Capillary-Based Displacement Flow Immunosensor

Abstract

We report on the preparation of a novel capillary flow immunosensor for 2,4,6-trinitrotoluene (TNT). Anti-TNT antibody is immobilized onto the silanized inner walls of an 800-µm-i.d. glass capillary using a heterobifunctional crosslinker, followed by saturating the capillary with fluorophore-labeled antigen. To perform the assay, an aliquot of TNT solution is injected into the capillary. TNT competes for the binding pocket of the fluorophore-labeled antigen, which is detected downstream. We have demonstrated that the sensitivity of the capillary immunosensor is improved by at least 2 orders of magnitude compared to that of conventional packed-column or membrane-based displacement flow immunosensors. The limit of detection (S/N > 3) for TNT using the capillary immunosensor is 15 pg/mL (7 fmol), and the linear dynamic range is 1-250 ng/mL. The entire assay can be performed in less than 3 min. During 15 injections of 100 ng/mL TNT solution, the signal reproducibility (RSD) was maintained to within 4%.The advantages of immunoassay as a selective, sensitive, and cost-effective method for screening clinical and environmental samples are now widely accepted. 1 Despite the success of the enzyme-linked immunosorbent assay (ELISA), 2 its end use is limited due to the requirement of trained personnel and long assay time. These limitations have sparked the development of immunosensors (antibody-based biosensors) using sandwich and competitive immunoassays at the surfaces of optical fibers, 3 planar waveguides, 4 surface acoustic waveguides, 5 plasmon resonance surfaces, 6 and piezoelectric devices. 7 Another class of immunosensors utilizes displacement immunoassays at the surface of antibody-coated beads 8 or membranes. 9 This report describes a novel capillary-based displacement continuous-flow immunosensor (capillary immunosensor hereafter) for the explosive 2,4,6-trinitrotoluene (TNT) which improves the sensitivity of the sensor by at least 2 orders of magnitude compared to that of the previously used packed-column 8 and membrane-based 9 flow immunosensors or to that of an ELISA using the same antibody reagents. 8b Microcapillaries offer the advantages of high surface-to-volume interaction, laminar flow, and reduced band-broadening effects compared to packed columns. 10 Although capillaries have proven to be very successful in separation science, 10 there are no reports of them being used in displacement flow immunosensors. Several recent reports do describe other types of capillary-based sensors: [11][12][13] reported on the development of "integrated chemical sensors" based on small capillaries; O'Neill et al. 12 reported on the use of a capillary fill device as an optical immunosensor using two glass sheets held apart by a gap of capillary dimensions and evanescent-based detection; Cosford and Kuhr 13 reported on the application of a capillary biosensor using a direct binding enzyme assay for glutamate; and Heineman et al. 14 reported on the use of capillary enzyme immunoassay with electrochemical detection for the...

Multianalyte Detection Using a Capillary-Based Flow Immunosensor

“…A flow rate of 100 l/min was used because we have previously demonstrated that at a flow rate lower than 100 l/min, the total assay time increases significantly (Ͼ15 min) and there is a loss of sensitivity for TNT detection due to peak broadening. At flow rates greater than 250 l/min, the displaced area is significantly lower, resulting in suboptimal sensitivity (23,24). Figure 2B represents the dynamic range of the TNT assay using the capillary immunosensor.…”

“…We have recently reported on the development of a displacement flow immunosensor for the detection of TNT 2 using a microcapillary and demonstrated that the sensitivity for direct TNT detection (i.e., no preconcentration of the sample) by the capillary immunosensor is better by at least an order of magnitude compared to any other analytical technique available for direct TNT detection (23,24). In this paper, we describe a dual-analyte immunoassay using capillarybased continuous flow immunosensors.…”

A capillary-based amperometric flow immunoassay for 2,4,6-trichlorophenol

“…In addition, capillary-based flow immunoassay provide supplementary advantages, such as high surface-to-volume ratio, laminar flow, reduced costs due to lower reagent consumption, reduced band-broadening effects compared to packed columns, and improved detection [59,60,61,62,63]. Moreover, electrochemistry generally offers a more sensitive detection method in comparison to UV and spectroscopy [60,64].…”

Non-aerosol detection of explosives with a continuous flow immunosensor

“…In the microcapillary system, a fused-silica microcapillary was cleaned with sodium hydroxide, then hydrochloric acid, followed by nitrogen to dry prior to antibody immobilization [26,27,28,29]. The microcapillary was then silanized with mercaptopropyl trimethoxysilane (Fluka, St Louis, MO, USA) followed by the heterobifunctional crosslinker, N-succinimidyl-4-maleimidobutyrate (GMBS, Fluka) and lastly the monoclonal anti-TNT or anti-RDX antibody.…”