Antibody-based Sensors Research Articles

Graphene-based synthetic peptide electrochemical sensor for colorectal cancer diagnosis

This work reports the development of an electrochemical sensor using graphene-peptide conjugates for detecting colorectal cancer (CRC) biomarker leucine-rich alpha-2 glycoprotein-1 (LRG1). To enable LRG1 quantification, we rationally designed peptides with dual graphene anchoring motifs for optimal orientation and binding activity when immobilized on a reduced graphene oxide (rGO) electrochemical transducer surface. The graphene nanomaterial provides several advantages such as high conductivity, large surface area, and excellent stability that can enhance the sensor's analytical performance metrics. Furthermore, the synthetic peptides offer benefits like smaller size, specificity, ease of modification and cost-effective production compared to traditional antibody receptors. Under optimized conditions, the peptide sensor exhibited high sensitivity of 22.3 μA/(ng/mL.cm2), low limit of detection (75 pg/mL LRG1 in serum), accuracy of 101.1 % spiked recovery, and precision within 6 % RSD. Testing with colonoscopy-classified patient serum specimens discriminated normal, precancerous adenomatous polyps and malignant carcinoma stages based on LRG1 overexpression. A 24 % elevation for adenomas and 103 % higher levels in CRC were observed. Validation with spiked plasma samples indicated 97–104 % recovery and <7 % RSD, proving accurate detection capability. Comparison to antibody-based sensors showed superior linear range, sensitivity, reproducibility, and faster assay time. This demonstrates the promise of computational peptide designing combined with advanced nanomaterials for electrochemical detection of CRC progression through serum protein biomarkers.

Design of Aptamer-Based Optical Nanosensors for the Detection of Interleukin-6

Interleukin-6 (IL-6) is an inflammatory cytokine that has been recognized as a potential target for various autoimmune diseases, chronic inflammatory conditions, cancers, and more. Dysregulation of IL-6 is implicated in the genesis and progression of these disease states. For these reasons, detection of IL-6 and related biomolecules in vivo is of clinical interest to researchers of cancer and other inflammatory diseases. Single-walled carbon nanotubes (SWCNT) possess unique near-infrared fluorescent properties. When functionalized with a molecular recognition element, SWCNT can act as optical sensors that respond sensitively and specifically to a target analyte. Both antibodies and DNA aptamers can serve as molecular recognition probes, but aptamers may hold advantages for certain sensing applications due to their smaller size, higher stability, and lower production cost. We functionalized SWCNT with an analyte-specific DNA aptamer, which resulted in a nanosensor which responds selectively to IL-6. In vitro testing revealed significant shifts in the center wavelength and intensity of SWCNT fluorescence in response to IL-6. We then conducted several studies to investigate the mechanism of this sensor response in order to compare against traditional antibody-based sensors prior to ex vivo clinical sample testing. Rapid, minimally invasive detection of IL-6 as a chronic disease biomarker in vivo through DNA aptamer-functionalized SWCNT holds promise for early disease diagnosis, observation of disease progression, and evaluation of response to therapy.

Simulation of BioGFET sensors using TCAD

We used a commercial TCAD simulator for the study of liquid-gate Graphene FETs as antibody-based sensors. To model the graphene layer, a zero-bandgap semiconductor with a linear density of states relationship in both the conduction and valence bands near the edge of the bands was adopted. The electrolyte which forms the liquid gate was also modeled as a semiconductor with tuned parameters to reproduce the behavior of a dilute PhosphateBuffer Saline (PBS) solution with pH = 7.4. Antibodies are anchored to the graphene surface and their effect is simulated with a charged cube box with a size similar to the one of the IgG antibody and a charge concentration similar to the charge usually carried by an anibody. The binding of the antibody with the antigen protein (Ab-Pr) complex is also represented in the simulator with a charged box carrying a different charge. We have calculated the ID-VFG curves for different numbers of antibodies anchored to the surface of the graphene and analyzed the behavior of the sensor as a function of the number of antibody-protein complexes.

Conjugated Polymers for Aptasensing Applications.

