Development Of Electrochemical Immunosensor Research Articles

Overview on the Development of Electrochemical Immunosensors by the Signal Amplification of Enzyme- or Nanozyme-Based Catalysis Plus Redox Cycling.

Enzyme-linked electrochemical immunosensors have attracted considerable attention for the sensitive and selective detection of various targets in clinical diagnosis, food quality control, and environmental analysis. In order to improve the performances of conventional immunoassays, significant efforts have been made to couple enzyme-linked or nanozyme-based catalysis and redox cycling for signal amplification. The current review summarizes the recent advances in the development of enzyme- or nanozyme-based electrochemical immunosensors with redox cycling for signal amplification. The special features of redox cycling reactions and their synergistic functions in signal amplification are discussed. Additionally, the current challenges and future directions of enzyme- or nanozyme-based electrochemical immunosensors with redox cycling are addressed.

Progress in Electrochemical Immunosensors with Alkaline Phosphatase as the Signal Label.

Electrochemical immunosensors have shown great potential in clinical diagnosis, food safety, environmental protection, and other fields. The feasible and innovative combination of enzyme catalysis and other signal-amplified elements has yielded exciting progress in the development of electrochemical immunosensors. Alkaline phosphatase (ALP) is one of the most popularly used enzyme reporters in bioassays. It has been widely utilized to design electrochemical immunosensors owing to its significant advantages (e.g., high catalytic activity, high turnover number, and excellent substrate specificity). In this work, we summarized the achievements of electrochemical immunosensors with ALP as the signal reporter. We mainly focused on detection principles and signal amplification strategies and briefly discussed the challenges regarding how to further improve the performance of ALP-based immunoassays.

Nanostructure-Based Electrochemical Immunosensors as Diagnostic Tools

Electrochemical immunosensors are affinity-based biosensors characterized by several useful features such as specificity, miniaturizability, low cost and simplicity, making them very interesting for many applications in several scientific fields. One of the significant issues in the design of electrochemical immunosensors is to increase the system’s sensitivity. Different strategies have been developed, one of the most common is the use of nanostructured materials as electrode materials, nanocarriers, electroactive or electrocatalytic nanotracers because of their abilities in signal amplification and biocompatibility. In this review, we will consider some of the most used nanostructures employed in the development of electrochemical immunosensors (e.g., metallic nanoparticles, graphene, carbon nanotubes) and many other still uncommon nanomaterials. Furthermore, their diagnostic applications in the last decade will be discussed, referring to two relevant issues of present-day: the detection of tumor markers and viruses.

Rapid fabrication of homogeneously distributed hyper-branched gold nanostructured electrode based electrochemical immunosensor for detection of protein biomarkers

In recent years, need for rapid, reliable, and low-cost protein biomarker screening has tremendously boosted research into the development of electrochemical immunosensors. In this line, here we report on rapid fabrication and proof-of-concept demonstration of a hyper-branched gold nanostructure (ns@gold) based electrochemical immunosensor for detection of protein biomarker at pg/mL level. We have optimized a simple direct electrochemical process to rapidly generate uniformly distributed high surface area ns@gold within 30 min. These 3D ns@gold show ∼ 8-times enhancement in conductivity and also a rapid surface functionalization via self-assembled monolayers (SAM) of mercaptoundecanoic acid within 45 min. Protein estimation data showed around 94 % immobilization efficiency on the ns@gold surface in comparison to 28.4 % on plain gold surface. A fast SAM formation and quick antibody immobilization steered the assay timing within 4 h. As a proof-of-concept, we used an antibody-antigen interaction for the detection of epithelial growth factor receptor (EGFR) protein. Voltammetric data for the target EGFR receptor resulted a linear working range of 10 pg/mL to 100 ng/mL with a low limit-of-detection of 6.9 pg/mL. High selectivity in detecting target proteins from a pool of non-target proteins using breast cancer cell lines was also demonstrated along with good stability and reusability.

Nanomaterials-based Electrochemical Immunosensors.

