Antibody Immobilization Research Articles

IMMUNOSENSOR BASED ON SCREEN-PRINTED GRAPHITE ELECTRODES MODIFIED WITH GOLD NANOPARTICLES AND SYNTHETIC MEMBRANE-LIKE SUBSTANCE FOR THE DETERMINATION OF CHLORAMPHENICOL

An electrochemical immunosensor based on screen-printed graphite electrodes has been developed for the determination of the antibiotic chloramphenicol in water and milk samples. It has been shown that the immobilization of chloramphenicol-specific antibodies in a liquid crystal layer of a membrane-like didodecyldimethylammonium bromide preserves the mobility and accessibility of active centers of antibodies, and the addition of gold nanoparticles improves electron transfer from the electrode surface to the redox centers of horseradish peroxidase used as a label. The limit of detection of chloramphenicol in water was 0.02 μg/L, in milk - 0.04 μg/L. The method can be used to determine residual amounts of chloramphenicol in animal products.

Tip-Enhanced Sub-Femtomolar Steroid Immunosensing via Micropyramidal Flexible Conducting Polymer Electrodes for At-Home Monitoring of Salivary Sex Hormones.

Noninvasive testing and continuous monitoring of ultralow-concentration hormones in biofluids have attracted increasing interest for health management and personalized medicine, in which saliva could fulfill the demand. Steroid sex hormones such as progesterone (P4) and β-estradiol (E2) are crucial for female wellness and reproduction; however, their concentrations in saliva can vary down to sub-pM and constantly fluctuate over several orders of magnitude. This remains a major obstacle toward user-friendly and reliable monitoring at home with low-cost flexible biosensors. Herein we introduce a 3D micropyramidal electrode architecture to address such challenges and achieve an ultrasensitive flexible electrochemical immunosensor with sub-fM-level detection capability of salivary sex hormones within a few minutes. This is enabled by micropyramidal electrode arrays consisting of a poly(3,4-ethylenedioxythiophene):poly(4-styrenesulfonate) (PEDOT:PSS) thin film as the coating layer and electrochemically decorated gold nanoparticles (AuNPs) to improve the antibody immobilization. The enhanced mass transport around the 3D tips provided by the micropyramidal architecture is discovered to improve the detection limit by 3 orders of magnitude, pushing it to as low as ∼100 aM for P4 and ∼20 aM for E2, along with a wide linear range up to μM. Accordingly, these hormones down to sub-fM in >1000-fold-diluted saliva samples can be accurately measured by the printed soft immunosensors, thus allowing at-home testing through simple saliva dilution to minimize the interfering substances instead of centrifugation. Finally, monitoring of the female ovarian hormone cycle of both P4 and E2 is successfully demonstrated based on the centrifuge-free saliva testing during a period of 4 weeks. This ultrasensitive and soft 3D microarchitected electrode design is believed to provide a universal platform for a diverse variety of applications spanning from accurate clinical diagnostics and counselling and in vivo detection of bioactive species to environmental and food quality tracing.

Development of potentiometric immunosensor for determination of live attenuated Varicella Vaccine: Potency and stability studies

Determination of Varicella vaccine's potency; containing live attenuated strain (Oka) of Varicella Zoster virus, has been limited to in vitro cell culture methods. In this study, a label free potentiometric biosensor has been developed for the first time and optimized to determine the content of varicella zoster virus. A passive ion-flux sensing platform has been developed using an anti-varicella monoclonal antibody and tetrabutyl ammonium bromide as a marker ion. The immunosensor has been optimized with respect to membrane diameter and concentration of the immobilized antibody. Linearity was achieved over a concentration range of 2.5–3.2 log PFU/dose with a LOD of 1.9 log PFU/dose. Potentiometric results were compared to the plaque-forming assay using the cell culture technique. The developed immunosensor was superior with respect to analysis time and cost without affecting critical analytical performance characteristics. Furthermore, in order to evaluate the stability indicating ability of the immunosensor, the effect of pH and temperature was investigated. Vaccine samples were subjected to forced degradation conditions; pH and elevated temperatures. Stability results showed the ability of immunosensor to differentiate between intact and degraded viral content. This would demonstrate the reliability of the immunosensor for evaluating the efficacy and stability of the vaccine.

