Immunosensing System Research Articles

A highly sensitive creatine kinase detection in human serum using 11-mercaptoundecanoic acid modified ITO-PET electrodes.

The enzyme creatine kinase (CK) is a biomarker that plays an extremely significant role in the early detection of cardiovascular disorders. Serum levels of CK are regularly monitored in patients with heart attacks, one of the most critical cardiovascular illnesses. In this study, a highly sensitive electrochemical immunosensor system was designed for the importance of early diagnosis of CK. This immunosensor system was developed by immobilizing 11- mercaptoundecanoic acid (11-MuA) on disposable indium tin oxide-polyethylene terephthalate (ITO-PET) electrodes. Electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and single frequency impedance (SFI) techniques were utilized throughout the immobilization process during the construction of the immunosensor. In addition, the proposed CK immunosensor system involves thorough analytical research, which may include linear determination range, repeatability, reproducibility, square wave voltammetry, storage capability, and regeneration. The suggested immunosensor was also characterized using scanning electron microscopy (SEM). The proposed immunosensor system demonstrated a broad dynamic range (0.1pg/mL - 100pg/mL), as well as a low limit of detection (LOD) and a low limit of quantification (LOQ) of 0.018pg/mL and 0.0394pg/mL, respectively. Finally, the immunosensor was tested on human serum samples, proving that it could be utilized in clinical situations.

A Rapid, Homogeneous, and Universal Electrochemical Immunosensing System Using an Antibody/Split-Enzyme Complex

Introduction Biosensors for point-of-care testing (POCT) must be rapid, user-friendly, and equipment-free. However, the conventional immunosensor principle employing the labelled secondary antibodies and the washing processes like enzyme-linked immunosorbent assay (ELISA) prevents the use for POCT applications. Our group has been developing antibody-enzyme complexes (AECs)1, which are composed of fragment antibodies and redox enzymes by applying a protein coupling module, SpyCatcher (SC)/SpyTag (ST). Although these AECs realized electrochemical immunosensors with rapid and sensitive detection2,3, but they still required magnetic bound/free (B/F) separation. We propose an ideal immunosensor employing AECs for POCT, which uses a protein-fragment complementation assay. This is based on the reconstitution of enzyme of the AEC which is inactivated by split technology and its enzymatic activity recovers in response to the target recognition by the AEC. To date, split pyrroloquinoline quinone-glucose dehydrogenase (PQQ-GDH) was reported and applied to the construction of AEC by means of genetic fusion with fragment antibodies4. However, the genetic fusion of antibodies is limited to the C-terminus of the enzyme to avoid loss of binding ability because the N-terminus of antibodies is located nearby the binding site of antibody. Moreover, the recombinant preparation of each protein fragment after splitting enzymes often accompanies the formation of the protein aggregates due to the exposure of the hydrophobic core of the enzyme. Here, we focused on a flavin adenine dinucleotide GDH (FAD-GDH) as a source for split enzyme, which is introduced to a loop of the enzyme, and employed SC and ST to construct universal split AECs. In addition, it is expected to prepare the split enzyme as soluble proteins because it is digested after the recombinant production. After the preparation of AECs, we applied the split AECs to electrochemical detection of biomarkers, C-reactive protein (CRP) and hemoglobin (Hb), and infectious virus, SARS-CoV-2. Methods First, we selected three split site candidates such as a loop exposed to the protein surface and a site which are not important for the protein structure. Then, tobacco etch virus (TEV) protease recognition sequence was inserted to each candidate site and SC was genetically fused to both terminals of FAD-GDH (SC-GDH-SC). ST was genetically fused to the C-terminus of single-chain variable fragments (scFv-ST). All the proteins were recombinantly produced in E. coli and purified. SC-GDH-SC was digested with TEV protease, and the preparation of split GDH was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Then, they were stoichiometrically mixed with the purified scFv-ST and incubated overnight at 4℃. The formation of split AEC was also evaluated by SDS-PAGE analysis. The binding ability and enzymatic activity caused by reconstitution upon target binding of the split AEC were evaluated. Finally, electrochemical detection system was constructed. The target was incubated with the split GDH, then mixed with the reaction solution containing a redox probe and glucose, and loaded onto a disposable electrode-printed chip. After incubation for 90 s, chronoamperometric measurement was performed. Results and discussion The result of SDS-PAGE analysis showed that all the split AECs were successfully prepared using the SC/ST system. Among the split site candidates, the GDH split between G407 and N408 showed the highest recovery of enzymatic activity after mixing with CRP. The enzymatic activity was not decreased even after protease digestion, which suggested that the GDH was not completely split into individual fragments in solution. This can also be confirmed by the binding ability of the split AEC, which showed enhanced affinity compared to scFv-ST due to the bivalent effect. However, the enzymatic activity of the split GDH was lowered in the absence of the target and recovered in response to the target concentration. When the different scFv-ST was used for the split AEC preparation, other targets were successfully detected, demonstrating the versatility of the system. As a result of electrochemical CRP detection, CRP concentration-dependent current increase was observed, and the linear range met the clinically required range. Conclusion We designed split AECs with high versatility, and applied it to the electrochemical detection of the protein biomarker. The detection can be completed within 5 min without any B/F separation, which can meet the criteria for POCT application. In addition, it was based on chronoamperometric measurement using GDH, a sensor configuration of a currently available glucose meter can be used for future applications. Reference 1Kimura et al., Anal. Chem., 2018, 90, 14500-14506. 2Kimura et al., Biosens. Bioelectron., 2021, 175, 112885. 3Miura et al., Talanta, 2021, 234, 122638. 4Guo et al., JACS, 2016, 138, 10108-10111.