Rapid and specific assaying of molecules that report on a pathophysiological condition, environmental pollution, or drug concentration is pivotal for establishing efficient and accurate diagnostic systems. One of the main components required for the construction of these systems is the recognition element (receptor) that can identify target analytes. Oligonucleotide switching structures, or aptamers, have been widely studied as selective receptors that can precisely identify targets in different analyzed matrices with minimal interference from other components in an antibody-like recognition process. These aptasensors, especially when integrated into sensing platforms, enable a multitude of sensors that can outperform antibody-based sensors in terms of flexibility of the sensing strategy and ease of deployment to areas with limited resources. Research into compounds that efficiently enhance signal transduction and provide a suitable platform for conjugating aptamers has gained huge momentum over the past decade. The multifaceted nature of conjugated polymers (CPs), notably their versatile electrical and optical properties, endows them with a broad range of potential applications in optical, electrical, and electrochemical signal transduction. Despite the substantial body of research demonstrating the enhanced performance of sensing devices using doped or nanostructure-embedded CPs, few reviews are available that specifically describe the use of conjugated polymers in aptasensing. The purpose of this review is to bridge this gap and provide a comprehensive description of a variety of CPs, from a historical viewpoint, underpinning their specific characteristics and demonstrating the advances in biosensors associated with the use of these conjugated polymers.

Advances in wearable electrochemical antibody-based sensors for cortisol sensing.

Cortisol is a crucial hormone involving many physiological processes. Hence, cortisol detection is essential. This review highlights the key progress made on wearable electrochemical sensors using antibodies. It covers the design, principle, and electroanalytical methodology for detecting cortisol noninvasively. This article also analyzes and collects the analytical performances of electrochemical cortisol sensors. The development of these sensors continues to face challenges such as biofouling, sample management, sensitivity, flexibility, stability, and recognition layer performance. It is also necessary to develop a sensitive electrode and material. This article also presents potential strategies for designing antibody electrodes and provides examples of sensing systems. Additionally, it discusses the challenges in translating research into practical applications.

Development of an Impedimetric Aptasensor for Detection of Progesterone in Undiluted Biological Fluids.

A cost-effective, deployable, and quantitative progesterone biosensor is desirable for regular progesterone sensing in biological and environmental samples to safeguard public health. Aptasensors have been shown to be affordable as compared to antibody-based sensors, but so far, none of the progesterone aptamers could detect it in undiluted and unprocessed biological samples. Thus, to select an aptamer suitable for biosensing in unprocessed biological samples, a modified magnetic bead-based approach with counter-selection in milk and serum was performed. G-quadruplex forming progesterone aptamers were preferentially screened through in silico, gold nanoparticle-based adsorption-desorption assay and circular dichroism spectroscopy. GQ5 aptamer showed extended stability and a high progesterone binding affinity (K D 5.29 ± 2.9 nM) as compared to any other reported progesterone aptamers (P4G11 and P4G13). Under optimized conditions, GQ5 aptamer was coated on the gold electrode to develop an impedimetric aptasensor (limit of detection: 0.53, 0.91, and 1.9 ng/mL in spiked buffer, undiluted milk, and serum, respectively, with the dynamic range of detection from 0.1 to 50 ng/mL in buffer and 0.1 to 30 ng/mL in both milk and serum). The aptasensor exhibited a very high level of κ value (>0.9) with ELISA to detect progesterone in milk and serum. The aptasensor could be regenerated three times and can be stored for up to 10 days at 4 °C. Therefore, GQ5 may be used to develop a portable impedimetric aptasensor for clinical and on-site progesterone sensing in various biological and environmental samples.