Rapid production of Plasmodium sporozoite detection paper dipstick assays using cellulose nanocrystals: Proof‐of‐concept for bio‐based, locally developed, point‐of‐care devices

AbstractEnhanced and rapid surveillance for diseases is critical to public health and meeting United Nations' Sustainable Development Goal for Good Health and Well‐being by allowing for targeted and accelerated prevention and control response strategies. Human malaria, caused by Plasmodium spp. and transmitted by mosquitoes is no exception. Advances in sustainable materials provide an opportunity to improve fast, sustainable, and equitable testing assays. Here, naturally abundant polymers and biomaterials, such as cellulose nanocrystals (CNCs) and chitosan, were used to increase antibody density deposition on the assay detection line when compared to traditional free antibody deposition, and thus the sensitivity, of easily assembled rapid tests designed to detect Plasmodium vivax infective (sporozoite) parasites in mosquitoes, a critical indicator of malaria transmission. The immobilization of antibodies onto chitosan‐coated CNCs allowed for antigen detection with a lower number of antibodies used in each test; likewise, the immobilization allowed to directly place the CNC‐Ab without the traditionally needed blockers layer on the paper like bovine serum albumin (BSA). This bio‐based prototype of a paper‐based dipstick assay shows a promising pathway for the development of rapid disease surveillance tools using sustainable and globally available materials.

Printed Capacitive Immunoassay for Detecting SARS-CoV-2 Viral Particles

The key to fight against a global pandemic such as COVID-19 is to have low-cost, reliable and fast response diagnostic tools. Electronic biosensors are preferred because of their ease of integration into current centralized health care networks and integration with modern point-of-care testing (POCT) devices. Printed electronic sensors provide a sensitive and reliable diagnostic platform to aid in controlling transmissible diseases. In this work, we demonstrate a fully printed capacitive biosensor. The sensor uses coplanar electrodes, coupled with capture antibodies immobilized on microporous Polyvinylidene-fluoride (PVDF) film to detect the SARS-CoV-2 spike protein in spiked buffer solutions. Antibody immobilization on PVDF surface is confirmed with confocal fluorescent imaging microscopy. Gold nanoparticle (GNP) tagged detection antibodies are also introduced to provide increased sensitivity. The gold nanoparticles provide a reflectance layer which leads to increased capacitance. This increased capacitance can be measured directly and has demonstrated the ability to screen for spiked samples with statistical significance. This fully printed capacitive immunoassay has the potential to be used as a transmissible disease screening and vaccine efficacy assessment tool for resource-limited areas.

A novel sandwich-type electrochemical immunosensor for sensitive detection of zearalenone using NG/PDDA/HNTs and Ti-MOF-KB composites for signal amplification

In this work, a novel sandwich-type electrochemical immunosensor based on signal amplification was developed for the ultrasensitive detection of zearalenone (ZEA). The composite consisting of poly(diallyldimethylammonium chloride) functionalized nitrogen doped graphene, halloysite nanotubes and toluidine blue (Tb/NG/PDDA/HNTs), was synthesized for the first time. Then it was modified with gold nanoparticles (AuNPs) to bind the secondary antibody (Ab2, 10 μg mL−1) and form the tracer label. In addition, ketjen black (KB) was doped into titanium-based metal–organic framework (Ti-MOF), which provided large specific surface area and employed as the sensing platform to increase the immobilization of the primary antibody (Ab1, 10 μg mL−1). This immunosensor showed a wide linear range for ZEA from 10 fg mL−1 to 100 ng mL−1 with a limit of detection (LOD) as low as 0.57 fg mL−1, which was below the maximum tolerable levels (50∼100 µg kg−1) set by the United Nations Food and Agriculture Organization (FAO).