A cost-effective and sensitive Au/6-mercaptohexanol/CNBr modified immunosensor approach for adiponectin analysis

This research aimed to develop a method for analyzing adiponectin using a label-free impedimetric indium tin oxide-polyethylene terephthalate (ITO-PET) immunosensing system. A simple and stable process to design a bioelectrode modified with gold (Au)/6-mercaptohexanol (6-MH)/cyanogen bromide (CNBr)/anti-adiponectin and its biocompatibility with adiponectin was investigated. The immunosensor performance, optimization tests, and immobilization steps were all monitored and analyzed using Electrochemical Impedance spectroscopy (EIS) and cyclic voltammetry (CV). Scanning Electron microscopy (SEM) examined the surface morphology of this biosensor during its immobilization process. Testing the developed biosensor with real human serum samples allowed for an evaluation of its clinical performance. The proposed immunosensor demonstrated high repeatability, reproducibility (RSD%: 4.37 %), long storage stability, and reusability (preserving 85 % of activity in the third regeneration step), along with a low limit of detection for adiponectin antigen and a wide range of linear values. The sensor suggested a linear detection range of 0.25 pg/mL to 500 pg/mL. The limit of detection (LOD) was found to be 0.20 pg/mL, and the limit of quantification (LOQ) was determined to be 0.66 pg/mL. It appears that the biosensor decorated with Au/6-MH/CNBr could be a good choice for the selective determination of adiponectin protein, according to the statistical data.

A fully integrated wireless microfluidic immunosensing system for portable monitoring of Staphylococcus aureus

The advanced devices that function fully without the need for external accessories are regarded as a pinnacle goal in the design and construction of modern ones. Staphylococcus aureus (S. aureus), a prominent human pathogen, is responsible for causing a wide variety of infections and chronic diseases. Herein, we present the first instance of a fully integrated wireless microfluidic immunosensing system (FIWMIS) capable of conducting point-of-care S. aureus monitoring in real samples of S. aureus-spiked commercial purified drinking water and S. aureus-spiked watermelon juice. The development of the proposed FIWMIS became a reality by conquering significant engineering hurdles in seamlessly integrating a microfluidic unit for liquid sample transport without the need of an external pump, an immunosensing unit for S. aureus monitoring, and an electronic control unit for signal conversion and wireless transmission. Such full integration culminated in a FIWMIS that upholds its pump-free, wireless, and low-cost characteristics for portable monitoring of S. aureus.

