Optical Immunosensors for the Efficient Detection of Target Biomolecules

Recently, immunosensors have attracted attention because they are widely applied for the detection of various pathogens. Among the commonly used immunosensors, the optical immunosensor features prominently as an effective tool for the quantification of the amount of antibodies, antigens, or haptens in complex samples with high sensitivity and specificity. However, very few studies provide comprehensive overviews of optical immunosensors. In this review, we present various methods and applications of optical immunosensors in pathogen detection. We introduced a concise definition of optical immunosensors and the principle of using them for detection. We subsequently discuss the main categories of optical immunosensors and their application to the detection of pathogens, as well as their advantages and limitations. Recent publications from 2006 to 2015 on variously designed optical immunosensors have also been updated. We conclude the review with a brief summary and discuss future directions of optical immunosensors.     

Immunosensors

The current trends and future aspects of the research and development of immunosensors are overviewed. A non-labelled immunosensor, whose selectivity depends on immunochemical affinity of an antigen for its corresponding antibody, has been developed as the basis for the potentiometric determination of an antigen, with an antibody-bound membrane or electrode. Non-labelled immunosensors for syphilis antibody, blood typing, human chorionic gonadotropin (HCG), and human serum albumin have been investigated. In contrast with non-labelled immunosensors, labelled immunosensors may be characterized by marked enhancement of sensitivity. Of these labelled immunosensors, enzyme immunosensors that use the chemical amplification of a labelling enzyme for sensitivity are promising. Enzyme immunosensors with an oxygen electrode have been developed to determine AFP, HCG, IgG and toxin. Bioaffinity sensors with a preformed metastable ligand-receptor complex, which are similar to the enzyme immunosensor have been found effective for the determination of thyroxine (T4), biotin, and insulin.     

用于肿瘤标志物检测的电化学免疫传感器

联合检测几种肿瘤标志物,在肿瘤早期诊断中具有重要的临床应用价值。随着纳米技术、流动注射分析技术、微流控技术以及丝网印刷术的迅猛发展,电化学免疫传感器可以在肿瘤标志物的检测中扮演越来越重要的角色。本文主要介绍了电化学免疫传感器的原理及其在肿瘤蛋白标志物检测中的应用情况,并介绍了纳米材料、流动注射分析、微流控等技术在肿瘤标志物免疫传感器中的运用,展望了电化学免疫传感器的前景。     

Optical immunosensing systems--meeting the market needs

Optical immunosensors and sensing systems are biosensors which produce a quantitative measure of the amount of antibody, antigen or hapten present in a complex sample such as serum or whole blood. The market needs for such devices and their associated instrumentation are reviewed. A brief history of the development of optical immunosensors is presented and the performance of the most well-developed optical immunosensors for meeting these market needs is reviewed. One device, the fluorescent capillary fill device (FCFD) is reviewed in detail with respect to it fulfilling the market needs for an optical immunosensor. Areas for the future development of such sensing systems are also discussed.     

Novel immunosensors

The development of practical immunosensors is an important topic for biosensor research. Recently the authors have demonstrated novel immuno-sensors called, respectively, the reactor-type enzyme immunosensor, the potentiometric sensing system for pathogenic microbes, the piezo-immunosensor, the pulse immunoassay, the bio-image sensor and the photofluctuation immunosensor. These six types of immunosensors based on novel principles are described.     

Detection of Bacillus thuringiensis Cry1Ab protein based on surface plasmon resonance immunosensor

Two novel surface plasmon resonance immunosensors were fabricated for detection of the Bacillus thuringiensis Cry1Ab protein and to demonstrate their performance in analyzing Cry1Ab protein in crop samples. Sensor 2 was modified by 1,6-hexanedithiol, Au/Ag alloy nanoparticles, 3-mercaptopropionic acid, and protein A (or not [sensor 1]), with Cry1Ab monoclonal antibody. As a result, both of the immunosensors exhibited satisfactory linear responses in the Cry1Ab protein concentration ranges of 10 to 500 ng ml⁻¹ and 8 to 1000 ng ml⁻¹, and the detection limits were 5.0 and 4.8 ng ml⁻¹, respectively. The immunosensors possessed good specificity and acceptable reproducibility. In addition, crop samples could be analyzed after a simple treatment. The transgenic crops could be easily identified from the conventional ones by the two immunosensors.     

