well-established LFIAs from the perspective of orientation manipulation of immobilized Abs on the nanoprobes or membranes. The preparation of excellent nanoprobes with Abs being oriented immobilized, consisting of the nanoprobe types, Ab types, and their conjugation chemistries, are reviewed. Followed by the introduction of efforts highlight the importance of directionally immobilized Ab on the membrane. The effects of Ab orientation on the analytical performance of LFIA platforms in terms of sensitivity, specificity, rapidity, reliability, cost-effectiveness, and stability are also summarized. Finally, the future development and challenges of Ab-oriented immobilization-assisted LFIAs are also discussed.

An integrated electrochemical immunosensor based on Pd-Ir cubic nanozyme and Ketjen black for ultrasensitive detection of circulating tumor cells.

Ultrasensitive detection of circulating tumor cells (CTCs) holds significant clinical importance in monitoring metastasis and therapeutic outcomes. In this study, we have developed a novel electrochemical sensing model based on nanomaterials for highly sensitive and specific determination of CTCs. A gold electrode co-modified with Ketjin black (KB) and Au nanoparticles (AuNPs) exhibits exceptional conductivity. By conjugating palladium-iridium cubic nanozyme (Pd-Ir CNE) with antibodies, we have created a detection probe capable of catalyzing hydrogen peroxide (H2O2), thereby amplifying the output signal and resulting in significantly enhanced current on the electrode for detecting CTCs. The constructed immunosensor has achieved a detection limit of 2 cell mL-1 for model MCF-7 cells. Furthermore, the as-constructed electrochemical immunosensor can accurately detect whole blood-spiked target CTCs, showing great promise for clinical applications in early cancer diagnosis and prognosis.

Ability of Antibodies Immobilized on Gold Nanoparticles to Bind Small Antigen Fluorescein.

The analytical applications of antibodies are often associated with their immobilization on different carriers, which is accompanied by a loss of antigen-binding activity for a sufficient proportion of the bound antibodies. In contrast to data on plain carriers, minimal data are available on the properties of antibodies on the surfaces of nanoparticles. Protein antigens have been predominantly investigated, for which space restrictions do not allow them to occupy all active sites of immobilized antibodies. This study considered a low-molecular-weight compound, fluorescein, as an antigen. Spherical gold nanoparticles with five different sizes, two differently charged forms of fluorescein, and three different levels of surface coverage by immobilized antibodies were tested. For gold nanoparticles with diameters from 14 to 35.5 nm with monolayers of immobilized antibodies, the percentage of molecules capable of binding carboxyfluorescein varied from 6% to 17%. The binding of aminofluorescein was more efficient; for gold nanoparticles with an average diameter of 21 nm, the percentage of active binding sites for the immobilized antibodies reached 27% compared with 13% for the carboxyfluorescein case. A fourfold reduction in the coverage of the nanoparticles' surface compared with that of the monolayer did not lead to reliable changes in the percentage of active binding sites. The obtained data demonstrate that an antigen's binding to immobilized antibodies is limited even for small antigens and depends on the size of the nanoparticles and the electrostatic repulsion near their surface.

A liquid crystal-based biosensor for sensitive detection of tumor necrosis factor-alpha.

Tumor necrosis factor-alpha (TNF-α) is a cytokine secreted by the macrophages and Th1 cells of the immune system in response to inflammation. Given its significance as a biomarker with elevated levels in physiological fluids in various conditions, there is an increasing demand for a simple and accurate TNF-α detection strategy. In this article, we present a liquid crystal (LC)-based biosensor developed for sensitive TNF-α detection. The biosensor operates as follows: TNF-α and detection antibodies (DAbs) form complexes during preincubation. These complexes then bind with the surface-immobilized capture antibodies (CAbs), facilitating the antigen-antibody reaction between the CAbs and the TNF-α/DAb complexes. This target recognition interaction alters the surface topography, disrupting the vertical orientation of LCs produced by dimethyloctadecyl[3-(trimethoxysilyl)-propyl]ammonium chloride. The orientational change in the LCs can be easily visualized with a polarized optical microscope, resulting in brighter images as TNF-α levels rise. Our results demonstrated a linear range of 5.00-500 pg/mL, with a limit of detection and limit of quantification being 1.08 and 3.56 pg/mL, respectively. Recovery experiments on diluted saliva samples produced reasonable results, with TNF-α recoveries ranging from 97.1% ± 2.58% to 107% ± 5.95%.