With the development of nanomaterials and sensor technology, nanomaterials-based electrochemical immunosensors have been widely employed in various fields. Nanomaterials for electrode modification are emerging one after another in order to improve the performance of electrochemical immunosensors. When compared with traditional detection methods, electrochemical immunosensors have the advantages of simplicity, real-time analysis, high sensitivity, miniaturization, rapid detection time, and low cost. Here, we summarize recent developments in electrochemical immunosensors based on nanomaterials, including carbon nanomaterials, metal nanomaterials, and quantum dots. Additionally, we discuss research challenges and future prospects for this field of study.

Suppressing Non-Specific Binding of Proteins onto Electrode Surfaces in the Development of Electrochemical Immunosensors.

Electrochemical immunosensors, EIs, are systems that combine the analytical power of electrochemical techniques and the high selectivity and specificity of antibodies in a solid phase immunoassay for target analyte. In EIs, the most used transducer platforms are screen printed electrodes, SPEs. Some characteristics of EIs are their low cost, portability for point of care testing (POCT) applications, high specificity and selectivity to the target molecule, low sample and reagent consumption and easy to use. Despite all these attractive features, still exist one to cover and it is the enhancement of the sensitivity of the EIs. In this review, an approach to understand how this can be achieved is presented. First, it is necessary to comprise thoroughly all the complex phenomena that happen simultaneously in the protein-surface interface when adsorption of the protein occurs. Physicochemical properties of the protein and the surface as well as the adsorption phenomena influence the sensitivity of the EIs. From this point, some strategies to suppress non-specific binding, NSB, of proteins onto electrode surfaces in order to improve the sensitivity of EIs are mentioned.

Enhanced conductivity of rGO/Ag NPs composites for electrochemical immunoassay of prostate-specific antigen

Electrode materials play a vital role in the development of electrochemical immunosensors (EIs), particularly of label-free EIs. In this study, composites containing reduced graphene oxide with silver nanoparticles (rGO/Ag NPs) were synthesized using binary reductants, i.e. hydrazine hydrate and sodium citrate. Due to the fact that graphene oxide (GO) was fully restored to rGO, and rGO stacking was effectively inhibited by insertion of small Ag NPs between the graphene sheets, the electrical conductivity of rGO/Ag NPs composites was significantly improved compared to rGO alone, with an enhancement factor of 346% at 40wt% of rGO. Moreover, the conducting path between rGO and Ag NPs formed because the structural defects in rGO were effectively repaired by decoration with Ag NPs. Subsequently, based on a screen-printed three-electrode system, a label-free EI for detecting prostate-specific antigen (PSA) was constructed using rGO/Ag NPs composites as a support material. The fabricated EIs demonstrated a wide linear response range (1.0–1000ng/ml), low detection limit (0.01ng/ml) and excellent specificity, reproducibility and stability. Thus, the proposed EIs based on rGO/Ag NPs composites can be easily extended for the ultrasensitive detection of different protein biomarkers.