A Versatile Method to Create Antibody/Split‐Enzyme Complexes and Its Application to a Rapid, Homogeneous, and Universal Electrochemical Immunosensing System

AbstractIn this study, a versatile method is developed to create an antibody/split‐enzyme complex for use in electrochemical immunosensors. SpyCatchers are genetically fused at each terminus of flavin adenine dinucleotide‐dependent glucose dehydrogenase (FAD‐GDH) and a protease recognition sequence is inserted into a loop region to prepare a soluble protein and the split GDH. SpyTag‐fused single‐chain variable fragments (scFvs) are employed, which leads to the simple preparation of antibody‐split enzyme complexes (split AECs). The addition of pentameric C‐reactive protein (CRP), as a representative multimeric protein, restored the GDH activity of the split AECs, as the two split AEC fragments formed active GDH when in close proximity. CRP in human serum is electrochemically detected within 5 min without any washing steps with a clinically relevant CRP detection range (0.03–1 mg dL−1). The detection system is expanded to detect hemoglobin, SARS‐CoV‐2 spike protein, and inactivated SARS‐CoV‐2 by exchanging the scFvs. These results show that the convenient preparation method of the split GDH and the split AEC by the insertion of the protease recognition sequence and the use of SpyCatcher/SpyTag can realize a rapid, homogeneous, and universal electrochemical detection system that can be integrated into a commercially available electrochemical glucose sensor.

Nanozyme-mediated signal amplification on g-C3N4/NaBiO3 Z-scheme heterojunction photoelectrode toward ultrasensitive photoelectrochemical immunoassay for prostate-specific antigen

A sandwich-type photoelectrochemical (PEC) immunosensor for prostate-specific antigen (PSA) detection was developed using the g-C3N4/NaBiO3 (CN/NBO) Z-scheme heterojunction as the photoelectrode and glutathione-Cu/Cu2O nanozyme (GSH-Cu/Cu2O NPs) as the signal amplifier. The unique Z-scheme charge-carrier migration pathway of the CN/NBO heterojunction significantly promotes the separation and migration of photogenerated electron-hole pairs, producing a high photocurrent response of the photoelectrode. GSH-Cu/Cu2O NPs with ascorbic acid oxidase (AAO) activity were introduced into the PEC immunosensing system through immunoreactions for signal amplification. Signal amplification was achieved through double photocurrent quenching of the GSH-Cu/Cu2O NPs on the photoelectrode. On the one hand, GSH-Cu/Cu2O NPs as AAO catalyzed ascorbic acid (AA), an electron donor in the PEC system, to produce dehydroascorbic acid (DHA), leading to a decrease in the photocurrent response. On the other hand, the GSH-Cu/Cu2O NPs prevented AA transfer from the detection solution to the interface of the photoelectrode, further aggravating the PEC signal quenching effect. Benefiting from the excellent performance of the Z-scheme heterojunction-based photoelectrode, the dual signal quenching effect of GSH-Cu/Cu2O NPs, the PEC immunosensor showed an ultralow detection limit of 5.1 × 10−15 g mL−1. By coupling a Z-scheme heterojunction-based photoelectrode with a nanozyme-mediated signal amplification strategy, this study provides a novel avenue for the construction of ultrasensitive and convenient biosensing systems.

Nano-immuno-conjugates inspired by hydrophilic perovskite fluorescent spheres and magnetic assisted for detection of hepatitis B surface antigen.

