Enzyme‐linked immunosorbent assay of Escherichia coli O157:H7 in surface enhanced poly(methyl methacrylate) microchannels

Abstract

A novel surface treatment method was developed to enhance polymer-based microchannel enzyme-linked immunosorbent assay (ELISA) for Escherichia coli O157:H7 detection. By applying an amine-bearing polymer, poly(ethyleneimine) (PEI), onto poly(methyl methacrylate) (PMMA) surface at pH higher than 11, PEI molecules were covalently attached and their amine groups were introduced to PMMA surface. Zeta potential analysis and X-ray photoelectron spectroscopy (XPS) demonstrated that the alkali condition is preferable for PEI attachment onto the PMMA surface. The amine groups on the PMMA surface were then functionalized with glutaraldehyde, whose aldehyde groups served as the active sites for binding the antibody by forming covalent bonds with the amine groups of the protein molecules. This surface modification greatly improved antibody binding efficiency and the microchannel ELISA for E. coli O157:H7 detection. Compared with untreated PMMA microchannels, approximately 45 times higher signal and 3 times higher signal/noise ratio were achieved with the PEI surface treatment, which also shortened the time required for cells to bind to the microchannel surface to approximately 2 min, much less than that usually required for the same ELISA carried out in 96-well plates. The detection in the microchannel ELISA only required 5-8 cells per sample, which is also better than 15-30 cells required in multi-well plates. With the high sensitivity, short assay time, and small reagent consumption, the microchannel ELISA can be economically used for fast detection of E. coli O157:H7.

Recent trends in the detection of pathogenic Escherichia coli O157 : H7

“…Direct and Indirect are the 2 types of ELISA frequently used to quantify E. coli O157 : H7, and other pathogenic bacteria 26,27 . The whole cell direct sandwich assay is very popular and when used in combination with signal enhancers such as multifunctional nano-composites is 4-5 orders of magnitude more sensitive than a standard ELISA.…”

Enhanced detection sensitivity of Escherichia coli O157:H7 using surface-modified gold nanorods

“…Conventional methods used to detect bacteria include colony counting, immunology-based enzyme-linked immunosorbent assays, and polymerase chain reaction, which are time consuming and require special sample pretreatment. [8][9][10] Biosensor-based detection techniques are sensitive, specific, and reliable, however, they require special chemical and fluidic attachments such as antibodies. 11 Emerging trends in nanotechnology open up materials to be applied in various aspects of biomedical applications, including antimicrobes, 12 cancer imaging and therapy, 13,14 drug or DNA delivery, 15,16 enzyme sensors, 17 UV protector, 18 and so forth.…”

Replication of DNA submicron patterns by combining nanoimprint lithography and contact printing

“…To modify the PMMA surface with PEI for DNA immobilization, pristine PMMA sheets were immersed in 100 mM borate buffer (pH 11.5) containing 1 wt% PEI at room temperature for 2 h [21]. After the reaction, they were immersed in DI water for 30 min, sonicated, and dried with nitrogen.…”