Special Issue for Electrochemical Immunosensors – State of the Art

There is a huge demand for fast, reliable and low-cost analytical systems for the detection and quantification of proteins with relevance in medicine, environmental pollutant monitoring, food safety, detection of foodborne pathogens and bioterrorism. Indeed, the development of biosensors fulfilling the requirements of adequate sensitivity and selectivity as well as convenience of use and cost is one of the grand scientific, engineering, and educational challenges of the 21st century. In this context, electrochemical immunosensors are considered particularly attractive analytical tools as a consequence of their inherent high sensitivity, simplicity of the operational procedure, easy availability and affordable cost of the required equipment together with possibility of miniaturization and suitability for in-field applications. Electrochemical immunosensors seek to exploit the great potentiality of immunoreactions in conjunction with electroanalytical techniques in a broad context facing a wide variety of analytical problems in medicine, biomedical research, drug discovery, environment, food, process industries, security and defense. The main goal is to provide a selective and fast response to the presence of a specific compound in a complex mixture of components, without perturbing the system. Interestingly, immunosensing using electroanalytical techniques is playing a more and more important role in protein analysis. Moreover, the growing research in advanced nanomaterials has impressively impacted also this field, by providing a great variety of novel, versatile and rationally designed nanostructured supports for transduction, signal generation and amplification. In parallel with these major advances in nanotechnology, the wide variety of immunoreagents available (commercial or produced by genetic recombination on demand conjugated with a variety of tags according to the required needs) and the versatility of design and modification offered by electrochemical substrates have also played major roles in the outstanding capabilities demonstrated by these electrochemical biosensors. The plethora of sophisticated approaches developed has exceeded even the experts' expectations in terms of functionality and resiliency. Due to the high demand of the world market and human interest for having devices able to provide the concentration of species in different complex samples, in a simple and fast way, a hard competition on this field has occurred among the researchers in recent years. In addition, the excellent and solid academic and practical multidisciplinary formation in sensors biotechnology, surface chemistry, advanced nanomaterials, and electrochemical transduction and characterization methods for immunodetection of these researchers amply justifies the unimaginable progress reached in such a short time and the fact that people working in this area do not dare to put limits on this field. The present Special Issue, which compiles 9 full papers, 2 short communications and 8 dedicated review articles, was authored by leading experts and pioneers in electrochemical immunosensors. These articles shed useful insights into the latest advances, current trends and future prospects in this exciting field. In particular, the selected contributions described the development of electrochemical immunosensing scaffolds to detect pollutants (polybrominated diphenyllethers, mycotoxins), clinical biomarkers (interleukin-8, estrogen and progesterone receptors), drugs (brombuterol), rotavirus and microbes and revise nicely the use of screen-printed electrodes, common and uncommon nanostructures, polymeric films, 3D-printing microfluidics and proximity ligation assays in the development of electrochemical immunosensors. Compiled contributions give also expert and updated overviews of this topic in multiplexed approaches, control of electron transfer, advantages compared with aptasensors and latest applications in real clinical practice. Therefore, one can envision further exciting applications of electrochemical immune-platforms for making ‘house-calls' in biomedical diagnostics and cancer theranostics, as inspectors for environmental monitoring, even in harsh working conditions, as well as alarm devices for food safety. The unique combination and integration of nanotechnology, micro and nanofluidics with immunoreactions and electrochemical analytical methodologies is expected to produce major advances in this area and open up new opportunities not only from the scientific point of view but even from a market perspective preparing point-of-care devices and in-situ alarm systems. Given the rapid development, interest and progress made in this field, the examples compiled in this Special Issue are just a small sample of those expected to come in this amazing field which future growth and success will rely on the abilities of the researchers to continue innovating and collaborating to address the existing challenges and opportunities. In the near future, advances in fundamental knowledge of new nanomaterials along with a focus on practical applications in real-world systems will drive electrochemical immunosensors to breakthroughs in many fields of social and economical relevance. As a logical consequence of the incessant flow of impressive ideas and innovations it looks like this field has the horsepower to keep on advancing for the foreseeable future.

Strategic Applications of Nanomaterials as Sensing Platforms and Signal Amplification Markers at Electrochemical Immunosensors

AbstractTremendous developments in electrochemical immunosensors have facilitated cost‐effective, rapid and robust detection of many analytes. Among the significant developments, electrochemical immunosensors capable of simultaneously detecting several analytes have begun to emerge. In addition, recent progress in materials science and engineering has yielded nanostructured materials of various geometries that were readily exploited to provide a compatible medium for many biomolecules in immunoassays. Applications of these nanostructured materials to electrochemical immunosensor developments have significantly improved sensor performance in terms of their electrical, chemical and transport properties. This review provides a survey of innovative immunosensor developments involving a range of nanomaterials exploited in designing sensing platforms and efficient detection markers to achieve substantial signal amplifications and thereby outstanding detection characteristics.

