Dual-cardiac marker capillary waveguide fluoroimmunosensor based on tyramide signal amplification.

The early diagnosis of acute myocardial infarction requires the determination of several markers in serum shortly after its incidence. The markers most widely employed are the isoenzyme MB of creatine kinase (CK-MB) and the cardiac troponin I (cTnI). In the present work, a capillary waveguide fluoroimmunosensor for fast and highly sensitive simultaneous determination of these markers in serum samples is demonstrated. The dual-analyte immunosensor was realized using glass capillaries internally modified with an ultrathin poly(dimethylsiloxane) film by creating discrete bands of analyte-specific antibodies. The capillary was then filled with a mixture of sample and biotinylated detection antibodies followed by reaction with streptavidin-horseradish peroxidase and incubation with a fluorescently labeled tyramide derivative to accumulate fluorescent labels onto immunoreaction bands. Upon scanning the capillary with a laser beam, part of the emitted fluorescence is trapped and waveguided through the capillary wall to a photomultiplier placed on one of its ends. The employment of tyramide signal amplification provided detection limits of 0.2 and 0.5 ng/mL for cTnI and CK-MB, respectively, in a total assay time of 30 min compared to 0.8 and 0.6 ng/mL obtained for the corresponding assays when the conventional fluorescent label R-phycoerythrin was used in a 65-min assay. In addition, the proposed immunosensor provided accurate and repeatable measurements (intra-assay and interassay coefficients of variation lower than 10%), and the values determined in serum samples were in good agreement with those obtained with commercially available enzyme immunoassays. Thus, the proposed capillary waveguide fluoroimmunosensor has all the required characteristics for fast and reliable diagnosis of acute myocardial infarction.

Bulk fluorescence light blockers to improve homogeneous detection in capillary-waveguide fluoroimmunosensors.

A simple approach that employs black drawing ink (BDI) as bulk fluorescence light blocker and improves considerably the homogeneous signal detection in capillary-waveguide fluoroimmunosensors is presented. The concept was proved using a capillary sensor configuration. Fluorescent molecules in the capillary were excited by a laser beam vertically to its axis and the emitted photons that were trapped and waveguided through the capillary wall were then collected. Two competitive fluoroimmunoassays, for rabbit gamma-globulins in buffer and thyroxine in human serum, respectively, were set-up to evaluate this approach. It was found that the presence of ink improved the specific to bulk fluorescence signal ratio by approximately 60-times without affecting the analyte-antibody binding reaction thus, facilitating homogeneous detection. The analytical characteristics of the two assays developed with the sensor operating in homogeneous detection mode were similar to those determined following the heterogeneous detection mode (i.e. after removal/washing of the immunoreaction mixture).

Capillary waveguide fluoroimmunosensor with improved repeatability and detection sensitivity.

An optical capillary waveguide fluoroimmunosensor based on glass capillaries internally coated with an ultrathin poly(dimethylsiloxane) (PDMS) film is presented. The evaluation of the capillaries developed was done in comparison with aminosilanized [3-(aminopropyl)triethoxysilane, APTES] glass and poly(methylpentene) (PMP) capillaries by immobilizing rabbit gamma-globulins on the internal capillary wall. Following reaction with (R)-phycoerythrin-labelled antibody, the capillary was scanned with a laser beam and the fluorescence waveguided through the capillary wall was detected by a photomultiplier placed at one of its ends. The capillaries developed provided considerably improved protein coating homogeneity (intracapillary coefficients of variation 2.9-6.6%) and repeatability (intercapillary coefficients of variation 2.1-5.0%) compared with APTES-treated ones (7.9-13.4 and 8.5-15.2%, respectively). With use of these capillaries in a sandwich-type immunosensor for the determination of rabbit gamma-globulins, the assay detection limit was improved eightfold (4.4 ng/mL) compared with that obtained using PMP capillaries (35.3 ng/mL), whereas the assay repeatability was improved threefold (intra-assay coefficients of variation 5.9-13.1%) compared with APTES-treated capillaries (15.6-36%).

Glycerin suppression of fluorescence self-quenching and improvement of heterogeneous fluoroimmunoassay sensitivity.

