Developing the portable CRP detection technologies that are suitable for point-of-care (POC) and primary care management is of utmost importance, and advancing the electrochemical immunosensors hold promise for POC implementation. Nevertheless, non-specific adsorption of numerous interfering proteins in complex biological media contaminates immunosensors, thereby restricting the reliability in detection efficacy. In this study, a three-dimensional flower-leaf shape amyloid bovine serum albumin/gold nanoparticles/polyaniline (AL-BSA/AuNPs/PANI) coating on the surface of the electrode was developed, which demonstrated strong anti-adsorption properties against bovine serum albumin, plasma, and cells. The immunosensor exhibited a good linear relationship to CRP response, featuring a detection limit of 0.09 μg/mL, consistent with clinical reference range. In addition, the CRP immunosensor demonstrated excellent specificity in other inflammation-related proteins and commendable anti-interference performance for CRP detection in plasma and whole blood tests. Importantly, by combining the development of a USB flash disk-type portable electrochemical workstation with a reagent-free mode, the developed CRP electrochemical immunosensor delivered ideal results in clinical samples. The anti-fouling performance, sensitivity and specificity of the immunosensor, as well as its flexible test modes in clinical samples, provide important scientific basis for developing POC detection technologies of vital biomarkers in complex biological media.

Bisphenol S (BPS) is a common pollutant in the environment and has posed a potential threat to aquatic animals and human health. To accurately assess the pollution level and ecological risk of BPS, there is an urgent need to establish simple and sensitive detection methods for BPS. In this study, BPS complete antigen was successfully prepared by introducing methyl 4-bromobutyrate and coupling bovine serum albumin (BSA). The monoclonal antibody against BPS (anti-BPS mAb) with high affinity (1: 256,000) was developed based on the BPS complete antigen, which showed low cross-reactivity with BPS structural analogues. Then, an electrochemical immunosensor was constructed to detect BPS using multi-walled carbon nanotubes and gold nanoflower composites as signal amplification elements and using anti-BPS mAb as the probe. The electrochemical immunosensor had a linear range from 1 to 250 ng⋅mL-1 and a limit of detection (LOD) down to 0.6 ng⋅mL-1. Additionally, a more stable and sensitive lateral flow immunoassay (LFIA) for BPS was developed based on iridium oxide nanoparticles, with a visual detection limit of 1 ng⋅mL-1, which was 10 times lower than that of classical Au-NPs LFIA. After evaluation of their stability and specificity, the reliability of these two methods were further validated by measuring BPS concentrations in the water and fish tissues. Thus, this study provides sensitive, robust and rapid methods for the detection of BPS in the environment and organisms, which can provide a methodological reference for monitoring environmental contaminants.

The fabrication of ordered nanoarray electrode (NAE) using UV imprinting and their application as electrochemical (EC) immunosensor is described in this study. Especially, the influence of the array density factors on the performance of NAE was characterized electrochemically and compared with flat-electrode. Low-density (hole: 200 nm, hole space = 600 nm), medium-density (hole: 200 nm, hole space = 400 nm), and high-density NAE (hole: 200 nm, hole space = 200 nm) which have the same active area were fabricated and their redox cycling was compared with empirical results. We observed that the high-density is the optimum NAE exhibiting the lowest charge transfer resistance and the highest redox cycling performance among all NAEs. Finally, to observe the effect of their EC performance as biosensor, an EC immunoassay was performed using Interleukine-6 (IL-6), and high-density NAE has lowest a low limit of detection (LOD) of 0.45 pg/mL compared with other NAEs (medium-density: 3.91 pg/mL, low-density: 5.87 pg/mL).

Exhaled breath electrochemical sensing is a promising biomedical technology owing to its portability, painlessness, cost-effectiveness, and user-friendliness. Here, we present a novel approach for target analysis in exhaled breath by integrating a comfortable paper-based collector into an N95 face mask, providing a universal solution for analyzing several biomarkers. As a model analyte, we detected SARS-CoV-2 spike protein from the exhaled breath by sampling the target analyte into the collector, followed by its detection out of the N95 face mask using a magnetic bead-based electrochemical immunosensor. This approach was designed to avoid any contact between humans and the chemicals. To simulate human exhaled breath, untreated saliva samples were nebulized on the paper collector, revealing a detection limit of 1 ng/mL and a wide linear range of 3.7-10,000 ng/mL. Additionally, the developed immunosensor exhibited high selectivity toward the SARS-CoV-2 spike protein, compared to other airborne microorganisms, and the SARS-CoV-2 nucleocapsid protein. Accuracy assessments were conducted by analyzing the simulated breath samples spiked with varying concentrations of SARS-CoV-2 spike protein, resulting in satisfactory recovery values (ranging from 97 ± 4 to 118 ± 1%). Finally, the paper-based hybrid immunosensor was successfully applied for the detection of SARS-CoV-2 in real human exhaled breath samples. The position of the collector in the N95 mask was evaluated as well as the ability of this paper-based analytical tool to identify the positive patient.

