Fiber-optic Immunosensor Research Articles

Effects of blocking buffers and plasma proteins on the protein C biosensor performance.

Protein C (PC) deficiency can lead to abnormal thrombus formation in blood vessels, obstructing oxygen and nutrient transport to various organs or tissues. Quantifying PC amount in blood plasma usually takes long time, is difficult and expensive due to its low concentration and other homologous proteins in it. A fiber-optic immunosensor has been under development for several years for the PC real-time assay. In this study, blocking buffers have been examined to reduce the noise caused by non-specific adsorption. Ethanolamine was found to reduce the noise significantly. The effect of various PC homologues (factors II, VII, IX, and X) on the biosensor performance was investigated. The sensor showed little cross reactivity with these homologues. Human serum albumin (HSA) in the sample decreased the signal intensity. However, PC could be still quantified in samples with HSA at the physiological level.

Assay procedure optimization of a rapid, reusable protein C immunosensor for physiological samples.

A fiber-optic immunosensor for quantifying the protein C (PC) amount in a plasma sample is being developed to provide accurate, fast, cost-effective diagnosis of heterozygous PC deficiency (0.5-2.5 microg ml(-1)). As a progress report on the sensor development, this paper focuses on optimizations of vanous assay steps. These include: (1) the primary antibody concentration; (2) the effect of the primary antibody leaching on the sensitivity; (3) the fluorophore to secondary antibody ratio; and (4) the sample and secondary antibody incubation times. The optimal primary antibody concentration for the PC sensor was determined to be 65 microg ml(-1); its leaching was minor, stabilized within 3 days, and the sensor sensitivity change after 30 days of storage was minor; sample and secondary antibody incubation times were reduced from 10 to 5 and from 5 to 3 min, respectively, while maintaining a reasonable signal-to-noise level. The immunosensor was also tested with (5) human serum albumin and human plasma and (6) with and without convective flow. High viscosity of human plasma decreased signal intensity, but clear signal discrimination was possible in the concentration range of interest. (7) Application of convective flow increased the signal intensity by increasing PC mass transport rate to the sensor surface. The optimized PC immunosensor provides a smaller (100 microl sample chamber) and faster (10-15 min) tool for PC detection in human plasma.

Portable fiber-optic immunosensor for detection of methsulfuron methyl

This fiber optic sensor, based on competitive immunoreactions between coating-haptens and free haptens in solutions with corresponding antibodies, was developed to determine the concentration of the free hapten, methsulfuron-methyl. The ovalbumin (OVA)-methsulfuron-methyl conjugate was immobilized on a microscope slide. Horseradish peroxidase (HRP) labeled goat anti-rabbit IgG was used to generate an optical signal. The portable optical device consisted of a 0.25-W tungsten-halogen light source and a photosensitive diode detector. A typical competitive-binding calibration curve was seen between 0.3 and 100 ng/ml of methsulfuron-methyl. The detection limit for methsulfuron-methyl was 0.1 ng/ml.

Fiber-optic immunosensor for mycotoxins.

Evanescent wave-based fiber-optic immunosensors were studied for the detection of fumonisins and aflatoxins in maize. Two formats, competitive and non-competitive, were used. A competitive format was used to measure fumonisin B1 (FB1) in both spiked and naturally contaminated maize samples. Fumonisin monoclonal antibodies were covalently coupled to an optical fiber and the competition between FB1 and FB1 labeled with fluorescein (FB1-FITC) for the limited number of binding sites on the fiber was assessed. The signal generated in the assay was inversely proportional to the FB1 concentration. For samples, the concentration causing an inhibition of binding by 50% (IC50) was dependent upon the clean-up procedure used. Simple dilution of methanolic maize extracts yielded an assay with an IC50 equivalent to 25 microg FB1 g(-1) maize with a limit of detection of 3.2 microg g(-1) maize. Affinity column clean-up yielded an assay with an IC50 equivalent to 5 microg FB1 g(-1) maize (limit of detection 0.4 microg FB1 g(-1)). An HPLC method and the immunosensor method agreed well for naturally contaminated maize samples except when large amounts of other fumonisins that cross-react with the immunosensor were present. The second sensor format, for the mycotoxin aflatoxin B1 (AFB1), was a non-competitive assay using the native fluorescence of this mycotoxin. Because the fluorescence of AFB1 itself was detected, the response of the sensor was directly proportional to the toxin concentration. The sensor, while capable of detecting as little as 2 ng ml(-1) of AFB1 in solution was technically not an immunosensor, since the attachment of aflatoxin specific antibodies was not required. Sensors of the formats described have the potential to rapidly screen individual maize samples but require coupling with a clean-up technique to be truly effective.

