The importance of interfacial design for the sensitivity of a label-free electrochemical immuno-biosensor for small organic molecules

An immuno-biosensing interface comprising a mixed layer of an oligo(ethylene glycol) (OEG) component, and an oligo(phenylethynylene) molecular wire (MW) is described. The OEG controls the interaction of proteins and electroactive interferences with the surface and the MW allows electrochemical communication to the underlying glassy carbon electrode. The layers are formed from in situ generated-aryl diazonium cations. To the distal end of the MW, a redox probe 1,1'-di(aminomethyl)ferrocene is attached followed by the surface bound epitope (the structural feature the antibody selectively recognizes) to which an antibody would bind. Association or disassociation of the antibody with the sensing interface causes a modulation of the ferrocene electrochemistry. X-ray photoelectron spectroscopy, cyclic voltammetry, and square wave voltammetry have been used to characterize the step-wise fabrication of the sensing interface. The influence of the molar ratio of the MW and OEG deposited onto the sensor interface was explored relative to the final sensor sensitivity. Five combinations of MW/OEG 1:0, 1:20, 1:50, 1:75 and 1:100 were tested on sensor sensitivity detection for a model analyte (biotin) free in solution, via a displacement assay. The ratio of 1:50 was found to give the highest sensitivity. At this ratio, good reproducibility (RSD 6.8%) and repeatability (RSD 9.6%) was achieved. This immuno-biosensor provides an intervention free immuno-biosensing platform for agriculture and biomedical samples.     

3D Active Stabilization System with Sub-Micrometer Resolution

Stable positioning between a measurement probe and its target from sub- to few micrometer scales has become a prerequisite in precision metrology and in cellular level measurements from biological tissues. Here we present a 3D stabilization system based on an optoelectronic displacement sensor and custom piezo-actuators driven by a feedback control loop that constantly aims to zero the relative movement between the sensor and the target. We used simulations and prototyping to characterize the developed system. Our results show that 95 % attenuation of movement artifacts is achieved at 1 Hz with stabilization performance declining to ca. 70 % attenuation at 10 Hz. Stabilization bandwidth is limited by mechanical resonances within the displacement sensor that occur at relatively low frequencies, and are attributable to the sensor's high force sensitivity. We successfully used brain derived micromotion trajectories as a demonstration of complex movement stabilization. The micromotion was reduced to a level of ～1 μm with nearly 100 fold attenuation at the lower frequencies that are typically associated with physiological processes. These results, and possible improvements of the system, are discussed with a focus on possible ways to increase the sensor's force sensitivity without compromising overall system bandwidth.     

Sensitive immobilization-free electrochemical DNA sensor based on isothermal circular strand displacement polymerization reaction

A highly sensitive electrochemical DNA sensor that requires no probe immobilization has been developed based on a target recycling mechanism utilizing a DNA polymerase with a strand displacement activity. The electrochemical detection is realized by taking advantage of the difference in diffusivity between a free ferrocene-labeled peptide nucleic acid (Fc-PNA) and a Fc-PNA hybridized with a complementary DNA, while the DNA polymerase-assisted target recycling leads to signal generation and amplification. The hybridization of the target DNA opens up a stem-loop template DNA with the Fc-PNA hybridized to its extruded 5' end and allows a DNA primer to anneal and be extended by the DNA polymerase, which results in sequential displacement of the target DNA and the Fc-PNA from the template DNA. The displaced target DNA will hybridize with another template DNA, triggering another round of primer extension and strand displacement. The released Fc-PNA, due to its neutral backbone, has much higher diffusivity towards a negatively charged electrode, compared to that when it is hybridized with a negatively charged DNA. Therefore, a significantly enhanced signal of Fc can be observed. The outstanding sensitivity and simplicity make this approach a promising candidate for next-generation electrochemical DNA sensing technologies.     