One-Stop Plasmonic Nanocube-Excited SERS Immunoassay Platform of Multiple Cardiac Biomarkers for Rapid Screening and Progressive Tracing of Acute Myocardial Infarction.

Rapid and precise acute myocardial infarction (AMI) diagnosis is essential for preventing patient death. In addition, the complementary roles of creatine kinase muscle brain (CK-MB) and cardiac troponin I (cTnI) cardiac biomarkers in the early and late stages of AMI demand their simultaneous detection, which is difficult to implement using conventional fluorescence and electrochemical technologies. Here, a nanotechnology-based one-stop immuno-surface-enhanced Raman scattering (SERS) detection platform is reported for multiple cardiac indicators for the rapid screening and progressive tracing of AMI events. Optimal SERS is achieved using optical property-based, excitation wavelength-optimized, and high-yield anisotropic plasmonic gold nanocubes. Optimal immunoassay reaction efficiencies are achieved by increasing immobilized antibodies. Multiple simultaneous detection strategies are implemented by incorporating two different Raman reports with narrow wavenumbers corresponding to two indicators and by establishing a computational SERS mapping process to accurately detect their concentrations, irrespective of multiple enzymes in the human serum. The SERS platform precisely estimated AMI onset and progressive timing in human serum and made rapid AMI identification feasible using a portable Raman spectrometer. This integrated platform is hypothesized to significantly contribute to emergency medicine and forensic science by providing timely treatment and observation.

A highly sensitive immunosensor based on nanochannel-confined nano-gold enhanced electrochemiluminescence for procalcitonin detection.

Sensitive detection of procalcitonin (PCT) in serum is crucial for the timely diagnosis and treatment of rheumatoid arthritis. In this work, an electrochemiluminescence (ECL) detection platform is developed based on in-situ growth of Au nanoparticles (AuNPs) in nanochannels and an analyte-gated detection signal, which can realize ECL determination of PCT with high sensitivity. Vertically ordered mesoporous silica films with amine groups and uniform nanochannel array (NH2-VMSF) is easily grown on the supporting indium tin oxide (ITO) electrode through electrochemical assisted self-assembly method (EASA). Anchored by the amino groups, AuNPs were grown in-situ within the nanochannels to catalyze the generation of reactive oxygen species (ROS) and amplify the ECL signal of luminol. An immuno-recognitive interface is constructed on the outer surface of NH2-VMSF, through covalent immobilization of PCT antibodies. In the presence of PCT, the immunocomplex will hinder the diffusion of luminol and co-reactants, leading to a gating effect and decreased ECL signals. Based on this principle, the immunosensor can detect PCT in the range from 10pg/mL to 100ngmL-1 with a limit of detection (LOD) of 7pgmL-1. The constructed immunosensor can also be used for detecting PCT in serum. The constructed sensor has advantages of simple fabrication and sensitive detection, demonstrating great potential in real sample analysis.

Optimization of the immunorecognition layer towards Brucella sp. on gold surface for SPR platform

A successful immunosensor is characterized by a proper antibody immobilization and orientation in order to enhance the antigen recognition. In this work, a thorough characterization of the antibody functionalized gold surface is performed to set up the best conditions to implement in an optical platform for the detection of Brucella sp. Two different strategies are evaluated, based on a random immobilization and on an oriented one: a direct antibody immobilization on carboxylic mixed polyethylene (PEG) self-assembled monolayer (SAM) or only carboxylic PEG SAM interface is compared to an oriented immobilization on a layer of protein G on the same PEG SAM interfaces. X-ray Photoelectron Spectroscopy (XPS), Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS) and contact angle (CA) are used to chemically characterize the gold functionalized surface and ToF-SIMS is also used to confirm the right antibody orientation. Optical characterization is applied to monitor the functionalization steps and fluorescence measurements are used to set up the proper experimental conditions and also to detect Brucella bacteria on the surface. Best results are obtained with a 10 ng/μl incubation solution of antibody immobilized, in an oriented way, on a mixed PEG SAM interface.