Aptamer-linked photonic crystal hydrogel sensor for rapid point-of-care detection of human immuno-deficiency virus-1 (HIV-1)

The detection of the human immunodeficiency virus-1 (HIV) at an early stage is vital and could be realized through its cell surface glycoprotein-120 (gp120) without virus preprocessing. Here, we present an ssDNA-aptamer-linked photonic crystal (APC) hydrogel sensor for HIV detection which is comprised of photonic crystals (PCs) made of polystyrene nanoparticles embedded in the polyacrylamide hydrogel. ssDNA aptamers specific for gp120 are crosslinked in the hydrogel which can selectively bind to gp120 by hydrogen bonding increasing the PCs particle spacing and swelling of the hydrogel. The binding response can be visually monitored as a color change due to the diffraction of light from PCs and can eventually be measured (1–1000 ng mL−1 of gp120) and 100 to 108 VP mL−1 of HIV by the Debye’s ring diameter or a UV/Vis spectrometer. APC-hydrogel can be regenerated by Tris-HCl and EDTA washing buffer system. The sensor demonstrates LOD of 7.1 ± 1.55 ng mL−1 for gp120 and 4 VP mL−1 for the whole HIV, a rapid response of 5 min, reusability up to 70 % (in fifth use), and recovery of 95.4 ± 0.1 % to 99.0 ± 0.2 % in plasma samples. The sensor is cost-effect and stable compared to antibody-based sensors and can be utilized to develop point-of-care testing (POCT) devices for HIV diagnosis.

A microfluidic paper analytical device using capture aptamers for the detection of PfLDH in blood matrices

BackgroundThe prevalence and death rate arising from malaria infection, and emergence of other diseases showing similar symptoms to malaria require the development of malaria-specific and sensitive devices for its diagnosis. To address this, the design and fabrication of low-cost, rapid, paper-based analytical devices (µPAD) using surface-immobilized aptamers to detect the presence of a recombinant malarial biomarker—Plasmodium falciparum lactate dehydrogenase (rPfLDH)—is reported in this study.MethodsTest zones on paper surfaces were created by covalently immobilizing streptavidin to the paper, subsequently attaching biotinylated aptamers to streptavidin. Aptamers selectively bound rPfLDH. The measurement of captured rPfLDH enzyme activity served as the means of detecting this biomarker. Enzyme activity across three replicate sensors was digitally quantified using the colorimetric Malstat assay.ResultsScreening of several different aptamers reported in the literature showed that aptamers rLDH7 and 2008s immobilized in this manner specifically recognised and captured PfLDH. Using rLDH7, the sensitivity of the µPAD sensor was evaluated and the µPAD sensor was applied for preferential detection of rPfLDH, both in buffered solutions of the protein and in spiked serum and red blood cell lysate samples. In buffered solutions, the test zone of the µPAD sensor exhibited a KD of 24 ± 11 nM and an empirical limit of detection of 17 nM, respectively, a limit similar to commercial antibody-based sensors exposed to rPfLDH. The specific recognition of 133 nM rPfLDH in undiluted serum and blood samples was demonstrated by the µPAD.ConclusionThe reported µPAD demonstrates the potential of integrating aptamers into paper-based malarial rapid diagnostic tests.Graphical The assembly of µPAD sensors using APTEC assay principles for the detection the malarial biomarker, lactate dehydrogenase enzymes from Plasmodium falciparum (PfLDH). The aptamers immobilized at the test zones capture the PfLDH in samples. After washing the unbound sample components from the zones, Malstat assay reagents are added for colour development, proportional to the amount of captured PfLDH.

Electrochemical Detection of Insulin with a Novel Protein-Based Artificial Receptor