Continuous-flow fluoro-immunosensor for paclitaxel measurement

A fluorescence-based continuous-flow immunosensor for sensitive, precise, accurate and fast determination of paclitaxel was developed. The sensor utilizes anti-paclitaxel antibody immobilized through its Fc region and crosslinked by dimethylpimelimidate to protein A attached covalently onto the silanized inner walls of a glass capillary column followed by saturation of the paclitaxel-binding sites with rhodamine-labeled paclitaxel. The assay is based on the displacement and detection downstream of the rhodamine-labeled paclitaxel, by a flow-through spectrofluorometer, as a result of the competition with paclitaxel introduced as a pulse into the stream of carrier buffer flowing through the system. The peak height of the fluorescence intensity profile of the displaced rhodamine-labeled paclitaxel was directly proportional to the concentration of paclitaxel applied and was a function of the carrier buffer flow rate. The sensitivity of the immunosensor response ranged from 0.31 relative fluorescence units (RFU)/ng/ml at a flow rate 0.1 ml/min to 0.52 RFU/ng/ml at 1 ml/min, while the lower detection limit ranged from 1 ng/ml at 0.1 ml/min to 4 ng/ml at 1 ml/min. The immunosensor response was very reproducible (RSD=4.8%; n=10) and linear up to 100 ng/ml. The assay time ranged from 2 min at 1 ml/min to 8 min at 0.1 ml/min. A technique developed to resaturate the antigen binding sites of the immobilized antibody with rhodamine-labeled paclitaxel was successful in regenerating the capillary column without affecting its performance, thus enhancing the economic viability of the immunosensor. The immunosensor was successfully applied for the determination of paclitaxel in human plasma.     

Needle-type lactate biosensor

A needle-type lactate biosensor has been developed for continuous intravascular lactate monitoring. The sensor employs poly(1,3-phenylenediamine) as the inner layer on the platinum electrode in order to eliminate the interference from oxidizable physiological substances. Cross-linking with glutaraldehyde was used for enzyme immobilization. Dithiothreitol was used as the stabilizer of lactate oxidase. PVC (polyvinyl chloride) was chosen as the external diffusion control membrane. Sensor performance was evaluated in vitro and the sensor shows a sensitivity of 10-15 nA/mM, and a linear range from 1 mM to at least 15 mM lactate. Evaluation of the sensor response in blood plasma showed similar sensitivity and linear range as indicated by the calibration curves obtained in buffer solution. The sensor has a short response time of approximately 1 minute. The sensors were operated continuously for 7 days in phosphate buffer containing solution with a concentration at the physiological lactate level. No significant change in sensor sensitivity and its linear range has been observed. Sensors show a minimum change in its performance when stored in buffer at 4 degrees C for at least 9 months.     

Irreversible enzyme-shuttle immunoassay

The concept of a competitive enzyme immunoassay that utilizes simultaneously the bound and the free analyte-enzyme conjugate (heterobifunctional conjugate) for signal generation in response to varying analyte concentrations in samples has been investigated. Two antigenic sites of the heterobifunctional conjugate are used in the assay for binding to immunoglobulins: the analyte derivative binds to an immobilized antibody, Ab(1), and the enzyme component binds to a spatially separated immobilized antibody, Ab(2). The analytical system is set up such that in the absence of analyte, the conjugate is predominantly bound in the compartment that contains Ab(1). With increasing concentration of native analyte in samples, an increasing concentration of native analyte in samples, an increasing amount of conjugate migrates to the second compartment that contains Ab(2). The enzyme bound in each compartment is used for signal generation. Mathematical models have been developed to determine the optimal conditions and to predict the performance of such dual-antibody systems. The theoretical predictions are supported by experimental results. The dual-antibody system has been compared with a conventional competitive enzyme immunoassay using the same reagents.     