Self-Assembly Multivalent Fluorescence-Nanobody Coupled Multifunctional Nanomaterial with Colorimetric Fluorescence and Photothermal to Enhance Immunochromatographic Assay.

The multimodal lateral flow immunoassay (LFIA) has provided accurate and reliable results for fast and immediate detection. Nonetheless, multimodal LFIA remains challenging to develop biosensors with high sensitivity and tolerance to matrix interference in agro-food. In this study, we developed a self-assembled multivalent fluorescence-nanobody (Nb26-EGFP-H6) with 16.5-fold and 30-fold higher affinity and sensitivity than a monovalent nanobody (Nb26). Based on the Nb26-EGFP-H6, we synthesized enhanced immune-probes Zn-CN@Nb26-EGFP-H6 by pyrolyzing and oxidizing an imidazolating zeolite framework-8 (ZIF-8) to obtain photothermal metal-carbon nanomaterials (Zn-CN) for immobilizing Nb26-EGFP-H6. The rough and porous structure of Zn-CN with a large surface area facilitates the enrichment and immobilization of antibodies. A trimodal lateral flow immunoassay (tLFIA) with colorimetric, fluorescent, and photothermal triple signal outputs was constructed for the detection of aflatoxin B1 (AFB1) in maize. Attractively, the Zn-CN-based tLFIA's multiplex guarantees accurate and sensitive detection of AFB1, with triple signal detection limits of 0.0012 ng/mL (colorimetric signals), 0.0094 ng/mL (fluorescent signals), and 0.252 ng/mL (photothermal signals). The sensitivity of the trimode immunosensor was 628-fold and 42-fold higher than that of the original Nb26-based ELISA (IC50) and the unimodal LFIA (LOD). This work provides an idea for constructing a sensitive, tolerant matrix and efficient and accurate analytical platform for rapidly detecting AFB1 in food.

Well-oriented immobilized immunoaffinity magnetic beads for detection of fumonisins in grains and feeds via pre-column automatic derivatization of high-performance liquid chromatography.

In this study, based on the high-throughput automatic sample pretreatment with immunoaffinity magnetic beads with oriented immobilized antibodies, grain and feed fumonisin (FB) content was detected using pre-column automatic derivatization of high-performance liquid chromatography (HPLC). The FB capacity of well-oriented antibody immunoaffinity magnetic beads was 1.5-1.8 times that of magnetic beads with randomly fixed antibody. This pre-column automatic derivatization method using an autosampler can reduce error from manual injection and improve detection efficiency. The spiked recoveries for three different concentrations in maize, husked rice, and pig feed under optimized conditions were 84.6-104.0% (RSD<9.3%). Our novel method was also applied to the analysis of FBs in 63 maize samples collected from the main maize-production regions in China. The results showed that as latitude increased, the contamination level of FBs tended to decrease. High temperature and high humidity are also more favorable for FB growth.

Nanodot zirconium trisulfide modified conducting thread: A smart substrate for fabrication of next generation biosensor