Extending the Shelf-Life of Immunoassay-Based Microfluidic Chips through Freeze-Drying Sublimation Techniques.

Point-of-care testing (POCT) platforms utilizing immunoassay-based microfluidic chips offer a robust and specific method for detecting target antibodies, demonstrating a wide range of applications in various medical and research settings. Despite their versatility and specificity, the adoption of these immunoassay chips in POCT has been limited by their short shelf-life in liquid environments, attributed to the degradation of immobilized antibodies. This technical limitation presents a barrier, particularly for resource-limited settings where long-term storage and functionality are critical. To address this challenge, we introduce a novel freeze-dry sublimation process aimed at extending the shelf-life of these microfluidic chips without compromising their functional integrity. This study elaborates on the mechanisms by which freeze-drying preserves the bioactivity of the immobilized antibodies, thereby maintaining the chip's performance over an extended period. Our findings reveal significant shelf-life extension, making it possible for these POCT platforms to be more widely adopted and practically applied, especially in settings with limited resources. This research paves the way for more accessible, long-lasting, and effective POCT solutions, breaking down previous barriers to adoption and application.

CRISPR/Cas12a-Powered Competitive Immunosorbent Assay for Small Molecules.

CRISPR/Cas systems are powerful tools for sensitive nucleic acid molecular diagnosis due to their specific nucleic acid recognition and high trans-cleavage activity and have also allowed for quantification of non-nucleic acid targets, relying on a strategy to convert the target detection to analysis of nucleic acids. Here, we describe a CRISPR/Cas12a-powered immunosorbent assay for sensitive small-molecule detection by using the antibody coated on the microplate to recognize the target and the small molecule-labeled active DNA (acDNA) to trigger the activity of CRISPR/Cas12a. In the absence of small-molecule targets, acDNA probes are captured by the antibody on the microplate and then activate Cas12a in catalytic trans-cleavage of fluorescent DNA reporters, generating strong fluorescence. The presence of small-molecule targets displaces the acDNA probes from the antibody, causing a decrease of acDNA probes on the microplate and reduction of activated Cas12a, so the fluorescence signal decreases, and small molecules can be detected by monitoring the fluorescence change. After systematically optimizing experimental conditions (e.g., Cas12a reaction), the proposed method achieved the detection of three model small molecules, biotin, digoxin, and folic acid, with low detection limits, and a flexible detection concentration range was obtained by simply changing the amount of acDNA probes and immobilized antibodies. The assay showed high selectivity and good applicability in complex media. The integration of the CRISPR/Cas12a system improves the analytical performance of immunoassay, broadening and facilitating its applications in rapid, simple, and sensitive small molecule analysis.

Immunoaffinity Chromatography for Protein Purification and Analysis.

Immunoaffinity chromatography (IAC) is a type of liquid chromatography that uses immobilized antibodies or related binding agents as selective stationary phases for sample separation or analysis. The strong binding and high selectivity of antibodies have made IAC a popular tool for the purification and analysis of many chemicals and biochemicals, including proteins. The basic principles of IAC are described as related to the use of this method for protein purification and analysis. The main factors to consider in this technique are also presented under a discussion of the general strategy to follow during the development of a new IAC method. Protocols, as illustrated using human serum albumin (HSA) as a model protein, are provided for the use of IAC in several formats. This includes both the use of IAC with traditional low-performance supports such as agarose for off-line immunoextraction and supports used in high-performance IAC for on-line immunoextraction. The use of IAC for protein analysis as a flow-based or chromatographic immunoassay is also discussed and described using HSA and a competitive binding assay format as an example. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Off-line immunoextraction by traditional immunoaffinity chromatography Basic Protocol 2: On-line immunoextraction by high-performance immunoaffinity chromatography Basic Protocol 3: Competitive binding chromatographic immunoassay.

Triple signal-enhancing sandwich-type electrochemical immunosensor based on Au@SNHC and PtPdCu/BNC for detection of Cardiac Troponin I.

