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.     

Open sandwich enzyme-linked immunosorbent assay for the quantitation of small haptens

The quantitation of low-molecular-weight haptens has been difficult with conventional sandwich immunoassays due to their small size. Many researchers have attempted to develop sandwich assays for haptens due to the significant advantages of the sandwich format over competitive assays including greater dynamic range, ease of automation, and sensitivity. Here we apply the open-sandwich ELISA (OS-ELISA), an immunoassay based on antigen-dependent stabilization of antibody variable regions (V(H) and V(L) domains), to hapten quantitation. Two fusion proteins, the high-affinity mutant V(H) domain from anti-4-hydroxy-3-nitrophenacetyl (NP) antibody B1-8 tethered with Escherichia coli alkaline phosphatase (V(H)(W33L)-PhoA) and the V(L) domain from the same antibody tethered with Streptococcus sp. protein G, were made. These fusion proteins when added together achieved Fv reassociation consequent to the addition of NP. Signal was generated in a direct relationship to the NP concentration with better sensitivity compared with competitive immunoassay, demonstrating this assay to be a quick noncompetitive alternative to the conventional assays for small compounds, such as environmental pollutants, drugs of abuse, and therapeutic drugs. With our previous demonstration that the OS-ELISA works well with large proteins, the OS-ELISA becomes the first practical immunoassay approach capable of quantifying any molecule regardless of their size.     

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.     

Novel poly-substituted aryl acridinium esters and their use in immunoassay

A new series of stable acridinium ester conjugates have been developed for use as non-isotopic labels in immunoassay. They have proved to be a flexible alternative to radioimmunoassay. We present data showing the successful development of immunoassays in sandwich, competitive and receptor formats. In addition, hydrophilic acridinium ester analogues have been synthesized, encapsulated in liposomes, and utilized as labels in immunoassay. The potential of this technology is discussed.     

Modeling of immunosensors under nonequilibrium conditions. I. Mathematic modeling of performance characteristics.

Immunosensors for the detection of small analytes that use analyte-enzyme conjugates as signal generators require special attention if operated under nonequilibrium conditions. If the size of the analyte and the analyte-enzyme conjugate differ substantially, the two antigens do not diffuse at the same rate. This can cause time-dependent shifts in the sensitivity of competitive immunoassays. Therefore, immunosensors operating at short incubation times require precise timing that meets closely the specifications for which the sensors were calibrated. As an example, we have analyzed kinetic binding curves for the quantitative determination of progesterone with an immobilized monoclonal antibody and a conjugate between horseradish peroxidase and progesterone as signal generator. Mathematical paradigms have been developed to simulate the diffusion, antigen-antibody complex formation, and competitive binding processes in this analytical system. Dose-response curves obtained under nonequilibrium conditions can vary substantially from those obtained at equilibrium of antigen-antibody interaction. The degree of this variation depends on the performance characteristics of the major components of the immunosensor. The developed mathematical solutions reflect experimental results and can be used to model optimal conditions for immunosensors operating under nonequilibrium conditions. In this paper (Part I), we report on the mathematical modeling of the interaction between analyte, analyte-enzyme conjugate, and an immobilized antibody. In Part II (W. Schramm and S.-H. Paek (1991) Anal. Biochem. 196), we present experimental results and compare them with the theoretical models.     

New antibodies immobilization system into a graphite-polysulfone membrane for amperometric immunosensors

Polysulfone membrane is used for the first time for the preparation of electrochemical immunosensors. A disposable immunosensor based on a porous conductor polymer graphite-polysulfone-electrode has been developed using a phase inversion technique for the determination of anti-rabbit IgG (anti-RIgG) as a model analyte. To construct the sensor, a conductor membrane was deposited on the surface of working graphite-epoxy composite (GEC) electrode. The membrane was characterized by SEM. This sensor was based on the competitive assay between free and labeled anti-RIgG for the available binding sites of immobilized rabbit IgG (RIgG). Incubation parameters were optimized in this work. The immunological reaction was detected using an enzymatic-labeling procedure (HRP enzyme) combined with the amperometric detection using H(2)O(2) as substrate and hydroquinone as mediator. This sensor shows stability during a week and a good reproducibility. The current was monitored amperometrically at -0.1 V versus SCE and this method showed a linear range of the anti-RIgG from 1 to 6 microg/ml. The detection limit was determined to be 0.77 microg/ml.     

Fabrication of a capillary immunosensor in polymethyl methacrylate.