The rampant hepatitis B virus (HBV) seriously endangers human health, and hepatitis B surface antigen (HBsAg) is its early diagnostic marker. Therefore, it is crucial to construct a fast and highly sensitive HBsAg detection method. Based on high-efficiency magnetic separation technology and fluorescent composite material labelling technology, an accurate, fast and sensitive fluorescent immunosensing system for HBsAg detectionwas developed. Immunomagnetic beads constructed from carboxyl-functionalized Fe3O4 nanoparticles (Fe3O4-COOH) with excellent magnetic response performance were used as efficient capture carriers for HBsAg. Immunofluorescence composite microspheres constructed based on ultra-stable polystyrene-coated CsPbBr3 perovskite nanocrystals (CPB@PSAA) with high hydrophilic properties, were excellent fluorescent markers for HBsAg. Using thissensitive sandwich fluorescence sensing system a good linear relationship within the range of 0.2-15ng/mL was established between HBsAg concentrationand fluorescence intensity with alimit of detection (LOD) of 0.05ng/mL. Thesystem obtained satisfactory results when tested on real human serum samples. The magnetic-assisted fluorescence immune-sandwich sensor system has broad application prospects in biomedicine such as rapid and early diagnosis and effective prevention of infectious diseases.

A dual mode immunosensing platform constructed on CdIn2S4 near-infrared electrochemiluminescence emitter and AuNPs@g-C3N4 for zearalenone sensing

In this study, a near-infrared (NIR) electrochemiluminescence (ECL) emitter CdIn2S4 was synthesized by a convenient hydrothermal strategy. The ECL properties of the obtained CdIn2S4 were examined in the presence of potassium persulfate coreactant at an excitation potential of − 1.25 V. The low trigger voltage of CdIn2S4 may be ascribed to its lower bandgap. This lower excitation potential could effectively reduce the side-effects induced by high voltages, while maintaining the biological activity of antibodides and antigens. The ECL features of CdIn2S4 were also investigated, demonstrating that CdIn2S4 exhibited excellent NIR characteristics, and surface state transitions led to the ECL emission. Importantly, CdIn2S4 was introduced into ECL and electrochemical impedance spectroscopy (EIS) to establish a dual-mode immunosensing interface. Meanwhile, AuNPs@g-C3N4 with excellent properties were used to promote electron transmission and increase the immobilization amount of zearalenone (ZEA) antibodies in the dual-mode sensing system. The developed immunosensing strategy displayed superior sensing performance in ZEA analysis. The detection limits of ZEA were 30 fg mL−1 (ECL model) and 10 fg mL−1 (EIS model), respectively. The desirable recovery results of ZEA in corn samples prove the application potential of the NIR emitter CdIn2S4 in developing a dual-mode immunosensing system for early real-time use.

Utilizing COVID-19 as a Model for Diagnostics Using an Electrochemical Sensor.

This paper reports a rapid and sensitive sensor for the detection and quantification of the COVID-19 N-protein (N-PROT) via an electrochemical mechanism. Single-frequency electrochemical impedance spectroscopy was used as a transduction method for real-time measurement of the N-PROT in an immunosensor system based on gold-conjugate-modified carbon screen-printed electrodes (Cov-Ag-SPE). The system presents high selectivity attained through an optimal stimulation signal composed of a 0.0 V DC potential and 10 mV RMS-1 AC signal at 100 Hz over 300 s. The Cov-Ag-SPE showed a log response toward N-PROT detection at concentrations from 1.0 ng mL-1 to 10.0 μg mL-1, with a 0.977 correlation coefficient for the phase (θ) variation. An ML-based approach could be created using some aspects observed from the positive and negative samples; hence, it was possible to classify 252 samples, reaching 83.0, 96.2 and 91.3% sensitivity, specificity, and accuracy, respectively, with confidence intervals (CI) ranging from 73.0 to 100.0%. Because impedance spectroscopy measurements can be performed with low-cost portable instruments, the immunosensor proposed here can be applied in point-of-care diagnostics for mass testing, even in places with limited resources, as an alternative to the common diagnostics methods.

Pt/Zn-TCPP Nanozyme-Based Flexible Immunoassay for Dual-Mode Pressure-Temperature Monitoring of Low-Abundance Proteins.