Cytokeratin 19 fragment antigen 21-1 (CYFRA21-1) is a protein fragment released into the bloodstream during the death of lung epithelial cells, serving as a predictive biomarker in diagnosing non-small cell lung cancer (NSCLC) and need to be accurately detected. Herein, a dual-responsive label-free electrochemical immunosensor was developed based on a three-dimensional ordered interconnecting macroporous carbon skeleton material modified with gold-cobalt nanoparticles (Au/Co NPs-3D MCF) to detect cytokeratin-19 fragment (CYFRA21-1). The three-dimensional ordered interconnect macroporous structure, by providing a high specific surface area and an electrochemically active area, not only enhances the electron transport channel and reduces mass transfer resistance, but also offers a confined region that elevates the collision frequency with the active site. In addition to exhibiting excellent biocompatibility for antibody binding, gold-cobalt nanoparticles contribute significantly to the overall robustness of the immunosensor. By capitalizing on the 3D network structure and collective effect of Au and Co NPs, the Au/Co NPs-3D MCF immunosensors exhibit exceptional response signals in both chronocurrent testing and square-wave voltammetry, allowing for a wide linear response range of 0.0001-100 ng/mL and a low detection limit. Moreover, the constructed immunosensor is capable of detecting CYFRA21-1 in human serum and has the potential for further extension to detect multiple biomarkers. This work opens up new avenues for the construction of other highly selective 3D network immunosensors.

BACKGROUND: Cardiac troponin I (CTnI) is demonstrated as one of the most promising disease biomarkers for early diagnosing acute myocardial infarction (AMI). To date, electrochemical immunosensors have been extensively studied in the field of cTnI determination. But highly accurate and sensitive cTnI detection by this method is still a challenge due to non-specific adsorption on electrode interfaces in complex human serum. As a result, it is necessary to develop an antifouling electrochemical immunosensor with high sensitivity for the detection of cTnI.
RESULTS: In this work, an antifouling electrochemical immunosensor was constructed based on vertically-aligned peptide layer consisting of Au nanoparticles (AuNPs) and amphiphilic CEAK16 peptide (CEAK16@AuNPs) for sensitive and accurate detection of cTnI in human serum. The vertically-aligned CEAK16@AuNPs interface provided a stable hydration layer originated from attraction of water molecules by amino acids on the hydrophilic side of the CEAK16, which effectively reduced non-specific adsorption and enhanced electron transfer rate. The cTnI immunosensor possessed great analytical performance with a wide range from 1 fg mL-1 to 1 μg mL-1 and a low detection limit of 0.28 fg mL-1 (S/N = 3). Additionally, the proposed CEAK16@AuNPs sensing interface showed excellent long-term antifouling performance and electrochemical activity that preserved 80 % of the initial signal after 20-days exposure in human serum samples. Consequently, the cTnI immunosensor displayed excellent detection accuracy compared to clinical methods and owned good selectivity, stability and reproducibility.
CONCLUSIONS: The development of this strategy provides a versatile tool for accurate quantitative cTnI analysis in real human serum, thus helping to achieve early AMI diagnosis effectively and holding the promising potentials for other immunosensor in disease diagnosis.