In present work, we report an eco-friendly, flexible and highly conducting cotton thread (CT) as a smart substrate for the development of biosensing platform towards ultrasensitive detection of swine flu serum amyloid A (SAA) biomarker. The biosensor was fabricated by optimized coating of CT with poly(3,4-ethylenedioxythiophene): poly(styrenesulfonate) (PEDOT:PSS) conductive ink followed by incorporation of nanodot zirconium trisulfide (nZrS3) which helped in enhancing the electrochemical properties and improving stability of PEDOT:PSS polymeric film. The fabricated nZrS3/PEDOT:PSS/CT electrode was then used for sequential immobilization of monoclonal antibodies of SAA (anti-SAA) and bovine serum albumin (BSA). The synthesized nanomaterials and fabricated electrodes were characterized through X-ray diffraction, Fourier-transform infrared spectroscopy, transmission electron microscopy, scanning electron microscopy and contact angle analyser techniques. The electrochemical response of the fabricated smart thread based biosensor (BSA/anti-SAA/ZrS3/PEDOT:PSS/CT) was recorded against SAA using chronoamperometry technique which revealed superior sensitivity {30.2 μA [log (μg mL−1)]−1 cm−2}, excellent lower detection limit (0.72 ng mL−1) and prolonged shelf life up to 48 days. The response of the biosensor was also validated by analysing the electrochemical response of SAA spiked serum samples and the obtained results showed good correlation with that of standard samples.

A-365 Carbohydrate-binding Module Proteins to Functionalize Paper for Lateral Flow Applications

Abstract Background Lateral flow (LF) tests have employed nitrocellulose membrane since their introduction. Refinement of nitrocellulose and LF production techniques have led to substantial improvements in reproducibility, sensitivity, clarity, and speed of results. The recent surge in demand for LF tests during the COVID-19 pandemic has drawn increased attention to the consequences of using non-biodegradable or non-recyclable materials in test production, requiring disposal via landfill. The nitrocellulose substrate is a critical component for substitution with an equivalent or superior material to address environmental concerns. Additionally, nitrocellulose and the specific molecular recognition reagents, typically antibodies, contribute significantly to the test cost, and whilst LF tests are less expensive than most other formats, challenges remain with respect to providing affordable tests with adequate performance globally in low resource environments. Despite attempts to substitute cellulose for nitrocellulose, principally for reducing cost and more latterly, environmental impact, cellulose is not widely used due to inferior performance, especially in relation to the clarity and quality of the signal line and reagent requirements. Here, we present preliminary data supporting the use of cellulose membrane (paper) as an alternative substrate to nitrocellulose for LF assays. We demonstrate that cellulose-binding module (CBM)-streptavidin fusion proteins for the immobilisation of capture antibodies on cellulose addresses the performance limitations, yielding sensitivity, clarity and speed of result matching current clinical and industry requirements for a qualitative SARS-CoV-2 antigen LF test. Methods We expressed and purified CBM-streptavidin fusion proteins from E.coli to a purity of &amp;gt;90%. Binding to biotinylated proteins and cellulose material was confirmed. An inhouse SARS-CoV-2 antigen assay was adapted for use with the CBM-streptavidin fusion protein on cellulose material. Using methods compatible with current LF test production, we conjugated and dried down 40nm gold (BBI) with anti-nucleocapsid antibodies (Hytest). The biotinylated anti-nucleocapsid capture antibody was dried down on a separate pad. CBM-streptavidin fusion protein was plotted on Whatman 43 paper at the test line. We evaluated assay performance using SARS-CoV-2 nucleocapsid protein and UV-inactivated SARS-CoV-2 virus (NIBSC) in artificial mucus. We further evaluated accelerated stability of the CBM-paper system at 45°C. Results Plotting of the CBM-fusion protein on the test line (0.3 mg/mL, 1 uL/cm) resulted in a tightly focused line, while the control line antibody, without CBM-fusion protein (1 mg/mL, 1 uL/cm), diffused from its deposition line. Additionally, we were able to substantially reduce the amount of capture antibody needed using the CBM-fusion protein system. The cellulose-based assay exhibited a runtime of 30 minutes and an analytical sensitivity of 10^3 pfu/mL. No significant changes in performance were observed after 9 weeks of incubation at 45°C, representing an approximate accelerated shelf-life of 12 months. Conclusion Our results indicate that paper-based tests match sensitivity, clarity, and speed of current qualitative antigen tests on nitrocellulose, with a shelf-life of at least one year. This preliminary investigation demonstrates the viability of using paper as a replacement for nitrocellulose when coupled with CBM-fusion proteins for antibody immobilisation, offering significantly reduced material cost and environmental impact.