Compensation of large motion sensor displacements during long recordings of limb movements

In motion capture applications using electromagnetic tracking systems the process of anatomical calibration associates the technical frames of sensors attached to the skin with the human anatomy. Joint centers and axes are determined relative to these frames. A change of orientation of the sensor relative to the skin renders this calibration faulty. This sensitivity regarding sensor displacement can turn out to be a serious problem with movement recordings of several minutes duration. We propose the “dislocation distance” as a novel method to quantify sensor displacement and to detect gradual and sudden changes of sensor orientation. Furthermore a method to define a so called fixed technical frame is proposed as a robust reference frame which can adapt to a new sensor orientation on the skin. The proposed methods are applied to quantify the effects of sensor displacement of 120 upper and lower limb movement recordings of newborns revealing the need for a method to compensate for sensor displacement. The reliability of the fixed technical frame is quantified and it is shown that trend and dispersion of the dislocation distance can be significantly reduced. A working example illustrates the consequences of sensor displacement on derived angle time series and how they are avoided using the fixed technical frame.     

A portable surface plasmon resonance sensor system for real-time monitoring of small to large analytes

Many environmental applications exist for biosensors capable of providing real-time analyses. One pressing current need is monitoring for agents of chemical- and bio-terrorism. These applications require systems that can rapidly detect small organics including nerve agents, toxic proteins, viruses, spores and whole microbes. A second area of application is monitoring for environmental pollutants. Processing of grab samples through chemical laboratories requires significant time delays in the analyses, preventing the rapid mapping and cleanup of chemical spills. The current state of development of miniaturized, integrated surface plasmon resonance (SPR) sensor elements has allowed for the development of inexpensive, portable biosensor systems capable of the simultaneous analysis of multiple analytes. Most of the detection protocols make use of antibodies immobilized on the sensor surface. The Spreeta 2000 SPR biosensor elements manufactured by Texas Instruments provide three channels for each sensor element in the system. A temperature-controlled two-element system that monitors for six analytes is currently in use, and development of an eight element sensor system capable of monitoring up to 24 different analytes will be completed in the near future. Protein toxins can be directly detected and quantified in the low picomolar range. Elimination of false positives and increased sensitivity is provided by secondary antibodies with specificity for different target epitopes, and by sensor element redundancy. Inclusion of more than a single amplification step can push the sensitivity of toxic protein detection to femtomolar levels. The same types of direct detection and amplification protocols are used to monitor for viruses and whole bacteria or spores. Special protocols are required for the detection of small molecules. Either a competition type assay where the presence of analyte inhibits the binding of antibodies to surface-immobilized analyte, or a displacement assay, where antibodies bound to analyte on the sensor surface are displaced by free analyte, can be used. The small molecule detection assays vary in sensitivity from the low micromolar range to the high picomolar.

     

A portable surface plasmon resonance sensor system for real-time monitoring of small to large analytes

Many environmental applications exist for biosensors capable of providing real-time analyses. One pressing current need is monitoring for agents of chemical- and bio-terrorism. These applications require systems that can rapidly detect small organics including nerve agents, toxic proteins, viruses, spores and whole microbes. A second area of application is monitoring for environmental pollutants. Processing of grab samples through chemical laboratories requires significant time delays in the analyses, preventing the rapid mapping and cleanup of chemical spills. The current state of development of miniaturized, integrated surface plasmon resonance (SPR) sensor elements has allowed for the development of inexpensive, portable biosensor systems capable of the simultaneous analysis of multiple analytes. Most of the detection protocols make use of antibodies immobilized on the sensor surface. The Spreeta 2000 SPR biosensor elements manufactured by Texas Instruments provide three channels for each sensor element in the system. A temperature-controlled two-element system that monitors for six analytes is currently in use, and development of an eight element sensor system capable of monitoring up to 24 different analytes will be completed in the near future. Protein toxins can be directly detected and quantified in the low picomolar range. Elimination of false positives and increased sensitivity is provided by secondary antibodies with specificity for different target epitopes, and by sensor element redundancy. Inclusion of more than a single amplification step can push the sensitivity of toxic protein detection to femtomolar levels. The same types of direct detection and amplification protocols are used to monitor for viruses and whole bacteria or spores. Special protocols are required for the detection of small molecules. Either a competition type assay where the presence of analyte inhibits the binding of antibodies to surface-immobilized analyte, or a displacement assay, where antibodies bound to analyte on the sensor surface are displaced by free analyte, can be used. The small molecule detection assays vary in sensitivity from the low micromolar range to the high picomolar.     