Fluorescent labels find wide application in immunoassays and immunosensors as well as in protein and DNA chips. However, the use of fluorescent labels in applications requiring high detection sensitivity is limited by fluorescence self-quenching observed when a relatively high number of fluorescent compounds is introduced in the recognition molecule. Here we describe a simple method that suppresses effectively fluorescence self-quenching observed when highly labeled antibodies are used as labels in immunoassays. This was achieved by treating the microtitration wells after the completion of the immunoassay with a glycerin solution followed by 15-min incubation of the emptied wells at 37 degrees C. The remedial action of this method on self-quenching was studied through a noncompetitive immunofluorometric assay for rabbit gamma-globulins employing a sheep anti-rabbit gamma-globulin antibody labeled with fluorescein at molar ratios ranging from 1.0 to 17.4. The glycerin/thermal treatment increased the fluorescence signal measured directly onto the solid surface by 9.2-117% for the antibodies with molar ratios of 1.0-17.4, compared with the values obtained prior to treatment. Furthermore, fluorescence self-quenching was completely removed for labeling ratios up to 14.0. The assay sensitivity was improved 2-4 times by the glycerin/thermal treatment when heavily fluoresceinated antibodies are used as labels (molar ratio >/=5.6). The proposed method resulted also in increased fluorescence signals when labels other than fluorescein were used and improved considerably the detection of protein spots on silicon dies.

Solid-phase fluoroimmunoassay for the determination of mesotrione--a novel triketone herbicide--in water with direct measurement of the fluorescence onto the solid support.

A straightforward, low-cost fluoroimmunoassay (FIA) for the determination of the new triketone herbicide mesotrione has been developed and optimized. The protein-mesotrione conjugate, immobilized on white opaque microtitration wells competes with the mesotrione in the sample or standard for the limited binding sites of a liquid phase anti-mesotrione antibody. The assay is based on the measurement of fluorescence intensity directly onto the solid support, using a fluorescein labeled second antibody and a fluorescence plate reader. To stabilize and enhance the fluorescence signal a glycerine-based treatment of the microtitration wells was included in the protocol. The detection limit of the assay is 40 ng 1(-1) (4 pg per well), the working range extends up to 9 microg l(-1), whereas the within and between run CVs are 0.7-4.2% and 2.1-5.5%, respectively. To evaluate the assay specificity, the cross-reactivities of two mesotrione metabolites: 4-methylsulfonyl-2-nitrobenzoic acid and 2-amino-4-methylsulfonyl-benzoic acid and several other compounds similar in structure to mesotrione such as: fomesafen, prosulfocarb, fluazinam, sulcotrione, 1,2-cyclohexanedione, 1,3-cyclohexanedione, 2-acetyl-1,3-cyclohexanedione were assessed. Most of the substances tested presented very low ( < 0.05%) cross-reactivity values with the exception of sulcotrione that cross-reacted 23% in the mesotrione assay. The assay was used for the determination of mesotrione in bottled natural waters fortified with the analyte and in a commercial herbicide formulation, namely CALLISTO.

Simultaneous determination of pesticides using a four-band disposable optical capillary immunosensor.

The development of a four-band capillary optical immunosensor for the simultaneous determination of mesotrione, hexaconazole, paraquat, and diquat is described. Four distinct bands (each corresponding to a different analyte) are created in the internal walls of a plastic capillary by immobilizing protein conjugates of the analytes. To perform the assay, the capillary is filled with a mixture of anti-analyte-specific antibodies together with a standard or sample containing the analyte(s). After a short incubation, a mixture of the appropriate second antibodies labeled with fluorescein is introduced into the capillary. To measure the fluorescence intensity bound onto each band, the capillary was scanned, perpendicularly to its axis, by a laser light beam. Part of the emitted photons were trapped into the capillary walls and waveguided to a photomultiplier placed at the one end of the capillary. The analytical characteristics of the assays of mesotrione, paraquat, diquat, and hexaconazole were as follows: detection limits of 0.04, 0.06, 0.09, and 0.10 ng/mL, respectively; dynamic ranges up to 9, 6, 12, and 15 ng/ mL, respectively, intra- and interassay CVs less than 10%. The analytical characteristics of the assays were comparable with those of the corresponding single-analyte fluoroimmunoassays performed in microtitration wells, proving the ability of the proposed immunosensor for reliable multianalyte determinations. Moreover, the combination of low-cost disposable plastic capillary tubes with the low consumption of reagents, the short assay time, and the multianalyte feature of the proposed immunosensor indicates its potential for environmental analysis.