A method for fabricating capillary-based immunosensors in a coupon milled from an inexpensive, commodity plastic (PMMA, plexiglass) is demonstrated. The key feature of the technique is the use of sol-gel technology to deposit a glass-like (Si [bond] OH) film on surfaces of the plastic capillary channels to facilitate antibody immobilization. The utility of this method was demonstrated in the context of continuous flow displacement immunosensors for the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). These sensors exhibited sensitivity to low microg/l RDX concentrations and peak-to-peak signal variations that were generally less than 10% for multiple injections at a single RDX concentration. The useful lifetime of the coupons in these experiments was greater than 10 h even after multiple exposures to high (1000 microg/l) RDX levels. This sensor platform has the physical characteristics needed for a portable field instrument: small, light-weight, and rugged.     

State of the art and recent advances in immunoanalytical systems

This article is an overview the state of the art and the recent developments in immunosensors. Homogeneous immunosensors, heterogeneous immunosensors, integrated immunosensors and biochip format immunosensors are presented, based on optical, electrochemical, magnetic or mechanical detection/transduction systems.     

Use of microsphere-antibody conjugates in microparticle-enhanced nephelometric immunoassay.

A new microparticle-enhanced nephelometric immunoassay has been recently described as a sensitive, accurate, and easy-to-perform competitive immunoassay for various analytes. As initially described, this test is based on the nephelometric quantification of the inhibition, by the antigen to be assayed, of immunoagglutination of microparticle-antigen conjugates. Its applicability as a competitive immunoassay is thus limited by the necessary availability of pure antigens to prepare microparticle-antigen conjugates. In this paper, we report an adaptation of this initial test, where microparticles are coated by monoclonal antibodies, eliminating the need for purified antigens. The new configurations of particle agglutination-based immunoassays described include use of these microparticle-antibody conjugates with microparticle-antigen conjugates, free antigen, and anti-mouse immunoglobulins antiserum. The feasibility of such configurations is studied with human chorionic gonadotropin hormone, human thyroid stimulating hormone, and human myoglobin as antigens. Capture of the analyte by microparticle-antibody conjugates is evidenced by inhibition of their agglutination with microparticle-antigen conjugates and by agglutination in a sandwich assay with a complementary monoclonal antibody or polyclonal antiserum. The use of a second xenogenic antibody enhances the agglutination process and increases the assay sensitivity. Microparticle-antibody conjugates may extend the applications of microparticle-enhanced nephelometric immunoassays to unavailable analytes.     

Chiral discrimination using an immunosensor

Based on the stereoselectivity of immunoglobulins, we have developed a new chiral sensor for the detection of low-molecular-weight analytes. Using surface plasmon resonance detection, enantiomers of free, underivatized alpha-amino acids can be monitored in a competitive assay by their interaction with antibodies specific for the chiral center of this class of substances. The sensitivity to the minor enantiomer in nonracemic mixtures exceeds currently available methods; therefore, such immunosensors can readily detect traces of enantiomeric impurities and are attractive for a range of applications in science and industry.     

Development of radioimmunoassay and enzyme immunoassays for luteinizing hormone in dromedaries (Camelus dromedarius).

Polyclonal antibodies against luteinizing hormone in dromedary (camLH) were raised in a rabbit and enabled the development of homologous immunoassays (radioimmunoassay, competitive enzymeimmunoassay (EIA) and sandwich EIA) for the measurement of circulating camLH in plasma. These assays were highly specific for camLH since neither dromedary follicle-stimulating hormone, growth hormone nor prolactin cross-reacted significantly. The lowest detection limit (0.08 ng/ml) was obtained with the sandwich EIA. In addition to its high specificity and sensitivity, this method does not require radiolabelled molecules or expensive laboratory facilities. It can be performed in the field using a portable, battery-powered plate reader.     

Electrochemical immunosensors for cancer biomarker with signal amplification based on ferrocene functionalized iron oxide nanoparticles

Ultrasensitive sandwich type electrochemical immunosensors for the detection of cancer biomarker prostate specific antigen (PSA) is described which uses graphene sheet (GS) sensor platform and ferrocene functionalized iron oxide (Fe(3)O(4)) as label. To fabricate the labels, dopamine (DA) was first anchored onto Fe(3)O(4) surface followed by conjugating ferrocene monocarboxylic acid (FC) and secondary-antibody (Ab(2)) onto Fe(3)O(4) through the amino groups of DA (DA-Fe(3)O(4)-FC-Ab(2)). The great amount of DA molecules anchored onto Fe(3)O(4) surface increased the immobilization of FC and Ab(2) onto the Fe(3)O(4) nanoparticle, which in turn increased the sensitivity of the immunosensor. GS used as biosensor platform increased the surface area to capture a great amount of primary antibodies (Ab(1)) and the good conductivity of GS enhanced the detection sensitivity to FC. Using the redox current of FC as signal, the immunosensor displays high sensitivity, wide linear range (0.01-40 ng/mL), low detection limit (2 pg/mL), good reproducibility and stability. In addition, this method could be extended to the immobilization of other interesting materials (fluorescence dyes) onto Fe(3)O(4) for preparing various kinds of labels to meet the different requirements in immunoassays.     