In this paper, we have developed a new electrochemical method to detect insulin with a protein-based artificial receptor. Diabetes is one of the most serious diseases all over the world. For the treatment of diabetes and maintenance of the patient's quality of life, the control of blood glucose level is important. Usually the blood glucose level in human body is regulated by insulin. Therefore, it is very important to know the status of both glucose and insulin in blood for the treatment of diabetes. Currently, it has become possible to measure the blood glucose level by patients themselves with bioelectrochemical sensors, however, there is no convenient method to measure insulin by patients themselves. In recent years, antibody-based and aptamer-based electrochemical insulin sensors have been developed. Although these sensors are highly sensitive, they may also detect physiologically inactive insulin such as partially digested or mutation insulin because the insulin recognition molecules of these sensors specifically recognize a part of insulin. However, it is desired to detect only active insulin for accurate evaluation of insulin which is functional in blood. Therefore, it is necessary to develop a novel sensor based on a new target recognition mechanism for the accurate evaluation of insulin. In our strategy, we utilized an insulin receptor, because the recognition by the insulin receptor is one of the essential processes for insulin to exhibit its functional role in blood. It has been reported that two domains in the insulin receptor, αCT segment and L1CR domain, recognize an insulin molecule by sandwiching format. However, the insulin receptor is typically buried in a cellular membrane and thus it is difficult to use the receptor with a native format for electrochemical detection. Hence, we have developed a novel artificial insulin receptor by connecting these two domains with a linker. We considered that this new artificial insulin receptor alters its structure in response to insulin recognition, and thus electrochemical properties of an electrode modified with the artificial receptor should reflect the structural change. In order to modify the electrode, we utilized three-time-repeats of a gold-binding peptide (GBP) that is known to assemble on a gold surface spontaneously. These domains were genetically fused and resulting artificial insulin receptors were termed as 3GαL and αL3G depending on their sequence of the domains, respectively. The novel artificial receptors were expressed by introducing plasmids encoding each recombinant gene to animal cells, then purified utilizing histidine-tag affinity. The expression and purification of each receptor was confirmed by SDS-PAGE. The ability of self-assembling on a gold surface was evaluated by QCM-D. The artificial receptor modified electrode was prepared by placing each protein solution on the gold surface of electrodes. The electrochemical response of the modified electrode to insulin was evaluated using electrochemical impedance spectroscopy (EIS). EIS measurement was performed in buffer solution in the presence of 5 mM [Fe(CN)6]3-/4- (the frequency range: 100 mHz to 100 kHz, potential: REST, amplitude: 10 mV). The comparisons of Nyquist plots revealed that the diameters of semicircle increased in the case of electrodes modified with the artificial receptors, when compared with an unmodified electrode. This is probably because the artificial receptors are proteins which inhibit interfacial electron transfer. When insulin was added to the solution, the diameters of semicircle further increased, while no drastic increase was observed when C-peptide was added. Comparison of the responses between electrodes modified with 3GαL and αL3G revealed that the electrode modified with 3GαL exhibited a better S/N ratio in response to insulin. In case of 3GαL, the side of αCT segment was immobilized on the gold surface and L1CR domain whose size is much larger than αCT segment kept rather free. In contrast, in case of αL3G, the side of larger domain of L1CR was already fixed and the smaller part of αCT segment kept rather free. Therefore, it was considered that the effect of structural change induced by the recognition of insulin on the electrochemical property of electrode should be more significant in the case of 3GαL than the case of αL3G. From these results, it has been concluded that insulin can be measured electrochemically with the electrode modified with the protein-based artificial receptors.

Label-Free Sensitive Detection of Steroid Hormone Cortisol Based on Target-Induced Fluorescence Quenching of Quantum Dots.

We discovered that several types of steroid hormones quench the fluorescence of quantum dots (QDs) at close proximity. Inspired by the finding, we developed a new type of biosensor for the sensitive detection of cortisol via direct fluorescence quenching of functionalized QD probes directly induced by the capture of target cortisol without additional reporter reagents. The detection selectivity was provided by cortisol-selective aptamers or anticortisol antibodies conjugated on the QD surfaces. With the magnetic nanoparticle labeling, the new sensing method enabled rapid cortisol sensing at physiologically relevant concentrations and yielded the detection limit of ∼1 nM for aptamer-based sensors and ∼100 pM for antibody-based sensors. We also evaluated the new detection method using saliva samples with an optimized sample preparation process under the assistance of magnetic manipulation. The result showed a satisfying recovery rate for spiked saliva tests. The facile sensing technology offers an appealing approach for the detection of steroid hormones in point-of-care settings.