Real-time monitoring of the strand displacement amplification (SDA) of human cytomegalovirus by a new SDA-piezoelectric DNA sensor system

Nucleic acid amplification has long been used in biosensor technologies, such as DNA sensors, DNA chips and microarrays, due to its advantage of high sensitivity in detecting target DNA. However, dynamic monitoring of nucleic acid amplifications with traditional DNA sensors in real-time is difficult since a constant temperature must be maintained during detection. Thus, the piezoelectric sensor, one type of traditional DNA sensor, is not applicable in real-time monitoring PCR due to the dramatic change in temperature that occurs during reaction. In this study, we introduced strand displacement amplification (SDA), an well-developed nucleic acid amplification technique that can work under conditions of constant temperature, into the development of a novel piezoelectric sensor. Using the new SDA-piezoelectric DNA sensor, we designed a stable system for liquid-phase detection, in which the crystal oscillator plate was fixed by an easily adjustable screw-threaded clamping mechanism and successfully applied the new sensor system to real-time SDA monitoring of human cytomegalovirus (HCMV). This new technique overcomes the shortcomings of traditional DNA sensors in real-time monitoring of nucleic acid amplification. The technique has proved to be a markedly simplified procedure with a number of advantages, such as higher sensitivity, better time efficiency, and the ability of dynamic real-time detection.     

Comparative Inhibitory Effects of Antigen and Antibody in the Staphylococcal Enterotoxin Solid-Phase Radioimmunoassay System

A solid-phase radioimmunoassay employing 125I-labeled enterotoxins and polystyrene tubes coated with specific antibody has been developed for assaying the relative concentrations of antibodies to staphylococcal enterotoxins A and B. Competitive binding occurs between tube-bound antibody and free antibody for binding sites on 125I-labeled enterotoxin. The sensitivity of the system is affected by the amount of antibody on the walls of the tubes, the concentration of 125I-labeled enterotoxin added to the system, and probably by the relative binding affinities of the bound and unbound antibodies. Antibody, 0.01 to 0.07 mug/ml, inhibited the uptake of 125I-labeled enterotoxin by 20%. Both the antibody and antigen solid-phase radioimmunoassay inhibition systems can be appropriately represented by either of the following two models: Loge (Y/1 - Y) = alpha0 + alpha1 LogeX and LogeY = beta0 + beta1 LogeX, where Y is bound activity, X is antigen or antibody concentration for inhibition, and alpha0, alpha1, beta0, and beta1 are regression coefficients. Estimates from the first model were slightly more precise for the antibody system, whereas the reverse was true for the antigen system.     

Disposable amperometric immunosensor system for rabbit IgG using a conducting polymer modified screen-printed electrode

A disposable and mediatorless immunosensor based on a conducting polymer (5,2':5'2"-terthiophene-3'-carboxylic acid) coated screen-printed carbon electrode has been developed using a separation-free homogeneous technique for the detection of rabbit IgG as a model analyte. Horseradish peroxidase (HRP) and streptavidin were covalently bonded with the polymer on the electrode and biotinylated antibody was immobilized on the electrode surface using avidin-biotin coupling. This sensor was based on the competitive assay between free and labeled antigen for the available binding sites of antibody. Glucose oxidase was used as a label and in the presence of glucose, H(2)O(2) formed by the analyte-enzyme conjugate was reduced by the enzyme channeling via HRP bonded on the electrode. The catalytic current was monitored amperometrically at -0.35 V vs. Ag/AgCl and this method showed a linear range of RIgG concentrations from 0.5 to 2 microg/ml with standard deviation +/-0.0145 (n=4). Detection limit was determined to be 0.33 microg/ml.     