A novel multiple amplification strategy for electrochemical immunoassay was developed elaborately. The realization of this strategy is based on the high efficiency catalysis of boron nitrogen double-doped carbon loaded trimetallic PtPdCu mulberry-like nanospheres (PtPdCu/BNC) and the satisfactory conductivity of gold nanoparticles modified with sulfur nitrogen co-doped hollow porous carbon (Au@SNHC). Single crystal anisotropic Pt octahedral seeds were synthesized with sodium citrate as shape-directing agent, and then three metals were grown in situ to prepare the trimetallic PtPdCu mulberry nanospheres, which had excellent utilization of atoms and a significant number of catalytic active centers. Boron nitrogen double-doped carbon (BNC) with abundant free electrons and high electron density micro-mesoporous can be used as the supported dispersion material of PtPdCu MBNSs, further catalytically enrich H2O2. Moreover, the etching vulcanization of small ZIF template and the co-doping of sulfur and nitrogen endow sulfur nitrogen co-doped hollow porous carbon (SNHC) with shorter the diffusion distance of charge carrier and lower mass transfer resistance. Under optimal conditions, the developed immunosensor exhibits good sensing performance with a detection limit of 4.27 fg⋅mL-1, and the wide linear dynamic range (10 fg⋅mL-1-100 ng⋅mL-1). This study provides a possible strategy for early diagnosis and postoperative monitoring of AMI.

A new ITO-based Aß42 biosensor for early detection of Alzheimer's disease.

In this study, a novel label-free impedimetric immunosensor was fabricated for rapid, selective, and sensitive quantitative analysis of Aß42 protein for use in the diagnosis of Alzheimer's disease. The immunosensor was fabricated using inexpensive and disposable indium tin oxide polyethylene terephthalate (ITO-PET) electrodes. After the electrodes were modified with 3-glycidoxypropyldimethoxymethylsilane (GPDMMS), the antibody specific to the Aß42 protein (anti-Aß42) was immobilized. The affinity interaction between anti-Aß42 and Aß42 in the immobilization steps in immunosensor fabrication and in the quantitation of Aß42 were analyzed using Electrochemical Impedance Spectroscopy (EIS) and Cyclic Voltammetry (CV) techniques. Additionally, the morphological changes occurring on the electrode surface during each immobilization step were imaged using scanning electron microscopy (SEM). The linear detection range of the immunosensor was determined as 1-100 pg/mL with the limit of detection value of 0.37 pg/mL. Analytical properties of the biosensor, including reproducibility, repeatability, storage stability, selectivity, and regeneration were investigated. The kinetic behavior of antibody-antigen complex formation was determined for the first time using single frequency impedance (SFI) analysis on an Aß42 biosensor. The potential for use of the immunosensor in clinical studies was demonstrated by analysis of Aß42 in commercially purchased human serum.

Sandwich-type electrochemical immunosensor based on CuFe2O4-Pd for cardiac troponin I detection.

A sandwich-type electrochemical immunosensor was designed by highly efficient catalytic cycle amplification strategy of CuFe2O4-Pd for sensitive detection of cardiac troponin I. CuFe2O4 with coupled variable valence metal elements exhibited favorable catalytic performance through bidirectional cycling of Fe2+/Fe3+ and Cu+/Cu2+ redox pairs. More importantly, Cu+ acted as the intermediate product of the catalytic reaction, promoted the regeneration of Fe2+ and ensured the continuous recycling occurrence of the double redox pairs, and significantly amplified the current signal response. Pd nanoparticles (Pd NPs) loaded on the surface of amino-functionalized CuFe2O4 (CuFe2O4-NH2) served as electrochemical mediators to capture labeled antibodies (Ab2), and also as co-catalysts of CuFe2O4 to further enhance the catalytic efficiency, thus improving the sensitivity of the electrochemical immunosensor. Under the optimal experimental conditions, the linear range was 0.001 ~ 100 ng/mL, and the detection limit was 1.91 fg/mL. The electrochemical immunosensor has excellent analytical performance, giving a new impetus for the sensitive detection of cTnI.

Release from persistent T cell receptor engagement and blockade of aryl hydrocarbon receptor activity enhance IL-6-dependent mouse follicular helper T-like cell differentiation in vitro.