Multi-analyte explosive detection using a fiber optic biosensor

A fiber optic immunosensor has been developed for simultaneous detection of the most common explosives, 2,4,6,-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). It employs a competitive immunoassay on the antibody-coated fiber optic probes, in which a fluorescent antigen competes with free antigen of unknown concentration for binding sites on the fiber surface. To achieve dual explosive detection, two α-TNT fiber probes and two α-RDX fiber probes are connected in series. The sample is mixed with fluorescent analogs, Cy5-ethylenediamine-trinitrobenzene (Cy5-EDA-TNB) and Cy5-ethylenediamine-RDX hapten (Cy5-EDA-RDH). Inhibition of the maximum signal in the presence of the sample is proportional to the concentration of the explosive. The detection limits for the multi-analyte assays are equivalent (6 ng/ml for both TNT and RDX) to those of the individual assays (5 ng/ml for both TNT and RDX). The standard curves for TNT and RDX represent a linear relationship between percent signal inhibition and the natural logarithm of analyte concentration in the multi-analyte format, as well as in single analyte assays, thus allowing a simple and precise method of quantification. There is minimal cross-reactivity for the two antigens in the multi-analyte immunosensor, so it is also an effective means in analyzing samples containing mixtures of RDX and TNT.

Detection of biomolecules in the near-infrared spectral region via a fiber-optic immunosensor

The design, development, and application of a fluorescent fiber-optic immunosensor (FFOI) procedure for the detection of antibody/antigen binding within the near-infrared (NIR) spectral region is reported. The technique was developed through the combined use of fiber-optics, semiconductor laser excitation, fluorescence detection, NIR dye, and immunochemical techniques. The antibody is immobilized on the FFOI's sensing tip and utilized as a recognition component for trace amounts of specific antigen. The FFOI is constructed to utilize antibody sandwich technique. Three individual immunoassays are reported. The first two assays utilize the FFOI and NN382, a commercial NIR dye, for the detection of human immunoglobulin G (IgG). In these assays, goat anti-human IgG antibody (GAHG) is immobilized on the sensitive terminal of the FFOI followed by the exposure of the antibody-coated terminal to human IgG. The probe is then introduced to GAHG labeled with NN382, generating a signal. The third assay utilizes the FFOI for the detection of trace amounts of Legionella pneumophila serogroup 1 (LPS1). In this assay, rabbit anti-LPS1 antibody is immobilized on the sensitive terminal of the FFOI followed by exposure to LPS1. The antigen-coated probe is then treated with monoclonal anti-LPS1 antibody followed by incubation with GAHG labeled with NN382. The assays are optimized to detect the corresponding antigen via the NIR–FFOI. Typical measurements are performed in 10–15 min. A 780-nm semiconductor laser provides the excitation of the immune complex and the resulting emission is detected by a 820-nm silicon photodiode detector. The intensity of the resulting fluorescence is directly proportional to the concentration of the antigen. Solutions of IgG and LPS1 with concentrations as low as 10 −11 M and 0.5 ng/ml, respectively, have been detected with a minimum interference.

Evanescent wave fiber optic fluorosensor: effect of tapering configuration on the signal acquisition

A comparative study of the signal acquisition from straight fiber and tapered optical fibers has been analyzed theoretically for the design of an evanescent wave fiber optic immunosensor. It has been shown that tapered optical fibers have higher sensitivity compared to straight fibers. Among the tapered fibers combination tapered fibers were also found to have higher sensitivity than continuous tapered fibers. Theoretical analysis indicates that an increase in signal is a result of three factors, namely, V number matching, an increase in evanescent absorption by the fluorescent medium, and an increase in fluorescence emission coupling into the fiber. Maximum sensitivity is obtained when the radius of the fiber is reduced to the V number matching radius within 1 cm from the start of the taper with rest of the fiber remaining untapered. Also, a higher signal level is obtained when the launching light fills the numerical aperture determined by the tapering ratio of the fiber. © 1998 Society of Photo-Optical Instrumentation Engineers. (S0091-3286(98)04301-3) Subject terms: evanescent wave; fiber-optic immunosensor; tapered fibers.