Pressure and temperature, as common physical parameters, are important for monitoring human health. In contrast, single-mode monitoring is prone to causing experimental errors. Herein, we innovatively designed a dual-mode flexible sensing platform based on a platinum/zinc-meso-tetrakis(4-carboxyphenyl)porphyrin (Pt/Zn-TCPP) nanozyme for the quantitative monitoring of carcinoembryonic antigen (CEA) in biological fluids with pressure and temperature readouts. The Pt/Zn-TCPP nanozyme with catalytic and photothermal efficiencies was synthesized by means of integrating photosensitizers into porous materials. The flexible sensing system after the antigen-antibody reaction recognized the pressure using a flexible skin-like pressure sensor with a digital multimeter readout, whereas the temperature was acquired via the photoheat conversion system of the Pt/Zn-TCPP nanozyme under 808 nm near-infrared (NIR) irradiation using a portable NIR imaging camera on a smartphone. Meanwhile, the dual-mode flexible sensing system was carried out on a homemade three-dimensional (3D)-printed device. Results revealed that the developed dual-mode immunosensing platform could exhibit good pressure and temperature responses within the dynamic range of 0.5-100 ng mL-1 CEA with the detection limits of 0.24 and 0.13 ng mL-1, respectively. In addition, the pressure and temperature were sensed simultaneously without crosstalk interference. Importantly, the dual-mode flexible immunosensing system can effectively avoid false alarms during the measurement, thus providing great potential for simple and low-cost development for point-of-care testing.

Microfluidic Immunosensor Platform for Sensitive Detection of Human Epidermal Growth Factor Receptor-2 Based on Enhanced Cathode Electrochemiluminescence of Bimetallic Nanoclusters.

In this work, a microfluidic immunosensor chip was developed by incorporating microfluidic technology with electrochemiluminescence (ECL) for sensitive detection of human epidermal growth factor receptor-2 (HER2). The immunosensor chip can achieve robust reproducibility in mass production by integrating multiple detection units in a series. Notably, nanoscale materials can be better adapted to microfluidic systems, greatly enhancing the accuracy of the immunosensor chip. Ag@Au NCs closed by glutathione (GSH) were introduced in the ECL microfluidic immunosensor system with excellent and stable ECL performance. The synthesized CeO2-Au was applied as a coreaction promoter in the ECL signal amplification system, which made the result of HER2 detection more reliable. In addition, the designed microfluidic immunosensor chip integrated the biosensing system into a microchip, realizing rapid and accurate detection of HER2 by its high throughput and low usage. The developed short peptide ligand NARKFKG (NRK) achieved an effective connection between the antibody and nanocarrier for improving the detection efficiency of the sensor. The immunosensor chip had better storage stability and sensitivity than traditional detection methods, with a wide detection range from 10 fg·mL-1 to 100 ng·mL-1 and a low detection limit (LOD) of 3.29 fg·mL-1. In general, a microfluidic immunosensor platform was successfully constructed, providing a new idea for breast cancer (BC) clinical detection.

Self-Powered Immunoassay of Norovirus in Human Stools by π-Electron-Rich Homojunction for Enhanced Charge Transfer.

Norovirus (NoV) stands as a significant causative agent of nonbacterial acute gastroenteritis on a global scale, presenting a substantial threat to public health. Hence, the development of simple and rapid analytical techniques for NoV detection holds great importance in preventing and controlling the outbreak of the epidemic. In this work, a self-powered photoelectrochemical (PEC) immunosensor of NoV capsid protein (VP1) was proposed by the π-electron-rich carbon nitride homojunction (ER-CNH) as the photoanode. C4N2 ring derived from π-rich locust bean gum was introduced into the tri-s-triazine structure, creating a large π-delocalized conjugated carbon nitride homojunction. This strategy enhances the C/N atomic ratio, which widens light utilization, narrows the bandgap, and optimizes the electronic band structure of carbon nitride. By introduction of a π-rich conjugated structure, p-type domains were induced within n-type domains to build the internal electric field at the interface, thus forming a p-n homojunction to boost carrier separation and transfer. The ER-CNH photoanode exhibited excellent photoelectric performance and water oxidation capacity. Since VP1 inhibits the water oxidation of the ER-CNH photoanode, the open-circuit potential of the as-prepared PEC immunosensor system was reduced for detecting NoV VP1. The self-powered PEC immunosensor achieved a remarkably low detection limit (∼5 fg mL-1) and displayed high stability and applicability for actual stool samples. This research serves as a foundation concept for constructing immunosensors to detect other viruses and promotes the application of self-powered systems for life safety.