Novel highly-performing immunosensor-based strategy for ochratoxin A detection in wine samples

The increasing concern about ochratoxin A (OTA) contamination of different food and feedstuffs demands high-performing detection techniques for quality assessment. Two indirect competitive enzyme-linked immunosorbent assay (ELISA) strategies were investigated for the development of OTA electrochemical immunosensors based on different OTA immobilisation procedures. Immunosensors based on avidin/biotin-OTA showed enhanced performance characteristics compared to those based on the adsorption of bovine serum albumin (BSA)-OTA conjugate. Performance of polyclonal (PAb) and monoclonal (MAb) antibodies against OTA was compared, showing at least one-order of magnitude lower IC(50) values when working with MAb. Alkaline phosphatase (ALP)- and horseradish peroxidase (HRP)-labelled secondary antibodies were evaluated. Both conjugates led to similar results when working with OTA standard solutions in buffer. However, whereas electroactive interferences present in spiked wine samples did not affect HRP-labelled immunosensors (4% slope deviation), they were likely oxidised at 0.225 V versus Ag/AgCl, the working potential for ALP-labelled immunosensors (25% slope deviation). Considering 80% of antibody binding as the limit of detection, values of 0.7 and 0.3 ng/mL for HRP- and ALP-labelled immunosensors respectively, validate these immunosensors as useful screening tools to assess OTA levels in wine.     

Electrochemical and chemiluminescent immunosensors for tumor markers

The determination of serum tumor markers plays an important role in clinical diagnoses for the patients with certain tumor-associated disease. Although many commercial kits have been applied in clinical immunoassays, conventional methods always have some disadvantages, resulting in the need of other new, efficient, and easily automated methods. Immunosensors, considered as a major development in immunochemical field, have attracted considerable attention. With the aim of rapid screening, many immunosensors that are small, semi-automated and portable are being developed. This brief review focuses on the current research of immunosensors for tumor markers based on the electrochemical and chemiluminescent detection with emphasis on recent advances, challenges, and trends. The works on series of novel immunosensors developed for the determination of tumor markers in our group in the last few years are also introduced.     

Microfluidic enzyme immunosensors with immobilised protein A and G using chemiluminescence detection

Affinity proteins were covalently immobilised on silicon microchips with overall dimensions of 13.1 x 3.2 mm, comprising 42 porous flow channels of 235 microm depth and 25 microm width, and used to develop microfluidic immunosensors based on horseradish peroxidase (HRP), catalysing the chemiluminescent oxidation of luminol/p-iodophenol (PIP). Different hydrophilic polymers with long flexible chains (polyethylenimine (PEI), dextran (DEX), polyvinyl alcohol, aminodextran) and 3-aminopropyltriethoxysilane (APTS) were employed for modification of the silica surfaces followed by attachment of protein A or G. The resulting immunosensors were compared in an affinity capture assay format, where the competition between the labelled antigen and the analyte for antibody-binding sites took place in the bulk of the solution. The formed immunocomplexes were then trapped by the microchip affinity capture support and the amount of bound tracer was monitored by injection of luminol, PIP and H2O2. All immunosensors were capable of detecting atrazine at the sub-microg l(-1) level. The most sensitive assays were obtained with PEI and DEX polymer modified supports and immobilised protein G, with limits of detection of 0.006 and 0.010 microg l(-1), and IC50 values of 0.096 and 0.130 microg l(-1), respectively. The protein G based immunosensors were regenerated with 0.4 M glycine-HCl buffer pH 2.2, with no loss of activity observed for a storage and operating period of over 8 months. To estimate the applicability of the immunosensors to the analysis of real samples, PEI and DEX based protein G microchips were used to detect atrazine in surface water and fruit juice, spiked with known amounts of the atrazine, giving recovery values of 87-102 and 88-124% at atrazine fortification levels of 0.5-3 and 80-240 microg l(-1), respectively.     