Fiber-optic immunosensor for measurement of myoglobin

A self-contained fiber-optic immunosensor was developed to measure the 16,500-Da protein myoglobin. The sensing element was constructed by entrapment of Cascade Blue-labeled antibody within polyacrylamide gel at the distal face of an optical fiber 300 microns in core diameter. The polyacrylamide gel composition was optimized to allow diffusion of myoglobin but to exclude hemoglobin and higher-molecular-mass proteins from the sensing area. The analytical signal was derived from fluorescence energy transfer between Cascade Blue and the heme group of myoglobin. Fluorescence quenching occurred when myoglobin bound to labeled antibody. The total amount of fluorescence quench was dependent on the antibody labeling conditions and the amount of antibody incorporated in the sensor gel matrix. Myoglobin concentrations > 5 nmol/L (83 micrograms/L) were measurable with response times of 15 to 130 min limited by diffusion into the sensing element. This report demonstrates the technical feasibility for a self-contained immunosensor to measure a protein analyte.

A compact fiber-optic immunosensor for Salmonella based on evanescent wave excitation

A compact fiber-optic evanescent-wave sensing system that features all-fiber optical design and red semiconductor-laser excitation has been developed and tested. A 2 × 2 fiber coupler directs the input light to the SMA-connected sensing fiber tip and the fluorescent signal back to a CCD fiber spectrophotometer. In this system, the fluorescent signal is confined in the fiber system so the signal-to-noise ratio is greatly improved and the system can be operated in ambient light conditions. A diode laser as the source has the advantages of small volume, ruggedness, low cost and stability; more importantly, since biological matrices demonstrate minimal fluorescent background at the laser wavelength of 650 nm, this system can reduce the background signal of non-essential biomolecules. To illustrate the biosensor's diagnostic capabilities, a sandwich immunoassay to detect Salmonella was developed. Tapered fiber tips with different shapes and treatments were studied and optimized. The system could detect Salmonella with a concentration as low as 10 4 colony-forming units per milliliter (CFU ml −1).

Evaluation of a fiber optic immunosensor for quantitating cocaine in coca leaf extracts

A fiber optic evanescent fluoroimmunosensor was used to rapidly detect and quantitate coaca alkaloids as cocaine equivalents in leaf extracts of five Erythroxylum species. A monoclonal antibody (mAb) made against benzoylecgonine (BE), a major metabolite of cocaine, was immobilized covalently on quartz fibers and used as the biological sensing element in the portable fluorometer. Benzoylecgonine-fluorescein (BE-FL) was used as the optical signal generator when it bound to the fiber. If present, cocaine competed for the mAb and interfered with the binding of BE-FL, thereby reducing the fluorescence transmitted by the fiber. Calibration curves were prepared by measuring (over 30 s) the rates of fluorescence increase in the absence, or presence of cocaine. Ethanol or acid extracts of dry coca leaves were assayed by this fiber optic biosensor, gas chromatography and fluorescent polarization immune assay. Biosensor values of cocaine content of leaves from five Erythroxylum species were not significantly different from gas chromatography values, but had higher variance. The biosensor assay was rapid and did not require cleanup of the crude leaf extracts. Cocaine in acid extracts was reduced significantly after 4 weeks at 23°C and after 3 weeks at 37°C. Fibers (mAb-coated), stored at 37°C in phosphate-buffered solution (0.02% NaN 3), gave stable responses for 14 days.

Design and development of a fiber-optic immunosensor utilizing near-infrared fluorophores

The design and application of a fluorescent fiber-optic immunosensor (FFOI) are reported. The FFOI is utilized for the detection of antibody/antigen binding within the near-infrared (NIR) spectral region. The technique is developed through the combined use of fiber-optic, semiconductor laser-excitation, fluorescence detection, NIR dye, and immunochemical techniques. The antibody is immobilized on the FFOI and utilized as a recognition component for trace amounts of specific antigen. The FFOI is constructed to utilize an antibody sandwich technique. The assay involves the immobilization of the capture antibody on the sensing tip of the FFOI followed by the exposure of the immobilized sensing tip to the antigen. The antigen-coated FFOI is then introduced to a second antibody previously labeled with the NIR dye. Typical measurements are performed in about 15 min. A semiconductor laser provides the excitation (780 nm) of the immune complex. The resulting emission is detected by a silicon photodiode detector (820 nm). The intensity of the resulting fluorescence is directly proportional to the concentration of the antigen. The sensitivity of the analysis reaches 10 ng/ml and the response time is 10-15 min.