Enhancement of Sensitivity for Retroreflection‐Based Biosensor by Controlling Polymer Brush on Janus Particles

AbstractAmong the methods for onsite detection of specific biomolecules, the use of retroreflective Janus particles (RJPs) is a simple and efficient method for biophotonic probes to detect biotarget molecules with spatioselectivity, which can make the RJPs align toward light sources after capturing the target biomolecules, enhancing retroreflection. Because RJP has two distinct regions with different properties, specific reagents can be easily functionalized through chemical reactions between the functional groups and biotarget molecules. The number of functional groups on RJP will be the critical parameter for efficiency of sensing ability. Here, clickable (azide) polymer brush is introduced on surface of RJP via different surface polymerization methods (grafting to and grafting from method). Azide groups allow for the easy modification of dibenzocyclooctyne‐linked biomolecules onto silica particles through click chemistry. RJPs are prepared with different quantities of azide functional groups on their surfaces via solvent volatilization method and metal deposition. Using a retroreflective immunosensing system, sensitive detection of target biomolecules is performed.

Graphene-Based Nanomaterial Electrochemical Biosensors for the Determination of the H3N2 Virus

Since influenza and COVID-19 cause similar symptoms, the need for selective, sensitive and practical systems for influenza A detection has been highlighted. For this reason, in this work, a graphene (Gr) based nanomaterial modified anti-hemagglutinine 3, from now on will be called Gr-antiH3, based disposible electrochemical influenza immunosensor was developed. In order to obtain the highest sensitivity, the effects of various Gr-based nanomaterials on developed electrochemical immunosensor responses were investigated. For this purpose, graphene oxide, Gr, Gr-platinum (Gr-Pt), Gr-gold and Gr-gold-platinum nanomaterials were synthesized and characterized via electrochemical impedance spectroscopy (EIS). As a result, since the best results were obtained with Gr-Pt, this nanomaterial was integrated into H3N2 immunosensor system as a modifier. The Gr-Pt modified immunosensor was shown to provide a linear range from 0.5 µg/mL to 7 µg/mL. The limit of detection was 0.38 µg/mL and limit of quantification was 1.28 µg/mL with a relative standard deviation of 3.05%. The developed biosensor was applied to H3N2 detection in synthetic saliva samples and promising results were obtained with recoveries of 100.41 ± 1.10%.

Polyaniline and polystyrene sulfonic acid modified on conductive biochar nanoparticle with strong signal enhancement for trace atrazine detection

A label and reagent free and cost effective immunosensing system was newly developed for detection of trace leveled atrazine in water. This was constructed by synthesis of polyaniline (PA) and polystyrene sulfonic acid (PSS) modified biochar (BC) nanoparticle composite (PA-PSS-BC) and coating them on screen printed electrode as conductive and electrochemically active layer, and covalently immobilizing anti-atrazine antibody on electrode for specific target binding. The low cost biochar from sugarcane was highly conductive and favorable for attaching PA and PSS due to large surface area and sp2 carbon structure. Crediting to dopant PSS, PA-PSS-BC achieved significant enhancement of redox current signal even in phosphate buffered saline of neutral pH compared to that of without PSS. And redox current of PA was extremely stable with negligible signal change even after twenty cycles repeated reactions owning to protection by BC. These essentially ensured sensing platform being sensitively performed in neutral solution with exemption of adding external redox reagent. As a result, fairly low concentrations of atrazine could be detected within 5 min, with limit of detection being 0.407 ng/L in a dynamic detection range varying from 1 to 100 ng/L. Moreover, the immunosensor exhibited reasonable signal recoveries for detecting trace leveled atrazine in real water samples, high selectivity, and good stability with dry and cold storage.

FeNC nanozyme-based electrochemical immunoassay for sensitive detection of human epidermal growth factor receptor 2.