A coated-tube radioimmunoassay for beta-thromboglobulin

A solid phase radioimmunoassay for beta-thromboglobulin is presented which has the advantage of easy technical performance. The antibody was absorbed onto polystyrene tubes and the separation of free and bound radiolabelled tracer was performed by simply sucking the tubes empty after incubation. The assay is precise and accurate but not particularly rapid, since an overnight incubation is needed. Maximum assay sensitivity was found to be 5.6 ng/ml beta-thromboglobulin for plasma samples. Preliminary studies showed that the sensitivity can be increased by the use of a more dilute antiserum. The plasma concentration of beta-thromboglobulin in 37 normal subjects was 17 +/- 4 ng/ml. The analytical performance of the assay corresponds to a commercial liquid-phase kit used for reference.     

A multifactorial screening strategy to identify anti-idiotypic reagents for bioanalytical support of antibody therapeutics

Antibodies are critical tools for protein bioanalysis; their quality and performance dictate the caliber and robustness of ligand binding assays. After immunization, polyclonal B cells generate a diverse antibody repertoire against constant and variable regions of the therapeutic antibody immunogen. Herein we describe a comprehensive and multifactorial screening strategy to eliminate undesirable constant region-specific antibodies and select for anti-idiotypic antibodies with specificity for the unique variable region. Application of this strategy is described for the therapeutic antibody Mab-A case study. Five different factors were evaluated to select a final antibody pair for the quantification of therapeutics in biological matrices: (i) matrix effect in preclinical and clinical matrices, (ii) assay sensitivity with lower limit of quantification goal of single-digit ng/ml (low pM) at a signal-to-background ratio greater than 5, (iii) epitope distinction or nonbridging antibody pair, (iv) competition with target and inhibitory capacity enabling measurement of free drug, and (v) neutralizing bioactivity using bioassay. The selected antibody pair demonstrated superior assay sensitivity with no or minimal matrix effect in common biological samples, recognized two distinct binding epitopes on the therapeutic antibody variable region, and featured inhibitory and neutralizing effects with respect to quantification of free drug levels.     

Electrochemical immunosensor for label free epidermal growth factor receptor (EGFR) detection

This paper presents an electrochemical immune sensor for label free detection of epidermal growth factor receptor (EGFR) by immobilizing anti-EGFR antibody (Anti-EGFRab) on dithiobissuccinimidyl propionate (DTSP) self-assembled monolayer (SAM) on gold (Au) electrode. Electrochemical studies show that increased surface concentration of redox moieties onto Anti-EGFRab/DTSP immuno-electrode leads to high electron transport and improved sensing performance. The antigen-antibody complex demonstrates a high association constant (5×10(12)L/mol) that results in high affinity of Anti-EGFRab to EGFR, confirming that the DTSP-SAM provides a conducive environment for anti-EGFR immobilization. The electrochemical response of EA/Anti-EGFRab/DTSP/Au electrode as a function of EGFR concentrations exhibits a linear range from 1pg/mL to 100ng/mL, a detection limit of 1pg/mL at a sensitivity of 2.02μAM(-1)at a regression coefficient of 0.99.     