Development of an ID-LC–MS/MS method using targeted proteomics for quantifying cardiac troponin I in human serum

BackgroundCardiac troponin is a complex protein consisting of the three subunits I, T and C located in heart muscle cells. When the heart muscle is damaged, it is released into the blood and can be detected. Cardiac troponin I (cTnI) is considered the most reliable and widely accepted test for detecting and confirming acute myocardial infarction. However, there is no current standardization between the commercial assays for cTnI quantification. Our work aims to create a measurement procedure that is traceable to the International System of Units for accurately measuring cardiac cTnI levels in serum samples from patients.MethodsThe workflow begins with immobilizing anti-cTnI antibodies onto magnetic nanoparticles to form complexes. These complexes are used to isolate cTnI from serum. Next, trypsin is used to enzymatically digest the isolated cTnI. Finally, the measurement of multiple cTnI peptides is done simultaneously using isotope dilution liquid chromatography–tandem mass spectrometry (ID-LC–MS/MS).ResultsThe maximum antibody immobilization was achieved by combining 1 mg of nanoparticles with 100 μg of antibody, resulting in an average of 59.2 ± 5.7 μg/mg of immobilized antibody. Subsequently, the anti-cTnI-magnetic nanoparticle complex was utilized to develop and validate a method for quantifying cTnI in human serum using ID-LC–MS/MS and a protein calibration approach. The analytical method was assessed regarding linearity and recovery. The developed method enables the quantification of cTnI from 0.7 to 24 μg/L (R > 0.996). The limit of quantification was 1.8 μg/L and the limit of detection was 0.6 μg/L. Intermediate precision was ≤ 9.6% and repeatability was 2.0–8.7% for all quality control materials. The accuracy of the analyzed quality control materials was between 90 and 110%. Total measurement uncertainties for target value assignment (n = 6) were found to be ≤ 12.5% for all levels.ConclusionsThe analytical method demonstrated high analytical performance in accurately quantifying cardiac troponin I levels in human serum. The proposed analytical method has the potential to facilitate the harmonization of cTnI results between clinical laboratories, assign target values to secondary certified reference materials and support reliable measurement of cTnI.Graphical

Boosting Electrochemiluminescence Immunoassay Sensitivity via Co-Pt Nanoparticles within a Ti3C2 MXene-Modified Single Electrode Electrochemical System on Raspberry Pi.

Point-of-care testing plays a crucial role in diagnostics within resource-poor areas, necessitating the utilization of portable and user-friendly devices. The adaptation of biosensors for point-of-care applications requires careful considerations, such as miniaturization, cost-effectiveness, and streamlined sample processing. In recent years, the electrochemiluminescence (ECL) immunoassay has gained significant attention due to its visual detection capabilities and ability to facilitate high-throughput analysis. However, the development of a practical and cost-effective ECL device remains a challenging task. This study presents the development of an integrated MXene-modified single-electrode electrochemical system (SEES) for visual and high-throughput ECL immunoassays incorporating a Raspberry Pi system. The SEES was designed by affixing a plastic sticker with multiple perforations onto a single carbon ink screen-printed electrode, which operates based on a resistance-induced potential difference. Leveraging the excellent adsorption and bioaffinity properties of the carbon ink screen-printed electrode, effective immobilization of antibodies was achieved. Furthermore, the incorporation of Co-Pt nanoparticles enhanced the ECL intensity and electron transfer kinetics, enabling the sensitive detection of SARS-CoV-2. The developed system comprised 18 individual reaction cells, allowing for simultaneous analysis while maintaining sample isolation. Impressively, the system achieved a remarkable minimum virus detection limit of 10-14 g mL-1, accompanied by a high R2 value of 0.9798. These findings highlight the promising potential of our developed system for efficient point-of-care testing in resource-limited settings.