Development of a highly specific chemiluminescence aptasensor for sulfamethazine detection in milk based on in vitro selected aptamers

Antibody-based sensors are the most widely used for screening antibiotic residues in food samples. However, these sensors always suffer from cross-reacting antibiotics which are able to bind the employed antibodies in a manner similar to the target, thus resulting in inaccurate quantification. In this study, we developed a highly specific and sensitive chemiluminescence aptasensor for sulfamethazine (SMZ) by employing the supernormal selectivity of a new selected aptamer. Four SMZ-specific aptamers (Kd ranging from 79 to 274 nM) were selected in vitro by MB-SELEX with an intentional counter selection to avoid the recognition other sulfonamides, which is impossible to achieve in the corresponding procedure of antibody production. The binding mechanism of aptamers for SMZ was further analyzed and clarified with molecular simulation and docking. Under optimum conditions, the chemiluminescence aptasensor based on the highest affinity aptamer named SA07 provided a limit of detection of 0.92 ng/mL with negligible cross-reactivity with the 27 tested sulfonamides. The developed aptasensor was applied to milk samples, and achieved good accuracy and precision. These results demonstrated that the integration of an aptamer and chemiluminescence in a sensor platform is a promising method for the sensitive, specific, rapid and accurate detection of antibiotic residues of interest in food samples.

Enhancing recombinant antibody performance by optimally engineering its format

Antibody-based sensors are now widely used in therapeutics, diagnostics, and in environmental monitoring. Recombinant antibodies are becoming integral parts of such devices due to their reported high affinities, their capacity for engineering to achieve highly defined performance characteristics and the fact that their production can be optimized to a significant degree. To aid as a model for the identification of important analyte binding residues within the antibody sub-structure and elucidate the docking characteristics of small molecules such as metabolites, illicit drugs, biotherapeutics (proteins, peptides and nucleic acids) or toxins towards the antibody, herein we report the binding of the harmful cyanobacterial-toxin, microcystin-leucine-arginine (MC-LR) to a single chain fragment variable (scFv) antibody fragment. Analysis of the binding of MC-LR to this scFv was used to identify key residues of interest and to show how ‘freely-available’ and ‘easily-accessible’ computer-based webservers can be utilized to initiate an investigation into the binding characteristics of interacting molecules.In this study, a detailed investigation of the sub-structure of the anti-MC-LR (scFv) was carried out and antibody/small-molecule binding interactions were analyzed. The profile elucidated using computational analysis revealed amino acids of importance in the complementarity determining region light chain region 3 (CDRL3) and framework region 3 (FR3) of the heavy chain. Important amino acid residues within CDRL3 and FR3 were mutated in vitro and sensitivity and binding profiles were examined. It was identified that phenylalanine (F) at position 91 and aspartate (D) at position 92 of the light chain region, and arginine (R) at position 66 in framework region 3 (FR3) of the heavy chain were nvolved in binding. The introduction of an auxiliary antibody domain to the variable heavy and variable light (scFv) to ascertain its influence on stability and binding was also examined.The strategy adopted provided a deeper knowledge of scFv sub-structure and identified the regions and amino acids essential to antibody/small-molecule binding.

Detection of Plasmodium Lactate Dehydrogenase Antigen in Buffer Using Aptamer-Modified Magnetic Microparticles for Capture, Oligonucleotide-Modified Quantum Dots for Detection, and Oligonucleotide-Modified Gold Nanoparticles for Signal Amplification.

To overcome the limitations associated with antibody-based sensors, we describe a proof-of-concept of an aptamer-based sandwich assay for detection of lactate dehydrogenase, an antigen associated with malaria. We show a detection limit of Plasmodium falciparum lactate dehydrogenase and Plasmodium vivax lactate dehydrogenase of 0.5 fmole in buffer, comparable to an antibody-based assay, using a magnetic particle-aptamer construct for capture and a quantum dot-aptamer construct for detection. We then demonstrate a detection limit of 10 amole (50-fold amplification) using oligonucleotide-functionalized gold nanoparticles to allow the conjugation of multiple quantum dots for each target antigen.

Aptamer-Based Microfluidic Electrochemical Biosensor for Monitoring Cell-Secreted Trace Cardiac Biomarkers.