Biomolecules/gold nanowires-doped sol-gel film for label-free electrochemical immunoassay of testosterone

A direct, rapid, and label-free electrochemical immunoassay method for testosterone has been described based on encapsulating testosterone antibody into polyvinyl butyral sol–gel film doped with gold nanowires. Gold nanowires prepared by using nanopore polycarbonate membrane were used to conjugate testosterone antibody onto the probe surface. The presence of gold nanowires provided a biocompatible microenvironment for biomolecules, greatly amplified the immobilized amount of biomolecules on the electrode surface, and improved the sensitivity of the immunosensor. In comparison with gold nanoparticle-conjugating probe, the gold nanowire-functionalized probe could avoid the leakage of biomolecules from the composite film, and enhanced the stability of the sensor. The performance and factors influencing the performance of the resulting immunosensor were investigated in detail. Under optimal conditions, the developed immunosensor exhibited a good linear relationship with testosterone ranging from 1.2 to 83.5 ng mL^− 1 with a detection limit of 0.1 ng mL^− 1 (at 3δ). Moreover, the proposed immunosensor exhibited high sensitivity, good reproducibility and long-term stability. The as-prepared immunosensors were used to analyze testosterone in human serum specimens. Analytical results suggest that the developed immunoassay has a promising alternative approach for detecting testosterone in the clinical diagnosis. Compared with the conventional ELISAs, the proposed immunoassay method was simple and rapid without multiple labeling and separation steps. Importantly, the route provides an alternative approach to incorporate gold nanowires into the solid matrix for biosensing application.     

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.     

Analysis of pigeon intestinal mucin allergens using a novel dot blot assay

Many glycoproteins contain multiple glycosylation sites that can present multi-valent epitopes for antigenic recognition. Release of the oligosaccharides results in loss of avidity of antibody binding, which has been overcome by reforming clustered ligands, usually by reductive amination of free reducing oligosaccharides to poly-amine groups. We have adapted this approach to hydrazinolytic release of O-linked chains of mucin glycoproteins and 'one-pot' microscale coupling to poly-L-lysine (PLL). The conjugated PLL adheres to nitrocellulose membranes through washing procedures required for antibody or lectin overlay and detection. We show evidence for the applicability of this technique using lectin and antibody reactivity to the oligosaccharides of pigeon intestinal mucins, which have been implicated in the allergic disease pigeon fanciers' lung.     

Rapid and sensitive biosensor for Salmonella

The rapid and sensitive detection of Salmonella typhymurium based on the use of a polyclonal antibody immobilized by the Langmuir-Blodgett method on the surface of a quartz crystal acoustic wave device was demonstrated. The binding of bacteria to the surface changed the crystal resonance parameters; these were quantified by the output voltage of the sensor instrumentation. The sensor had a lower detection limit of a few hundred cells/ml, and a response time of < 100 s over the range of 10(2)-10(10) cells/ml. The sensor response was linear between bacterial concentrations of 10(2)-10(7) cells/ml, with a sensitivity of 18 mV/decade. The binding of bacteria was specific with two binding sites needed to bind a single cell. The sensors preserve approximately 75% of their sensitivity over a period of 32 days.     

Supramolecular formation of antibodies with viologen dimers: utilization for amplification of methyl viologen detection signals in surface plasmon resonance sensor

Monoclonal antibodies for 1-(carboxypentyl)-1'-methyl-4,4'-bipyridinium dichloride have been prepared. The complex formation of one of the antibodies, 10D5, with viologen dimer has been studied by a biosensor technique based on surface plasmon resonance. The dissociation constants of the complex between antibody 10D5 and methyl viologen or viologen dimer are found to be (2.0 +/- 0.2) x 10 (-7) and (1.5 +/- 0.5) x 10 (-7) M, respectively. Enhancement of response signal intensities in SPR is observed by the addition of the antibody solution to the viologen dimer-antibody complex indicating the formation of linear supramolecules between the antibody and viologen dimer. Amplification of methyl viologen sensing processes is realized by the inhibition of the complex formation between antibodies and viologen dimer-antibody complexes by methyl viologen and signal enhancement due to the supramolecular formation of the antibody and viologen dimer. The sensitivity in this system is found to be 2 orders larger than that obtained in the simple addition of methyl viologen to the antibody immobilized to the surface of the sensor chip.