Theproof-of-concept of sensitive electrochemical immunoassay for the quantitative monitoring of human epidermal growth factor receptor 2 (HER2)is reported. The assay is carried out on iron nitrogen-doped carbon (FeNC) nanozyme-modified screen-printed carbon electrode using chronoamperometry. Introduction of target HER2 can induce the sandwiched immunoreaction between anti-HER2 monoclonal antibody-coated microplate and biotinylated anti-HER2 polyclonal antibody. Thereafter, streptavidin-glucose oxidase (GOx) conjugate is bonded to the detection antibody. Upon addition of glucose, 3,3',5,5'-tetramethylbenzidine (TMB) is oxidized through the produced H2O2 with the assistance of GOx and FeNC nanozyme. The oxidized TMB is determined via chronoamperometry. Experimental results revealed that electrochemical immunosensing system exhibited good amperometricresponse, and allowed the detection of target HER2 as low as 4.5 pg/mL. High specificity and long-term stability are acquired with FeNC nanozyme-based sensing strategy. Importantly, our system provides a new opportunity for protein diagnostics.

A novel construction strategy of photoelectrochemical immunosensor for detecting neuron-specific enolase: Sensing mode integrating photoanode and photocathode

In the realm of photoelectrochemical (PEC) analysis, it is customary to employ a lone photoanode or photocathode system for analyte detection. Nonetheless, such a single detection scheme inherently carries some deficiencies. While photoanode-based PEC immunoassay methods do exhibit conspicuous photocurrent responses and heightened sensitivity, they do suffer from inadequate resistance to interference when detecting in real sample detection. Photocathode-based analysis methods are capable of effectively surmounting the limitations of photoanode-based analysis methods, but their stability is poor. Based on the above reasons, this paper reports a novel immunosensing system combining ITO/WO3/Bi2S3 photoanode and ITO/CuInS2 photocathode. The system that incorporates both photoanode and photocathode exhibits a steady and discernible photocurrent, possesses robust resistance to external interference, and has effectively accomplished the quantification of NSE in the linear range from 5 pg/mL to 30 ng/mL. Remarkably, the detection limit has been determined to be 1.59 pg/mL. Besides the notable advantages of satisfactory stability, exceptional specificity, and outstanding reproducibility, the sensing system also introduces an innovative approach to fabricate PEC immunosensors.

Photoelectrochemical immunoassay for thyroglobulin on nanogold-functionalized BiVO4 photoanode coupling with enzymatic biocatalytic precipitation

Methods derived from photoelectrochemical (PEC) have been constructed for immunoassays, but most involve the split-type immunoreaction modes, and thus easily cause unpredictable intermediate precision. Herein, we innovatively designed an integrated PEC immunosensing platform for the quantitative monitoring of thyroglobulin (TG) on the gold nanoparticles (AuNPs)-functionalized BiVO4 photoanode coupling with enzymatic biocatalytic precipitation (EBCP). This sensing system could simultaneously implement the immunoreaction and photocurrent measurement. Anti-TG capture antibodies were modified onto AuNPs-decorated BiVO4 photoelectrode. A sandwich-type immunoreaction was carried out in the presence of target TG using horseradish peroxidase (HRP)-conjugated anti-TG detection antibody. The carried HRP molecules catalyzed 4-chloro-1-naphthol (4-CN) to generate an insoluble benzo-4-chlorohexadienone product on the photoanode in the presence of peroxide hydrogen, thereby decreasing the photocurrent. Under optimal conditions, the PEC immunosensors gave good photocurrent responses toward target TG within the dynamic range of 0.01–10 ng mL−1 at a detection limit of 7.6 pg mL−1. Good repeatability and precision, high specificity and acceptable storage stability were acquired during the measurement. No significant differences were encountered for screening 15 human serum specimens between the developed PEC immunoassay and commercially available enzyme-linked immunosorbent assay (ELISA) method for the detection of target TG. Significantly, PEC immunosensing system offers promise for simple and cost-effective analysis of disease-related biomarkers.