Development of immunosensors using carbon nanotubes

With increasing reports on bioterrorism, avian flu, and other bio-threats, rapid and real time detection methods are highly warranted. Studies on developing highly sensitive immunosensors aiming at the early detection and clinical diagnoses of various diseases including cancer are undertaken all over the globe. Carbon nanotubes (CNTs) have been widely discussed as materials with enormous potential for a wide range of in vivo and in vitro bioapplications, ranging from drug delivery to highly sensitive biosensors, owing to their superior electronic and mechanical properties along with nanoscale dimensions. Though a lot of attention has been drawn toward carbon nanotubes for the past 15 years in academia and to a certain extent in industry, CNT-based immunosensors and other applications are still in the nascent stage, and there are many challenges to be overcome for the successful commercialization of the concepts. This article highlights on the recent developments and the possible impacts of carbon nanotube based immunosensors.     

A novel microfluidic immunoassay system based on electrochemical immunosensors: an application for the detection of NT-proBNP in whole blood

Electrochemical immunosensors have attracted great interest in the search for a selective, simple and reliable system for molecular recognition. Presently, electrochemical immunosensors have been widely studied for biomedical molecular's detection, but the regeneration of these immunosensors has restricted their wide application. To prepare a regeneration-free immunosensor, which may be more suitable for clinical determination, a repeatable immunoassay system was developed based on an electrochemical immunosensor with magnetic nanoparticles, biotin-avidin system (BAS) and Fab antibodies for the heart failure markers aminoterminal pro-brain natriuretic peptides (NT-proBNP). At the same time, a microfluidic system was combined into the proposed system, which enabled continuous determination. Using NT-proBNP as a model system, the proposed immunosensor exhibited rapid and sensitive amperometric response to NT-proBNP with good selectivity, stability, and a wide linear range (0.005-1.67 ng/mL and 1.67-4 ng/mL with a detection limit of 0.003 ng/mL under optimal conditions). Importantly, the proposed immunosensor was also suitable for the detection of other proteins and provided new opportunities for disease diagnosis.     

Protein immunosensor using single-wall carbon nanotube forests with electrochemical detection of enzyme labels

Vertically aligned arrays of single-wall carbon nanotubes (SWNT forests) on pyrolytic graphite surfaces were developed for amperometric enzyme-linked immunoassays. Improved fabrication of these SWNT forests utilizing aged nanotube dispersions provided higher nanotube density and conductivity. Biosensor performance enhancement was monitored using nanotube-bound peroxidase enzymes showing a 3.5-fold better sensitivity for H2O2 than when using fresh nanotubes to assemble the forests, and improved detection limits. Absence of improvements by electron mediation for detection of H2O2 suggested very efficient electron exchange between nanotubes and enzymes attached to their ends. Protein immunosensors were made by attaching antibodies to the carboxylated ends of nanotube forests. Utilizing casein/detergent blocking to minimize non-specific binding, a detection limit of 75 pmol mL(-1) (75 nM) was achieved for human serum albumin (HSA) in unmediated sandwich immunosensors using horseradish peroxidase (HRP) labels. Mediation of the immunosensors dramatically lowered the detection limit to 1 pmol mL(-1) (1 nM), providing significantly better performance than alternative methods. In the immunosensor case, the average distance between HRP labels and nanotube ends is presumably too large for efficient direct electron exchange, but this situation can be overcome by electron mediation.     

An electrochemical immunosensor for aflatoxin M1 determination in milk using screen-printed electrodes

The production and assembling of disposable electrochemical AFM1 immunosensors, which can combine the high selectivity of immunoanalysis with the ease of the electrochemical probes, has been carried out. Firstly immunoassay parameters such as amounts of antibody and labelled antigen, buffer and pH, length of time and temperature of each steps (precoating, coating, binding and competition steps) were evaluated and optimised in order to set up a spectrophotometric enzyme-linked immunosorbent assay (ELISA) procedure. This assay exhibited a working range between 30 and 160 ppt in a direct competitive format. Then electrochemical immunosensors were fabricated by immobilising the antibodies directly on the surface of screen-printed electrodes (SPEs), and allowing the competition to occur between free AFM1 and that conjugated with peroxidase (HRP) enzyme. The electrochemical technique chosen was the chronoamperometry, performed at -100 mV. Furthermore, studies of interference and matrix effects have been performed to evaluate the suitability of the developed immunosensors for the analysis of aflatoxin M1 directly in milk. Results have shown that using screen-printed electrodes aflatoxin M1 can be measured with a detection limit of 25 ppt and with a working range between 30 and 160 ppt. A comparison between the spectrophotometric and electrochemical procedure showed that a better detection limit and shorter analysis time could be achieved using electrochemical detection.     