Immunochemical characterization of type a botulinum neurotoxin in its purified and complexed forms

Immunochemical reactivities of type A botulinum neurotoxin to polyclonal antibodies raised against the neurotoxin complex toxoid have been investigated using enzyme-linked immunosorbent assay (ELISA) and with a fiberoptic immunosensor. The complex contains a group of complexing proteins in addition to the neurotoxin itself. Neurotoxin specific immunoglobulin G (IgG), purified from the IgG raised against the complex using an affinity column, showed a two-fold increase in reactivity with purified neurotoxin compared to the neurotoxin in the complex. Antibodies against the whole complex reacted approximately five times better with the complex than with the neurotoxin, suggesting that the detection of the neurotoxin complex may be more sensitive. Considering the fact that the amount of the complexing proteins and neurotoxin appears to be in a 4:1 ratio, a five-fold higher reactivity could suggest a 25-fold higher detectability of the neurotoxin in the complex. ELISA binding curves of complexing proteins and purified neurotoxin with antibodies raised against the whole complex indicated the complexing proteins to have significantly higher antigenicity. Furthermore, IgG fraction with or without the neurotoxin specific antibodies reacted almost equally to the neurotoxin complex, again suggesting higher immunogenicity of the complexing proteins. Increased binding of the complexing proteins versus the purified neurotoxin to antibodies against the complex and thus immunogenicity was also observed in the binding curves generated using a fiberoptic immunosensor.

Quantitative Analysis of 2,4-Dichlorophenoxyacetic Acid in Water Samples by Two Immunosensing Methods

Two sensitive methods based on a monoclonal anti-2,4-dichlorophenoxyacetic acid (2,4-D) antibody, a flow injection immunoanalysis (FIIA), and a fiber optic immunosensor are described and evaluated for the quantitative determination of 2,4-D in water samples. Detection limits for 2,4-D of ∼0.03 μg/L with FIIA and 0.2 μg/L with the fiber optic immunosensor can be achieved, i.e. well below the maximum concentration permitted, in the case of FIIA, by the EC Drinking Water Directives of 0.1 μg/L. The midpoints of the 2,4-D test are found at concentrations of 0.3 μg/L with FIIA and 10 μg/L with the fiber optic immunosensor. Measurable concentrations range from 0.03 to 3 μg/L with FIIA and from 0.2 to 100 μg/L with the fiber optic immunosensor. The FIIA did not require concentration or cleanup steps for ground and surface water samples. In addition, validation experiments with GC/MS showed a good correlation of the data for the FIIA system. However, both tests can be applied as alarm systems for monitoring poten...

Antibody characteristics for a continuous response fiber optic immunosensor for theophylline

A self-contained fiber optic immunosensor was developed to measure continuously theophylline concentrations. The analytical signal was derived from the non-radiative energy transfer quench of fluorescence following binding of a fluorescence donor, B-phycoerythrin labeled theophylline, to an energy acceptor, Texas Red labeled antibody. Increases in free theophylline analyte concentrations resulted in a shift in antibody binding equilibrium between labeled and unlabeled theophylline that elicited a proportional increase in the fluorescence. The selection criteria for a monoclonal antibody as a molecular recognition element, and the optimization of labeling conditions to maximize the dynamic range and minimize sensor response time are described. Under one or more Texas Red labeling conditions, five antibody clones exhibited significant quenching when mixed with labeled analyte and also demonstrated 95% or greater reversible binding to labeled analyte. Two clones failed to exhibit fluorescence quenching when mixed with labeled analyte. The response time of the indicator chemistry system was dependent on the dissociation rate constant of the antibody. The equilibrium response time of intact sensors was limited by analyte diffusion across the containment membrane.

Detection of protein A produced by Staphylococcus aureus with a fiber-optic-based biosensor.

Staphylococcus aureus is a pathogen important in causing human infections and intoxication. A sensitive fiber-optic that produces evanescent waves was developed for the detection of protein A, a product secreted only by S. aureus. In the immunosensor, a 40-mV argon-ion laser that generated laser light at 488 nm was used together with plastic optical fiber and antibodies to protein A were physically adsorbed onto the fiber. The principle of the detection involved a sandwich immunoassay with fluorescein isothiocyanate conjugated with anti-(protein A) immunoglobulin G to produce signals of the antigen-antibody reaction. The detection limit was 1 ng of protein A per milliliter. The fiber-optic immunosensor could be used for rapid and specific detection of S. aureus in clinical specimens and foods.