Follicular helper T (Tfh) cells are crucial for humoral immunity. Dysregulation of Tfh cell differentiation can cause infectious, allergic, and autoimmune diseases. To elucidate the molecular mechanisms underlying Tfh cell differentiation, we attempted to establish an in vitro mouse model of Tfh cell differentiation in the absence of other cell types. Various cytokines and cell surface molecules are suggested to contribute to the differentiation. We found that stimulating naïve CD4+ T cells with immobilized antibodies to CD3, ICOS, and LFA-1 in the presence of soluble anti-CD28 antibody, IL-6, and antibodies that block IL-2 signaling for 3 days induced the expression of Bcl6 and Rorc(γt), master regulator genes of Tfh and Th17 cells, respectively. TGF-ß significantly enhanced cell proliferation and Bcl6 and Rorc(γt) expression. An additional 2 days of culture without immobilized antibodies selectively downregulated Rorc(γt) expression. These cells produced IL-21 and promoted B cells to produce IgG antibodies. Adding the aryl hydrocarbon receptor (AhR) antagonist CH-223191 to the T cell culture further downregulated Rorc(γt) expression without significantly affecting Bcl6 expression, and upregulated expression of a key Tfh marker, CXCR5. Although their CXCR5 expression levels were still not high, the CH-223191-treated cells showed chemotactic activity towards the CXCR5 ligand CXCL13. On the other hand, AhR agonists upregulated Rorc(γt) expression and downregulated CXCR5 expression. These findings suggest that AhR activity and the duration of T cell receptor stimulation contribute to regulating the balance between Tfh and Th17 cell differentiation. Although this in vitro system needs to be further improved, it may be useful for elucidating the mechanisms of Tfh cell differentiation as well as for screening physiological or pharmacological factors that affect Tfh cell differentiation including CXCR5 expression.

A polyaniline functionalized NiFeP nanosheet array-based electrochemical immunosensor using Au/Cu2O nanocubes as a signal amplifier for the detection of SARS-CoV-2 nucleocapsid protein.

Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which emerged as a novel pathogen in 2019. The virus is responsible for a severe acute respiratory syndrome outbreak, affecting the respiratory system of infected individuals. COVID-19 is a super amplifier of basic diseases, and the disease with basic diseases is often more serious. Controlling the spread of the COVID-19 pandemic relies heavily on the timely and accurate detection of the virus. To resolve the problem, a polyaniline functionalized NiFeP nanosheet array-based electrochemical immunosensor using Au/Cu2O nanocubes as a signal amplifier is fabricated for the detection of SARS-CoV-2 nucleocapsid protein (SARS-CoV-2 NP). Polyaniline (PANI) functionalized NiFeP nanosheet arrays are synthesized as an ideal sensing platform for the first time. PANI is coated on the surface of NiFeP by electropolymerization to enhance biocompatibility, beneficial for the efficient loading of the capture antibody (Ab1). Significantly, Au/Cu2O nanocubes possess excellent peroxidase-like activity and exhibit outstanding catalytic activity for the reduction of H2O2. Therefore, Au/Cu2O nanocubes combine with a labeled antibody (Ab2) through the Au-N bond to form labeled probes, which can effectively amplify current signals. Under optimal conditions, the immunosensor for the detection of SARS-CoV-2 NP shows a wide linear range of 10 fg mL-1-20 ng mL-1 and a low detection limit of 1.12 fg mL-1 (S/N = 3). It also exhibits desirable selectivity, repeatability, and stability. Meanwhile, the excellent analytical performance in human serum samples confirms the practicality of the PANI functionalized NiFeP nanosheet array-based immunosensor. The electrochemical immunosensor based on the Au/Cu2O nanocubes as a signal amplifier demonstrates great potential for application in the personalized point-of-care (POC) clinical diagnosis.

PdPtRu trimetallic nanozymes and application to electrochemical immunosensor for sensitive SARS-COV-2 antigen detection.

Herein, a ternary PdPtRu nanodendrite as novel trimetallic nanozyme was reported, which possessed excellent peroxidase-like activity as well as electro-catalytic activity on account of the synergistic effect between the three metals. Based on the excellent electro-catalytic activity of trimetallic PdPtRu nanozyme toward the reduction of H2O2, the trimetallic nanozyme was applied to construct a brief electrochemical immunosensor for SARS-COV-2 antigen detection. Concretely, trimetallic PdPtRu nanodendrite was used to modify electrode surface, which not only generated high reduction current of H2O2 for signal amplification, but also provided massive active sites for capture antibody (Ab1) immobilization to construct immunosensor. In the presence of target SARS-COV-2 antigen, SiO2 nanosphere labeled detection antibody (Ab2) composites were introduced on the electrode surface according sandwich immuno-reaction. Due to the inhibitory effect of SiO2 nanosphere on the current signal, the current signal was decreased with the increasing target SARS-COV-2 antigen concentration. As a result, the proposed electrochemical immunosensor presented sensitive detection of SARS-COV-2 antigen with linear range from 1.0 pg/mL to 1.0 µg/mL and limit of detection down to 51.74 fg/mL. The proposed immunosensor provide a brief but sensitive antigen detection tool for rapid diagnosis of COVID-19.