Immunosensors for detection of pesticide residues

Immunosensors are biosensors that use antibodies or antigens as the specific sensing element and provide concentration-dependent signals. There is great potential in the applications of immunosensing technologies for rapid detection of pesticide residues in food and environment. This paper presents an overview of various transduction systems, such as electrochemical, optical, piezoelectric, and nanomechanics methods, which have been reported in the literature in the design and fabrication of immunosensors for pesticide detection. Various immobilization protocols used for formation of a biorecognition interface are also discussed. In addition, techniques of regeneration, signal amplification, miniaturization, and antibodies are evaluated for the development and applications of these immunosensors. It can be concluded that despite some limitations of the immunosensing technologies, these immuosensors for pesticide monitoring are becoming more and more relevant in environmental and food analysis.     

Advances in ovarian cancer diagnosis: A journey from immunoassays to immunosensors

This review focuses on the technological advancements, challenges and trends in immunoassay technologies for ovarian cancer diagnosis. Emphasis is placed on the principles of the technologies, their merits and limitations and on the evolution from laboratory-based methods to point-of-care devices. While the current market is predominantly associated with clinical immunoassay kits, over the last decade a major thrust in development of immunosensors is evident due to their potential in point-of-care devices. Technological advancements in immunosensors, extending from labeled to label-free detection, with and without mediators, for enhancing proficiencies and reliability have been dealt with in detail. Aspects of the utilisation of nanomaterials and immobilization strategies for enhancing sensitivity and altering the detection range have also been addressed. Finally, we have discussed some distinct characteristics and limitations associated with the recently commericalised technologies used for quantitation of relevant ovarian cancer markers.     

Highly sensitive near-simultaneous assay of multiple "lean meat agent" residues in swine urine using a disposable electrochemiluminescent immunosensors array

Nowadays, β(2)-agonists are abused illegally as "lean meat agents" for food-producing animals, and cause increasing food-safety accidents in some countries. Due to their hazard to the human health, "lean meat agents" are banned in most countries and required to be routinely monitored. We herein report a disposable electrochemiluminescent immunosensors array for near-simultaneous assay of multiple β(2)-agonist residues in swine urine, by using ractopamine and salbutamol as the models. In this investigation, a screen-printed carbon electrodes array was assembled and acted as the substrate of the immunosensors array. Then the immunosensors array was constructed by site-selectively immobilizing the antigens of ractopamine and salbutamol on the working electrodes of array. After the competitive immuno-binding, with the aid of a homemade single-pore-four-throw switch, the electrochemiluminescent signals of the two β(2)-agonists were sequentially detected using a non-array detector. The limits of detection for ractopamine and salbutamol were 8.5 and 17pg/mL, respectively, which were much lower than those of the most previous reports. Compared with other routine methods based on chromatography and ELISA, this method is more suitable for screening of multiple β(2)-agonists in quantities of samples, owing to its merits of low cost, user-friendliness and high throughput, and shows great promise in food safety and agonist surveillance.     

Recent trends in antibody based sensors

This review details recent advances in the fields of immunosensors and closely related immunoassays in the past decade, together with a discussion of possible future trends. Immunosensors can be classified by the way in which they transduce the signal produced upon the formation of an antibody antigen complex. Recent advancements to these methods of detection and transduction are discussed in detail, with particular focus on electrochemical, optical, piezoelectric and magnetic based sensors. The varying applications of these sensors are also discussed. Some of the most significant advances include development of immunosensors for the continuous monitoring of analytes, point of care (PoC) devices, with lower unit costs, automation, reusability and ease of use. Immunosensor technology has advanced at a prolific rate since its conception and has grown into a diverse area of ongoing research.     