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.     

Direct electron transfer catalysed by enzymes: application for biosensor development

The ability to catalyse an electrode reaction via direct (mediatorless) electron transfer has been demonstrated for a number of redox enzymes. In the case of mediatorless electron transfer, the electron is transferred directly from the electrode to the substrate molecule via the active site of the enzyme, or vice versa. The electron itself is the second substrate for the reaction. An important point characterizing bioelectrocatalysis is the catalytic removal of the reaction over-voltage. Therefore the enzyme attached to the electrode is able to catalyse electrode reaction and forms a 'molecular transducer'. The substrate can be detected by potentiometric measurement of the removal of reaction over-voltage. The enzyme laccase is able to catalyse the reaction of oxygen electroreduction. Therefore a laccase molecular layer attached to the electrode surface forms an oxygen transducer. The formation of the layer results in a change of the electrocatalytic feature of the electrode. Laccase label coupled with either ligand or receptor allows the detection of ligand-receptor complex formation/dissociation on the electrode surface. The detection is virtually reagentless. The substrates for the reaction are molecular oxygen and the electron itself. Numerous reagentless immunosensors of different formats (competitive, displacement and sandwich) have been developed, as well as the reagentless detection system for immunofiltration/immunochromatography.     

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.     

Effects of particle characteristics on magnetic immunoassay in a thin channel

The effects of size and porosity of particles on magnetic immunoassay in a thin channel were studied. Experimental parameters were investigated and compared using a model immunoassay complex of carcinoembryonic antigen (CEA)/anti-CEA. The rate constant for the affinity reaction between functional particles increased as the size of magnetic nanoparticles (800-80 nm) decreased. The affinity reaction between functional particles had no significant effect on the sizes of microparticles (1.0-4.4 μm) at commonly used thin channel flow-rates of 0.001-0.025 ml/min. Competitive and sandwich reactions of CEA/anti-CEA were studied for CEA detection. Microparticles of different porosities produced similar linear ranges of detection and limits of detection. The limits of detection for CEA were 0.29 pg/ml and 0.21 pg/ml for competitive and sandwich reactions, respectively. The linear ranges of detection were from 0.49 pg/ml to 4.9 ng/ml for both competitive and sandwich reactions. The detection limits were lower, and the linear ranges were wider than those of literature. There was a 9% difference in CEA detection measurements between competitive and sandwich magnetic immunoassay. The measurements of two magnetic immunoassays differed by less than 13% from the ELISA reference measurements. The running time was less than 30 min. Magnetic immunoassay in a thin channel has great potential for biochemical analysis and immunoassay-related applications.     

Immunosensors in medical diagnostics--major hurdles to commercial success.

In the last few years the format of commercial immunoassays has dramatically changed. Automated instruments running up to 180 samples per hour and dry strip or film immunochemistry products are now available. These changes have been precipitated by the need for immunoassays that require less time and skill to perform. To date, no immunosensor technology has been successfully commercialized although a few are purported to be on the brink of being released for sale. Here, B. Manning and T. Maley evaluate the major factors affecting the success of immunosensors.     

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.     

A competitive enzyme hybridization assay for plasma determination of phosphodiester and phosphorothioate antisense oligonucleotides.

An enzyme competitive hybridization assay was developed and validated for determination of mouse plasma concentrations of a 15mer antisense phosphodiester oligodeoxyribonucleotide and of two phosphorothioate analogs. Assays were performed in 96-well microtiter plates. The phosphodiester sense sequence was covalently bound to the microwells. The 5'-biotinylated antisense sequence was used as tracer. The principle of the assay involves competitive hybridization of tracer and antisense nucleotide to the solid phase-immobilized sense oligonucleotide. Solid phase- bound tracer oligonucleotide was assayed after reaction with a streptavidin-acetylcholinesterase conjugate, using the colorimetric method of Ellman. As in competitive enzyme immunoassays, coloration was inversely related to the amount of analyte initially present in the sample. The limit of quantification was 900 pM for phosphodiester antisense oligonucleotide using a 100 microl volume of plasma without extraction. Cross-reactivity was negligible after a four base deletion in either the 3'or 5'position. The assay was simple and sensitive, suitable for in vitro screening of oligonucleotide hybridization potency in biological fluids and for measuring the plasma pharmacokinetics of phosphorothioate and phosphodiester sequences.