Fiber-Optic Immunosensor for the Detection of Fumonisin B1

A fiber-optic immunosensor was developed to measure the mycotoxin fumonisin B1 (FB1). Monoclonal antibodies produced against FB1 were covalently bound through a heterobifunctional silane to an etched 800 μm core optical fiber. An evanescent wave effect was utilized to excite fluorescein isothiocyanate labeled FB1 (FB1-FITC) molecules near the surface of the fiber. A direct competitive assay was used to measure FB1 concentrations with the following steps: saturation of antibody binding sites by FB1-FITC, competition of FB1 and FB1-FITC with displacement of the labeled toxin, and resaturation of binding sites by FB1-FITC. The signal generated in the assay was found to be inversely proportional to the FB1 concentration. This sensor exhibited a working range of 10−1000 ng of FB1/mL, an IC50 of 70 ng/mL, and a limit of detection of 10 ng/mL. These values compared favorably with those for currently available ELISA techniques. The sensor exhibited cross-reactivity with fumonisin B2 but did not react with hydrol...

Fiber optic immunosensor for polychlorinated biphenyls.

A fiber optic immunosensor, consisting of a quartz fiber coated with partially purified polyclonal anti-PCB antibodies (Abs), was used to detect polychlorinated biphenyls (PCBs). The optical signal was generated by the binding and subsequent fluorescein (FL) conjugate of 2,4,5-trichlorophenoxybutyrate (TCPB) to the Ab-coated fiber. The Abs, immobilized on the fiber, bound TCPB-FL with high affinity, saturably and reversibly. TCPB and PCBs competed with TCPB-FL for binding and decreased fluorescene in a concentration-dependent manner, with a detection limit of 10 ppb TCPB. The biosensor detected 1 ppm Aroclors, but not polychlorinated pesticides, polychlorinated phenols, or trichlorobenzene. It was more selective for TCPB at 1 ppm than the four tested Aroclors (i.e. 1016, 1232, 1250, and 1262) by 1.4-, 2.2-, 5.5-, and 3-fold, respectively. It detected Aroclor 1242 added to river waters and standard reference soil samples with reasonable accuracy. Advantages of this biosensor are its fast detection time (seconds to minutes), high sensitivity, and reversibility. 37 refs., 11 figs., 3 tabs.

USA — Measurement with a self-contained fiber-optic immunosensor: In ANAL. CHEM. (66/10 (1675–1682) 1994)) J.R. Astles and W.G. Miller of the Medical College of Virginia report on Measurement of free phenytoin in blood with a self-contained fiber-optic immunosensor

USA — Measurement with a self-contained fiber-optic immunosensor: In ANAL. CHEM. (66/10 (1675–1682) 1994)) J.R. Astles and W.G. Miller of the Medical College of Virginia report on Measurement of free phenytoin in blood with a self-contained fiber-optic immunosensor

Construction and biomedical application of immunosensors based on fiber optics and enhanced chemiluminescence

The main scheme of the measuring cell as well as the sensor device, the analysis conditions, and the results of the quantitative determination of several antigens are presented. Special attention is paid to the immobilization of one of the immunocomponents on the fiber optic surface and three approaches were investigated: (1) covalent binding of the immunocomponent with the help of glutaraldehyde to the surface preliminary treated by aminopropilethoxysilane, (2) direct binding of the immunocomponent to silicon oxide surface by BrCN, and (3) usage of a special membrane closely connected to the fiber optics. The investigations showed that the level of immobilized biomolecules may be achieved almost two fold more by the second approach in comparison with the first one. Good results can also be received by the usage of the special membrane. The sensitivity of the fiber optic immunosensors intended for the determination of the antigens selected was comparable with that made by the enzyme-linked immunosorbent assay (ELISA) method. But in all cases the rate of analyses by immunosensors was much higher than that of the ELISA method, which is the. main advantage of such types of immunosensors.

Measurement of free phenytoin in blood with a self-contained fiber-optic immunosensor.

We describe the measurement of the anticonvulsant drug phenytoin (5,5-diphenylhydantoin) in human blood and plasma using a self-contained fiber-optic immunosensor which gave a reversible response to changes in concentration. No regenerative treatment of the immunosensor was required between measurements. The analytical signal depended on the degree of energy transfer from B-phycoerythrin labeled with phenytoin to Texas Red labeled anti-phenytoin antibody. Dextran 70K was added to the reagent system to equalize the oncotic pressure across the encapsulation membrane. A gas chromatography reference method was used to measure free drug concentrations. Regression analysis for plasma samples gave the relationship (sensor) = 1.02 (reference) + 0.07 microM; Syx = 1.00 microM; r = 0.953; sensor mean 4.45 microM; reference mean 4.31 microM. Sensor performance in plasma and whole blood was essentially equivalent. We demonstrate the feasibility of measuring free phenytoin directly in blood and suggest that the sensor design is generally applicable for the measurement of other haptens in blood.