Continual monitoring of secreted biomarkers from organ-on-a-chip models is desired to understand their responses to drug exposure in a noninvasive manner. To achieve this goal, analytical methods capable of monitoring trace amounts of secreted biomarkers are of particular interest. However, a majority of existing biosensing techniques suffer from limited sensitivity, selectivity, stability, and require large working volumes, especially when cell culture medium is involved, which usually contains a plethora of nonspecific binding proteins and interfering compounds. Hence, novel analytical platforms are needed to provide noninvasive, accurate information on the status of organoids at low working volumes. Here, we report a novel microfluidic aptamer-based electrochemical biosensing platform for monitoring damage to cardiac organoids. The system is scalable, low-cost, and compatible with microfluidic platforms easing its integration with microfluidic bioreactors. To create the creatine kinase (CK)-MB biosensor, the microelectrode was functionalized with aptamers that are specific to CK-MB biomarker secreted from a damaged cardiac tissue. Compared to antibody-based sensors, the proposed aptamer-based system was highly sensitive, selective, and stable. The performance of the sensors was assessed using a heart-on-a-chip system constructed from human embryonic stem cell-derived cardiomyocytes following exposure to a cardiotoxic drug, doxorubicin. The aptamer-based biosensor was capable of measuring trace amounts of CK-MB secreted by the cardiac organoids upon drug treatments in a dose-dependent manner, which was in agreement with the beating behavior and cell viability analyses. We believe that, our microfluidic electrochemical biosensor using aptamer-based capture mechanism will find widespread applications in integration with organ-on-a-chip platforms for in situ detection of biomarkers at low abundance and high sensitivity.

Strategy for making a superior Quenchbody to proteins: effect of the fluorophore position.

Antibody-based sensors have made outstanding contributions to the fields of molecular biology and biotechnology. Our group recently developed a novel powerful fluorescent immunosensor strategy named Quenchbody (Q-body), which has been applied to the detection of a range of antigens in a rapid, simple, and sensitive manner. However, there were some Q-bodies whose fluorescence response was limited, especially for detecting protein antigens. With the aim of improving this issue, here we made twelve types of Q-bodies incorporated with different number and position of TAMRA fluorophore in the single chain Fv of HyHEL-10, an anti-hen egg lysozyme antibody, as a model. By measuring the fluorescence intensity and its antigen dependency, it was revealed that VL-VH type Q-bodies labeled at a non-CDR loop region of the VL shows the highest fluorescence response. This position locates close to the quenching Trp35 in VL, while it is far from Trp residues in the bound antigen. This result clearly suggests the importance of dye position to maximize the fluorescence quenching and antigen-dependent de-quenching. The discovery may open a way to make many other Q-bodies with superior response.

Myelin basic protein immunosensor for multiple sclerosis detection based upon label-free electrochemical impedance spectroscopy

A novel highly sensitive impedimetric Myelin Basic Protein (MBP) immunosensor for the determination of a Multiple Sclerosis (MS) autoantibody, Anti-Myelin Basic Protein (Anti-MBP) was developed by immobilization of MBP on Gelatin and Gelatin–Titanium Dioxide (TiO2) modified platinium electrode. Cyclic voltammetric (CV) and Electrochemical Impedance Spectroscopic (EIS) methods were employed in determination of the electrode responses and applicability. Gelatin-MBP and gelatin–TiO2-MBP electrodes were prepared by chemical immobilization of the substrates onto the platinium electrodes. The formal potentials of MBP confined on gelatin-MBP and gelatin–TiO2-MBP surfaces are estimated to be 195 and 205mV, respectively. Thus, a little more reversible electron transfer reaction occurs on the gelatin–TiO2-MBP immunosensor surface. The peak separations of MBP (150mV and 110mVs−1 at 100mVs−1) and the asymmetric anodic and cathodic peak currents indicate that the electron transfer between Anti-MBP and gelatin-MBP/gelatin–TiO2-MBP immunosensor is quasireversible. Control samples containing a nonspecific human immunoglobulin G (hIgG) antibody were also studied, and calibration curves were obtained by subtraction of the responses for specific and nonspecific antibody-based sensors. Gelatin-MBP and gelatin–TiO2-MBP immunosensors have detection limit of 0.1528ngml−1 and 0.1495ngml−1 respectively. This immunosensor exhibits high sensitivity and low response times (58s for gelatin-MBP and 46s for gelatin–TiO2-MBP immunosensor). The developed label-free impedimetric immunosensors also provide a simple and sensitive detection method for the specific determination of Anti-MBP in human cerebrospinal fluid (CSF) and serum samples.