Signal-On Near-Infrared Photoelectrochemical Aptasensors for Sensing VEGF165 Based on Ionic Liquid-Functionalized Nd-MOF Nanorods and In-Site Formation of Gold Nanoparticles

The low photon energy and deep penetrating ability of near-infrared (NIR) light make it an ideal light source for a photoelectrochemical (PEC) immunosensing system. Absorption wavelengths of the metal-organic frameworks (MOFs) can be regulated by adjusting the metal ions and the conjugation degree of the ligands. Herein, an ionic liquid with a large conjugated structure was synthesized and was used as a ligand to coordinate with Nd ions to prepare Nd-MOF nanorods with a band gap of 1.26 eV. The Nd-MOF rods show a good photoabsorption property from 200 to 980 nm. A PEC platform was constructed by using Nd-MOF nanorods as the photoelectroactive element. A detachable double-stranded DNA labeled with alkaline phosphatase (ALP), which is specific to VEGF165, was immobilized onto the PEC sensing interface. After blocking unspecific active sites with bovine albumin, an NIR PEC aptasensing system was developed for VEGF165 detection. After being incubated in a mixture of VEGF165, l-ascorbic acid 2-phosphate (magnesium salt hydrate) (AAP), and chloroauric acid, the aptamers for VEGF165 were detached from the PEC aptasensing interface, thus resulting in the decrease of the charge-transfer resistance and the increase of the photocurrent response. The shedding of the aptamers also makes the ALP approach the electrode surface, thus catalyzing the reduction of AAP to produce ascorbic acid (AA). Subsequently, AA reduces in situ chloroauric acid to produce AuNPs on the Nd-MOF-based sensing interface. With the excellent conductivity and localized surface plasmon resonance effect, the AuNPs can accelerate the separation of electron-hole pairs generated from Nd-MOF nanorods, thus promoting the photoelectric conversion efficiency and achieving signal amplification. Under optimized conditions, the PEC responses were linearly related to the VEGF165 concentrations in the range of 0.01-100 ng mL-1 and exhibit a low detection limit of 3.51 pg mL-1 (S/N = 3). VEGF165 in human serum samples was detected by the NIR PEC aptasensor. Their concentrations were found to be well consistent with that obtained from ELISA. Furthermore, the PEC aptasensor demonstrated recoveries from 96.07 to 103.8%. The relative standard deviations were within 5%, indicating good accuracy and precision. The results further verify its practicability for clinical diagnosis.

Highly Sensitive Photoelectrochemical Immunosensor Based on Organic Multielectron Donor Nanocomposite as Signal Probe.

Organic photoelectric materials with conjugated electron-rich structures and good biocompatibilities have broad application prospects in biosensors. Herein, we report a promising organic photoelectric multielectron donor nanocomposite for highly sensitive PEC immunoassays. Specifically, the organic multielectron donor nanocomposite (DA-ZnTCPP-g-C3N4) was prepared from dopamine (DA, polyphenol hydroxyl structure substance), zinc tetracarboxylate porphyrin (ZnTCPP, large p-π conjugated heterocyclic compound), and two-dimensional graphene-like nitrogen carbide (g-C3N4) via an amidation reaction. With a multielectron donor structure and photoelectricity, this nanocomposite can achieve sensitization by self-structure without the addition of an electron donor in the test solution. It was utilized to label the carcinoembryonic detection antibody as a immuno-probe (Ab2-DA-ZnTCPP-g-C3N4). Meanwhile, the glassy carbon electrode electrodeposited with gold nanoparticles anchoring the capture antibody was used as a PEC immunomatrix (Ab1/DpAu/GCE). The enhanced PEC current, "signal on", was confirmed by the immunosensor via sandwich immunorecognition of a carcinoembryonic antigen (CEA). Under optimal conditions, the as-prepared sensing platform displayed high sensitivity for CEA with a dynamic linear response range from 10 fg·mL-1 to 1 mg·mL-1 and a lower detection limit of 3.6 fg·mL-1. This organic nanocomposite showed good sensitivity and stability in an immunosensing system with a low background. This strategy affords a promising approach for biological applications of organic photoelectric materials.