Proximity Ligation Assay‐induced Structure‐switching Hairpin DNA toward Development of Electrochemical Immunosensor

AbstractA novel electrochemical immunosensor was designed for sensitive detection of mycotoxins (aflatoxin B1, AFB1, used in this case) by coupling proximity ligation assay‐induced conformation switch of hairpin DNA with the antigen‐antibody reaction. The assay was carried out by anti‐AFB1 antibody‐conjugated DNA1 (mAb‐DNA1), AFB1‐BSA‐labeled DNA2 (AFB1‐DNA2) and hairpin DNA. Each hairpin included a stem of 6 base pairs and a 6 nucleotide (nt) loop with the labelled ferrocene tag at the 3′ end, which was immobilized on the electrode via self‐assembly of the terminal thiol moiety at the 5′ end. The proximity ligation assay was carried out via the specific antigen‐antibody reaction between mAb‐DNA1 and AFB1‐DNA2 to form an omega‐like DNA junction. Thereafter, the junction hybridized with hairpin DNA to open the probe, thus resulting in the ferrocene tag far away from the electrode for the decreasing of the redox current. Upon target AFB1 introduction, the competitive‐type immunoassay was executed between the analyte and AFB1‐DNA2 for mAb‐DNA1. Under the optimal conditions, the electrochemical signal was indirectly proportional to target AFB1 concentration, and allowed the detection of target AFB1 at a concentration as low as 3.2 pg mL−1. Our strategy also exhibited high selectivity, good reproducibility and precision. Importantly, the accuracy of this methodology was validated for analysis of spiked or naturally contaminated peanut samples, giving results matched well with those obtained from commercialized available AFB1 ELISA kit.

Copper-doped titanium dioxide nanoparticles as dual-functional labels for fabrication of electrochemical immunosensors

Constructions of versatile electroactive labels are key issues in the development of electrochemical immunosensors. In this study, copper-doped titanium dioxide nanoparticle (Cu@TiO2) was synthesized and used as labels for fabrication of sandwich-type electrochemical immunosensors on glassy carbon electrode (GCE). Due to the presence of copper ions, Cu@TiO2 shows a strong response current when coupled to an electrode. The prepared nanocomposite also shows high electrocatalytic activity towards reduction of hydrogen peroxide (H2O2). The dual functionality of Cu@TiO2 enables the fabrication of immunosensor using different detection modes, that is, square wave voltammetry (SWV) or chronoamperometry (CA). While Cu@TiO2 was used as labels of secondary antibodies (Ab2), carboxyl functionalized graphene oxide (CFGO) was used as electrode materials to immobilize primary antibodies (Ab1). Using human immunoglobulin G (IgG) as a model analyte, the immunosensor shows high sensitivity, acceptable stability and good reproducibility for both detection modes. Under optimal conditions, a linear range from 0.1pg/mL to 100ng/mL with a detection limit of 0.052pg/mL was obtained for SWV analysis. For CA analysis, a wider linear range from 0.01pg/mL to 100ng/mL and a lower detection limit of 0.0043pg/mL were obtained. The proposed metal ion-based enzyme-free and noble metal-free immunosensor may have promising applications in clinical diagnoses and many other fields.

Electrochemical immunosensors for detection of cancer protein biomarkers.

Bioanalytical methods have experienced unprecedented growth in recent years, driven in large part by the need for faster, more sensitive, more portable ("point of care") systems to detect protein biomarkers for clinical diagnosis. Electrochemical detection strategies, used in conjunction with immunosensors, offer advantages because they are fast, simple, and low cost. Recent developments in electrochemical immunosensors have significantly improved the sensitivity needed to detect low concentrations of biomarkers present in early stages of cancer. Moreover, the coupling of electrochemical devices with nanomaterials, such as gold nanoparticles, carbon nanotubes, magnetic particles, and quantum dots, offers multiplexing capability for simultaneous measurements of multiple cancer biomarkers. This review will discuss recent advances in the development of electrochemical immunosensors for the next generation of cancer diagnostics, with an emphasis on opportunities for further improvement in cancer diagnostics and treatment monitoring. Details will be given for strategies to increase sensitivity through multilabel amplification, coupled with high densities of capture molecules on sensor surfaces. Such sensors are capable of detecting a wide range of protein quantities, from nanogram to femtogram (depending on the protein biomarkers of interest), in a single sample.

