Single-Step Sandwich Immunoreaction in a Square Glass Capillary Immobilizing Capture and Enzyme-linked Antibodies for Simplified Enzyme-linked Immunosorbent Assay

Abstract

To simplify the complicated operation steps and to minimize sample and reagent amounts for enzyme-linked immunosorbent assays (ELISA), we developed a square glass capillary immunosensor containing both covalently immobilized capture antibodies and physically adsorbed enzyme-linked antibodies. The immobilization of capture antibodies (anti-human IgG) was carried out by the treatment of 3-aminopropyltriethoxy silane, glutaraldehyde, and protein-A, followed by affinity capture of the antibody. In contrast, the enzyme-linked antibodies (alkaline phosphatase (ALP)-linked anti-human IgG) were physically adsorbed on the four corners of the capillary with the aid of polyethylene glycol (PEG) acting as a scaffold. A nanoliter volume of antigen (human IgG)-containing sample solution was introduced via capillary action. This addition resulted in the release and diffusion of ALP-linked anti-human IgG into the bulk solution. This event led to a 20-min single-step sandwich immunoreaction at the inner wall of capillary; the reaction was detected through the reaction with fluorescein diphosphate (FDP) which generated a fluorescent product, fluorescein. Using this technique, we obtained an intra-capillary precision with a coefficient of variation of 9.7%. In addition, the specificity study showed that the human IgG capillary immunosensor did not respond to rabbit IgG. Quantitative analysis was possible within the response range of 10 - 5000 ng mL(-1) anti-human IgG. This capillary immunosensor can act as a single analytical unit or can be integrated into a capillary array for multiple bioanalysis.

Fabrication and packaging of a mass-producible capillary-assembled microchip for simple and multiplexed bioassay

“…As an initial demonstration, we have successfully attained the multiplexed glycosidase enzyme assay. In addition to its use for enzyme activity assays, the CAs-CHIP-3G microdevice could also be used for other bioassays by integrating other capillary biosensors that we have developed previously for various analytes such as ions [27,28], metabolites [30,31], and proteins [37]. This inexpensive, reliable, and easy-to-use bioanalytical microdevice could be extended for colorimetric assay with the smartphone camera as the detector which has more potential for point-of-care or point-of-use applications.…”

“…We have developed various glass capillary biosensors to be integrated into CAs-CHIP, to measure various analytes such as proteins [25][26][27][28], ions [29,30], enzymes [30,31], and metabolites [32,33] Among many capillary biosensors developed, dry reagentrelease capillary (dRRC) which allows spontaneous dissolution of analytical reagents and selective reaction to give fluorescence response by sample introduction via capillary action, played an important role for the simplicity in capillary biosensor preparation and biosensing [30]. However, both the first (CAs-CHIP-1G) [23,30,31] and second (CAs-CHIIP-2G) [35] generations of CAs-CHIP requires the meticulous one-by-one capillary embedding onto a PDMS microchannel.…”

Advancements in Capillary-Assembled Microchip (CAs-CHIP) Development for Multiple Analyte Sensing and Microchip Electrophoresis

“…46 Multi-step ELISA requires a longer analysis time because of the multiple washing steps and incubation intervals; therefore, we have made several efforts to decrease the number of steps by using the combination of RRC with antibodies immobilized on the inner surface of the capillary. [47][48] Despite these efforts, a purely single-step and robust immunosensing mechanism needs to be developed for real sample application for eventual integration with other biosensing capillaries in a single CAs-CHIP.…”

Sensitive “capillary ELISA” via vapor-phase surface modification

“…Also in the POC applications, various versions of capillary tube-based immunoassay systems were developed taking advantage of its simple structural feature [10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25]. Rectangular-shaped capillary tubes were used to perform enzyme immunoassay for various types of biomarkers [18,[22][23][24]. When the well-known surface chemistry based on 3-aminoprolytriethoxysilane (APTES) and glutaraldehyde (GA) were used to install antibody in the capillary tube, the immunoassay results produced 10 ng/mL level detection capability [24].…”