Comparison of different carbon ink based screen-printed electrodes towards amperometric immunosensing

The aim of the present work was to make amperometric immunosensors based on the principle of enzyme-linked immunosorbent assay (ELISA). For this purpose, screen-printed electrodes (SPEs) were fabricated using various carbon inks (commercially available inks Gwent, Acheson, Eltecks and two homemade inks PSG & PVCG) to determine the best ink in realizing immunosensors. Amperometric immunosensors made by different carbon inks were compared with standard ELISA in terms of total assay time, amount of biological materials used and sensitivity of detection. A model system containing rabbit anti-mouse immunoglobulin G (RαMIgG) as the capturing antibody, mouse IgG (MIgG) as antigen and alkaline phosphatase conjugated RαMIgG as revealing antibody was used. In these studies, 1-naphthyl phosphate was used as substrate. The experiments done include electrochemical characterization of electrodes, optimization of dilutions of antibodies, immobilization of antibody on the electrode were carried out. The minimum detection limit for the best results of MIgG determination were obtained on screen-printed electrode made by Gwent carbon ink and PSG carbon ink, with a detection limit of 1.0 and 2.0 ng/ml respectively. The time required for detection of mouse IgG was 40 min for SPEs. By using the conventional spectrophotometric method (ELISA method), the minimum detection limit for the MIgG (antigen) detection was 50 ng/ml and the time required for analysis was found to be 140 min.     

Highly sensitive amperometric immunosensors for microcystin detection in algae

The presence of cyanobacterial toxins in water and algae pose a health hazard for animals and humans, due to their tumour-promoting activity and carcinogen effects. The use of simple, rapid and reliable tools for routine analysis is becoming a necessity. With this purpose, our group has developed two electrochemical immunosensors for the detection of microcystin-LR (MC-LR) based on the affinity between this cyanotoxin and the corresponding monoclonal and polyclonal antibodies. A competitive direct enzyme-linked immunosorbent assays (ELISAs) was designed and, after validation of the approach on microtiter wells, screen-printed graphite electrodes were used as supports. Colorimetry was used to optimise the experimental parameters and to compare the performance of monoclonal and polyclonal antibodies. Afterwards, electrochemical measurements were performed at -200 mV (versus Ag/AgCl) using 5-methyl-phenazinium methyl sulfate (MPMS) as mediator for horseradish peroxidase (HRP), the enzymatic label of the competitor. The IC(50) values were 0.10 and 1.73 microgL(-1) for MAb and PAb, respectively. Whereas Mab provided higher sensitivities, the reproducibility was better when using PAb. The developed amperometric immunosensors were applied to the analysis of cyanobacterial samples from the Tarn River (Midi-Pyrénées, France) and the presence of MC was confirmed by the colorimetric protein phosphatase inhibition (PPI) assay and high performance liquid chromatography (HPLC). The limits of detection attained from the calibration curves and the results obtained for the real samples demonstrate the potential use of the immunosensors as screening tools for routine use in the assessment of water quality and the control of toxins in algae.     

New amperometric and potentiometric immunosensors based on gold nanoparticles/tris(2,2'-bipyridyl)cobalt(III) multilayer films for hepatitis B surface antigen determinations

Two generic, fast, sensitive and novel electrochemical immunosensors have been developed. Initially, a layer of plasma-polymerized Nafion film (PPF) was deposited on the platinum electrode surface, then positively charged tris(2,2'-bipyridyl)cobalt(III) (Co(bpy)(3)(3+)) and negatively charged gold nanoparticles were assembled on the PPF-modified Pt electrode by layer-by-layer technique. Finally, hepatitis B surface antibody (HBsAb) was electrostatically adsorbed on the gold nanoparticles surface. Electrochemical behavior of the {Au/Co(bpy)(3)(3+)}(n) multilayer film-modified electrodes was studied. Cyclic voltammetry, electrochemical impedance spectroscopy (EIS) were adopted to monitor the regular growth of the multilayer films. The performance and factors influencing the performance of the resulting immunosensors were studied in detail. The multilayer film-modified immunosensor was used for hepatitis B surface antigen (HBsAg) determination via the amperometric and potentiometric immunosensor systems, and both systems provided the same linear ranges from 0.05 to 4.5 microg/mL with different detection limits for the amperometric system 0.005 microg/mL and for the potentiometric system 0.015 microg/mL. The immunosensors were used to analyse HBsAg in human serum samples. Analytical results of clinical samples show that the developed immunoassay is comparable with the enzyme-linked immunosorbent assays (ELISAs) method, implying a promising alternative approach for detecting HBsAg in the clinical diagnosis. In addition, the multilayer films also showed better stability for 1 month at least.