The synthesis and characterization of biotin-silver-dendrimer nanocomposites as novelbioselective labels

This paper presents a synthesis of a novel nanoparticle label with selective biorecognitionproperties based on a biotinylated silver-dendrimer nanocomposite (AgDNC). Two types oflabels, a biotin-AgDNC (bio-AgDNC) and a biotinylated AgDNC with a poly(ethylene)glycolspacer (bio-PEG-AgDNC), were synthesized from a generation 7 (G7) hydroxyl-terminatedethylenediamine-core-type (2-carbon core) PAMAM dendrimer (DDM) by anN,N′-dicyclohexylcarbodiimide (DDC) biotin coupling and aNaBH4 silver reduction method. Synthesized conjugates were characterized by several analyticalmethods, such as UV–vis, FTIR, AFM, TEM, ELISA, HABA assay and SPR. Theresults show that stable biotinylated nanocomposites can be formed either withinternalized silver nanoparticles (AgNPs) in a DMM polymer backbone (‘typeI’) or as externally protected (‘type E’), depending on the molar ratio of thesilver/DMM conjugate and type of conjugate. Furthermore, the selective biorecognition function ofthe biotin is not affected by the AgNPs’ synthesis step, which allows a potentialapplication of silver nanocomposite conjugates as biospecific labels in variousbioanalytical assays, or potentially as fluorescence cell biomarkers. An exploitation of thepresented label in the development of electrochemical immunosensors is anticipated.

Comparative study of thiolated Protein G scaffolds and signal antibody conjugates in the development of electrochemical immunosensors

To achieve a high efficiency of analyte capture by a capture antibody attached to an electrochemical immunosensor, we have immobilised an analyte-specific antibody on a self-assembled layer of recombinant Protein G that was thiolated with succinimidyl-6-[3′-(2-pyridyldithio)-propionamido] hexanoate (LC-SPDP). Then two techniques were employed for conjugating a second antigen-specific antibody to alkaline phosphatase (mAb2-AP) using either LC-SPDP or the biotin–streptavidin interaction as the mode of cross-linking the antibody and enzyme. After characterising the two mAb2-AP preparations (mAb2-(LC-SPDP)-AP and mAb2-(Biotin-SA)-AP), they were each used as the signal antibody for immunosensors formatted for two-site immunoassays where the capture antibody was attached to a Protein G-(LC-SPDP) scaffold on gold electrodes. The antibodies and assays were specific for the clinically important hormone, human chorionic gonadotrophin (hCG). Protein G-(LC-SPDP) provided a stable scaffold, while mAb2-(LC-SPDP)-AP and mAb2-(Biotin-SA)-AP performed well as the signal antibodies. Immunosensors with mAb2-(Biotin-SA)-AP were characterised by a limit of detection of 216 IU L −1 for hCG and a linear response up to approximately 2000 IU L −1. Conversely, immunosensors with mAb2-(LC-SPDP)-AP exhibited a limit of detection of 240 IU L −1 and a linear response up to 4000 IU L −1.

Development of Electrochemical Immunosensor for Progesterone Analysis in Milk

A disposable electrochemical biosensor, based on a screen‐printed carbon electrode (SPE) coated with a progesterone (prog)–BSA conjugate, has been prepared and evaluated for measuring progesterone in cows' milk. The immunosensor was employed in an indirect competitive assay format involving anti‐progesterone monoclonal antibody and anti‐species antibody labeled with the enzyme alkaline phosphatase (AP). Differential pulse voltammetry (DPV) and amperometry were used as electrochemical means to detect the product of the enzymatic reaction [p‐aminophenol (p‐AP)]. Amperometric detection was carried out at +350 mV vs. Ag/AgCl reference electrode. The DPV detection was in the potential range of +100 to +500 mV vs. Ag/AgCl reference electrode. Progesterone was detectable in milk matrix by sigmoidal curve (variable slope) method from 13 to 189 ng/mL and from a linear calibration between 16 and 256 ng/mL with an associated limit of detection (LOD) of 3 ± 2 ng/mL progesterone. The use of DPV improved the accuracy of our measurements over conventional amperometric detection by electrode background correction. Errors were significantly lower by this method and conditions. Assays can be performed directly in full fat milk, with a C.V. of 4% and total analysis time of less than 30 minutes.