Guided mode resonance immunosensor for label-free detection of pathogenic bacteria Pseudomonas aeruginosa

Photonic biosensors are promising platforms for the rapid detection of pathogens with the potential to replace conventional diagnostics based on microbiological culturing methods. Intricately designed sensing elements with robust architectures can offer highly sensitive detection at minimal development cost enabling rapid adoption in low-resource settings. In this work, an optical detection scheme is developed by structuring guided mode resonance (GMR) on a highly stable, transparent silicon nitride (SiN) substrate and further biofunctionalized to identify a specific bacteria Pseudomonas aeruginosa. The resonance condition of the GMR chip is optimized to have relatively high bulk sensitivity with a good quality factor. The biofunctionalization aims at oriented immobilization of specific antibodies to allow maximum bacteria attachment and improved specificity. The sensitivity of the assays is evaluated for clinically relevant concentrations ranging from 102 to 108 CFU/mL. From the calibration curves, the sensitivity of the chip is extracted as 0.134nm/Log10 [concentration], and the detection modality possesses a favorably good limit of detection (LOD) 89 CFU/mL. The use of antibodies as a biorecognition element complemented with a good figure of merit of GMR sensing element allows selective bacteria identification compared to other non-specific pathogenic bacteria that are relevant for testing physiological samples. Our developed GMR biosensor is low-cost, easy to handle, and readily transformable into a portable handheld detection modality for remote usage.

Boron-doped diamond nanosheet volume-enriched screen-printed carbon electrodes: a platform for electroanalytical and impedimetric biosensor applications

This paper focuses on the development of a novel electrode based on boron-doped diamond nanosheet full-volume-enriched screen-printed carbon electrodes (BDDPE) for use as an impedimetric biosensor. Impedimetric biosensors offer high sensitivity and selectivity for virus detection, but their use as point-of-care devices is limited by the complexity of nanomaterials’ architecture and the receptor immobilisation procedures. The study presents a two-step modification process involving the electroreduction of diazonium salt at the BDDPE and the immobilisation of antibodies using zero-length cross-linkers for a selective impedimetric biosensor of Haemophilus influenzae (Hi). The incorporation of diamond nanosheets into BDDPE leads to enhanced charge transfer and electrochemical behaviour, demonstrating greatly improved electrochemically active surface area compared with unmodified screen-printed electrodes (by 44% and 10% on average for [Ru(NH3)6]Cl2 and K3[Fe(CN)6], respectively). The presented sensing system shows high specificity towards protein D in Hi bacteria, as confirmed by negative controls against potential interference from other pathogens, with an estimated tolerance limit for interference under 12%. The Hi limit of detection by electrochemical impedance spectroscopy was 1 CFU/mL (measured at − 0.13 V vs BDDPE pseudo-reference), which was achieved in under 10 min, including 5 min sample incubation in the presence of the analyte.Graphical abstract

Strategies and Applications of Graphene and Its Derivatives-Based Electrochemical Sensors in Cancer Diagnosis.

Graphene is an emerging nanomaterial increasingly being used in electrochemical biosensing applications owing to its high surface area, excellent conductivity, ease of functionalization, and superior electrocatalytic properties compared to other carbon-based electrodes and nanomaterials, enabling faster electron transfer kinetics and higher sensitivity. Graphene electrochemical biosensors may have the potential to enable the rapid, sensitive, and low-cost detection of cancer biomarkers. This paper reviews early-stage research and proof-of-concept studies on the development of graphene electrochemical biosensors for potential future cancer diagnostic applications. Various graphene synthesis methods are outlined along with common functionalization approaches using polymers, biomolecules, nanomaterials, and synthetic chemistry to facilitate the immobilization of recognition elements and improve performance. Major sensor configurations including graphene field-effect transistors, graphene modified electrodes and nanocomposites, and 3D graphene networks are highlighted along with their principles of operation, advantages, and biosensing capabilities. Strategies for the immobilization of biorecognition elements like antibodies, aptamers, peptides, and DNA/RNA probes onto graphene platforms to impart target specificity are summarized. The use of nanomaterial labels, hybrid nanocomposites with graphene, and chemical modification for signal enhancement are also discussed. Examples are provided to illustrate applications for the sensitive electrochemical detection of a broad range of cancer biomarkers including proteins, circulating tumor cells, DNA mutations, non-coding RNAs like miRNA, metabolites, and glycoproteins. Current challenges and future opportunities are elucidated to guide ongoing efforts towards transitioning graphene biosensors from promising research lab tools into mainstream clinical practice. Continued research addressing issues with reproducibility, stability, selectivity, integration, clinical validation, and regulatory approval could enable wider adoption. Overall, graphene electrochemical biosensors present powerful and versatile platforms for cancer diagnosis at the point of care.

