Plastic ELISA-on-a-Chip Based on Sequential Cross-Flow Chromatography

Abstract

A plastic chip that can perform immunoassays using an enzyme as signal generator, i.e., ELISA-on-a-chip, was developed by incorporating an immunostrip into channels etched on the surfaces of the chip. To utilize an analytical concept of cross-flow chromatography, the chip consisted of two cross-flow channels in the horizontal and vertical directions. In the vertical channel, we placed a 2-mm-wide immunostrip for cardiac troponin I (cTnI), which was identical to a conventional rapid test kit except for the utilization of an enzyme, horseradish peroxidase (HRP), as tracer. An enzyme substrate supply channel and a horizontal flow absorption pad compartment were transversely arranged on each lateral side of the signal generation pad of the strip, respectively. Upon application of a sample containing cTnI, it migrated vertically through the membrane strip by capillary action, and antigen-antibody binding occurred. After 15 min, the horizontal flow was initiated by the addition of a chromogenic substrate solution for HRP into the supply channel and by partial superimposition of the horizontal flow absorption pad onto the signal generation pad. A color signal proportional to the analyte concentration was produced on this pad, measured after 5 min as optical densities using a digital camera-based detector, and quantified by integration of the densities under the peak after normalization. Its calibration curve indicated that the detection limit of the chip was approximately 0.1 ng/mL and its quantification limit was 0.25 ng/mL. In measuring blindly prepared samples, the chip performance correlated with that of a reference system, Beckman Coulter Access, within 2.5-fold discrepancy at the detection limit.

An ELISA-on-a-Chip Biosensor System for Early Screening of Listeria monocytogenes in Contaminated Food Products

“…Labeling of Antibody with Horseradish Peroxidase: Mouse monoclonal antibody LZF7 (HyTest, Turku, Finland) which recognizes the outer membrane fraction and intact cells of L. monocytogenes was chemically conjugated to horseradish peroxidase (HRP; EDM chemicals, Gibbstown, NJ), via a cross-linker following a protocol described elsewhere. 22 Briefly, the antibody (2.65 nM) was first reduced using 10 mM dithiotheritol (Pierce, Rockford, IL), at 37 o C for 1 h and the excess reagent was removed on a Sephadex G-15 gel column (10 mL volume). HRP (26.45 nM) was activated with a 25-fold molar excess of 661.29 nM succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (Pierce, Rockford, IL), and the excess reagent was separated on the same gel column.…”

“…Analytical Procedure: The procedure used to detect and analyze the presence of L. monocytogenes was previously described. 22,24 Briefly, standard samples of L. monocytogenes cells (100 µL) in 10 mM phosphate buffer containing 140 mM NaCl, pH 7.4, (PBS) was applied to the sensor and the vertical flow was maintained for 15 min to complete the immune reactions. The horizontal flow absorption pad was then connected to the lateral side of the signal generation pad, and 150 µL 3,3',5,5'-tetramethylbenzidine for membranes (TMB-M; Moss, Pasadena, ML) was supplied into the substrate supply pot.…”

“…The color signals on the captured image were quantified along the center line of the immuno-strip in the vertical direction using software as described elsewhere. 22 The analysis was repeated three times for the same sample and, to establish the calibration curve, the mean values for each sample were plotted against the analyte concentration.…”

“…[19][20][21] However, this chromatographic assay has not been able to achieve a low detection limit (e.g., 10 5 cells/mL) of the target analyte in samples compared to ELISA, which is the traditional immunoassay. 22 Due to this inferior performance, this technique may not be able to detect the target microorganism at the concentration levels it exists in certain food products. Moreover, the law demands that food products must be screened at a level where even a single cell of the food-poisoning bacterium must be detected.…”

Immuno-chromatographic Analysis for HPV-16 and 18 E7 Proteins as a Biomarker of Cervical Cancer Caused by Human Papillomavirus

“…21 This suggests that the detection capability of the immuno-chromatographic assay may not be sufficient for clinical applications, and a more sensitive analytical method such as ELISA-based rapid testing 22 may need to be employed in the future. Furthermore, variations in the level of the E7 protein as a function of the progress of the virally infected disease should also be clinically determined.…”