Molecular PINCERs® for biomarker analysis and their potential application in hepatitis C diagnosis

Molecular PINCERs(®) are antibody-based protein sensors that allow rapid and sensitive detection of specific proteins in solution. The sensors allow quick detection of a target, with sensitivity and specificity approaching that of conventional immunoassays, but require no sample manipulation other than mixing of the test sample with sensors. Combining the PINCER assay with immunoprecipitation allows quick and sensitive detection of anti-HCV immunoglobulin (Ig)G and IgM in human serum. This improvement is achieved through binding of anti-HCV IgG and IgM from serum to HCV-antigen-immobilized microspheres, followed by quantification of the IgG and IgM using molecular PINCERs. The PINCER-based assay is cost-effective, sensitive and has potential for clinical diagnostic use.

Effect of buried potential barrier in label-less electrochemical immunodetection of glutathione and glutathione-capped gold nanoparticles

The influence of potential barriers, introduced to the immunoglobulin-based sensory films, on voltammetric signals of a redox ion probe has been investigated. Films with positive and negative barriers have been examined by depositing charged self-assembled thiol monolayers as the basal layers of a sensory film. The studies performed with monoclonal anti-glutathione antibody-based sensors using ferricyanide ion probe have shown stronger sensor response to the layer components, as well as to the glutathione-capped gold nanoparticles acting as the antigen, for films with positive potential barrier buried deep in the film than for negative barrier films. The larger changes in differential resistance, peak separation and peak heights observed for films with positive barrier have been attributed to different depth and width of the charge distributions in these films. A buried positive barrier with narrow charge distribution width provides the best conditions for film stability and prevents fouling (less ion-exchanges with the medium). This conclusion has been confirmed by calculations of the electric field distribution and potential profiles in immunosensing films performed by numerical integration of Poisson equation for Gaussian distributions of fixed charges of covalently bound components. The proposed fixed-charge model can aid in rapid evaluation of sensory films in sensor development work. The implications of potential barriers in sensory film design are discussed.

Antibody-Based Sensors: Principles, Problems and Potential for Detection of Pathogens and Associated Toxins

Antibody-based sensors permit the rapid and sensitive analysis of a range of pathogens and associated toxins. A critical assessment of the implementation of such formats is provided, with reference to their principles, problems and potential for ‘on-site’ analysis. Particular emphasis is placed on the detection of foodborne bacterial pathogens, such as Escherichia coli and Listeria monocytogenes, and additional examples relating to the monitoring of fungal pathogens, viruses, mycotoxins, marine toxins and parasites are also provided.

Labeless Immunosensor Assay for the Stroke Marker Protein Neuron Specific Enolase Based upon an Alternating Current Impedance Protocol

This paper describes the development and characterization of a label-less immunosensor for neuron-specific enolase (NSE) and its interrogation using an ac impedance protocol. Commercial screen-printed carbon electrodes were used as the basis for the sensor. Poly(1,2-diaminobenzene) was electrodeposited onto the sensors--and this modified surface was then sonochemically ablated to form an array of micropores. A second electropolymerization step was then used to deposit conductive polyaniline within these pores to give a microarray of polyaniline protrusions with diameters of several mum. This array was then utilized as a substrate to immobilize a biotinylated antibody for NSE using a classical avidin-biotin approach. Electrodes containing the antibodies were exposed to solutions of NSE and interrogated using an ac impedance protocol. The real component of the impedance of the electrodes was found to increase with increasing concentration of antigen. Control samples containing a nonspecific IgG antibody were also studied and calibration curves obtained by subtraction of the responses for specific and nonspecific antibody-based sensors, thereby accounting for and eliminating the effects of nonspecific adsorption of NSE. A linear relationship between the concentration of NSE in buffer solutions from 0 to 50 pg mL(-1) and the impedimetric response was observed.