Development of a high-performance label-free electrochemical immunosensor for early cancer diagnosis using anti-CEA/Ag-MOF/GO/GCE nanocomposite

In order to detect carcinoembryonic antigen (CEA) as a tumor marker in lung cancer for early cancer diagnosis, this study aimed to develop a label-free electrochemical immunosensor based on the immobilization of an Anti-CEA antibody on a metal-organic framework (MOF)-graphene oxide nanocomposite modified glassy carbon electrode (Anti-CEA/Ag-MOF/GO/GCE). Ag-MOF/GO nanocomposite was prepared on the GCE surface using the ultrasonic irradiation method, and Anti-CEA antibody was subsequently immobilized on the surface. Analysis of the crystal structure and morphology of the modified electrode using FE-SEM and XRD revealed that the correct combination of GO nanosheets and Ag-MOF nanoparticles produced a high surface area to trap the antibodies. Electrochemical tests utilizing the CV and DPV methods revealed that the immunosensor's sensitivity, stability, and selectivity were improved by Anti-CEA/Ag-MOF/GO/GCE. Results showed that, with a detection limit of 0.005 ng/mL, the change in the reduction peak current was inversely correlated with the logarithm concentration of CEA in the range of 10-3 to 5000 ng/mL. The suggested CEA immunosensor's applicability in a human serum sample was investigated, and findings of analytical studies via standard addition technique for both ELISA and DPV assays revealed that significant agreement existed between the outcomes of the two assays. Additionally, the recoveries ranged from 99.00% to 99.25%, and all relative standard deviations (RSDs) for the sample detections were below 5.01%, indicating satisfactory accuracy in results measured with the proposed CEA immunosensor, indicating that the prepared CEA immunosensor in this study can be used in clinical applications and human fluids.

Achieving enhanced sensitivity and accuracy in carcinoembryonic antigen (CEA) detection as an indicator of cancer monitoring using thionine/chitosan/graphene oxide nanocomposite-modified electrochemical immunosensor

The current study has focused on electrochemical immunosensing of carcinoembryonic antigen (CEA) employing an immobilized antibody on a thionine, chitosan, or graphene oxide nanocomposite modified glassy carbon electrode (anti-CEA/THi–CS–GO/GCE) as an indicator of cancer monitoring. THi–CS–GO nanocomposites were made using ultrasonication, and analyses of their morphology and crystal structure using SEM, FTIR, and XRD showed that thionine and chitosan molecules were intercalated with stacking interactions with both the top and bottom of GO nanosheets. Electrochemical experiments revealed anti-CEA, THi–CS–GO/GCE to have exceptional sensitivity and selectivity towards CEA compounds. The detection limit value was established to be 0.8 pg/mL when it was discovered that variations in the decrease peak current were directly proportional to the logarithm concentration of CEA over a wide range from 10-3 to 104 ng/mL. Results of testing the immunosensor's application capability for detecting CEA in a sample of human serum show that ELISA and DPV results are very congruent. The produced immunosensor demonstrated adequate immunosensor precision in determining CEA in prepared genuine samples of human serum and clinical applications.