To advance cervical cancer diagnostics, we propose a state-of-the-art label-free electrochemical immunosensor designed for the simultaneous detection of multiple biomarker proteins (p16INK4a, p53, and Ki67). This immunosensor is constructed using a polyethyleneimine-coated gold nanoparticles/2D tungsten disulfide/graphene oxide (PEI-AuNPs/2D WS2/GO) composite-modified three-screen-printed carbon electrode (3SPCE) array. The 2D WS2/GO hybrid provides a large specific surface area for supporting well-dispersed PEI-AuNPs and adsorbed redox-active species, enhancing overall performance. The PEI-AuNPs-decorated 2D WS2/GO composite not only improves electrode conductivity but also increases the antibody loading capacity. Redox-active species, including Cd2+ ions, 2,3-diaminophenazine (DAP), and methylene blue (MB), serve as distinct signaling compounds to quantitatively detect the cervical cancer biomarkers p16INK4a, p53, and Ki67, respectively. Additionally, the immunosensor demonstrates the detection with high sensitivity, good storage stability, high selectivity, and acceptable reproducibility. This immunosensor demonstrates a good linear relationship with the logarithm of protein concentrations. Additionally, the immunosensor also demonstrates high sensitivity, good storage stability, high selectivity, and acceptable reproducibility. Our promising results and the successful application of the immunosensor in detecting three tumor markers in human serum highlight its potential for clinical diagnosis of cervical cancer.

Three-dimensional (3D) network provide a promising platform for construction of high sensitive electrochemical immunosensor due to the benefits of high specific surface area and electron mobility. Herein, a sensitive label-free electrochemical immunosensor based on Au nanoparticles modified Ni-B nanosheets/graphene matrix was constructed to detect diethylstilbestrol (DES). The 3D network not only could increase the electron transport rate and surface area, but also could provide confinement area, which is conducive to increases the collision frequency with the active site. Moreover, Au NPs also have good biocompatibility, which is beneficial for ligating antibodies. Benefiting from the 3D network structure and Au collective effect, the electrochemical immunosensor possess sterling detection ability with wide linear response range (0.00038-150 ng/mL) and low detection limit (31.62 fg/mL). Moreover, the constructed immunosensor can also be extend to detect DES in Tap-water and river water. This work may provide a novel material model for the construction of high sensitive immunosensor.

Human epididymis protein 4 (HE4), a diagnostic biomarker of ovarian cancer, is crucial for monitoring the early stage of the disease. Hence, it is highly important to develop simple, inexpensive, and user-friendly biosensors for sensitive and quantitative HE4 assays. Herein, a new sandwich-type electrochemical immunosensor based on Prussian blue (PB) as a signal indicator and functionalized metal-organic framework nanocompositesas efficient signal amplifiers was fabricated for quantitative analysis of HE4. In principle, ketjen black (KB) and AuNPs modified on TiMOF (TiMOF-KB@AuNPs) could accelerate electron transfer on the electrode surface and act as a matrix for the immobilization of antibodies via cross-linking to improve the determination sensitivity. The PB that covalently binds to labeled antibodies endows the biosensors with intense electrochemical signals. Furthermore, the concentration of HE4 could be indirectly detected by monitoring the electroactivity of PB. Benefiting from the high signal amplification ability of the PB and MOF nanocomposites, this strategy displayed a wide linear range (0.1-80 ng mL-1) and a lower detection limit (0.02 ng mL-1). Hence, this study demonstrated great promise for application in clinical ovarian cancer diagnosis and treatment, and provided a new platform for detecting other cancer biomarkers.

An electrochemical immunoassay system was developed to detect CA-125 using a glassy carbon electrode (GCE) modified with MXene, graphene quantum dots (GQDs), and gold nanoparticles (AuNPs). The combined MXene-GQD/AuNPs modification displayed advantageous electrochemical properties due to the synergistic effects of MXene, GQDs, and AuNPs. The MXene-GQD composite in the modified layer provided strong mechanical properties and a large specific surface area. Furthermore, the presence of AuNPs significantly improved conductivity and facilitated the binding of anti-CA-125 on the modified GCE, thereby enhancing sensitivity. Various analytical techniques such as FE-SEM and EDS were utilized to investigate the structural and morphological characteristics as well as the elemental composition. The performance of the developed immunosensor was assessed using electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), square wave voltammetry (SWV), and differential pulse voltammetry (DPV). Under optimized conditions in a working potential range of -0.2 to 0.6 V (vs. Ag/AgCl), the sensitivity, linear range (LR), limit of detection (LOD), and correlation coefficient (R2) were determined to be 315.250 µA pU.mL-1/cm2, 0.1 to 1 nU/mL, 0.075 nU/mL, and 0.9855, respectively. The detection of CA-125 in real samples was investigated using the developed immunoassay platform, demonstrating satisfactory results including excellent selectivity and reproducibility.