Chemiluminescence Imaging ELISA Using an Imprinted Polymer as the Recognition Element Instead of an Antibody

Abstract

An imaging assay analogous to competitive enzyme immunoassays has been developed using a molecularly imprinted polymer instead of an antibody. The antigen 2,4-dichlorophenoxyacetic acid (2,4-D) was labeled with tobacco peroxidase, and the chemiluminescence reaction of luminol was used for detection. Microtiter plates (96 or 384 wells) were coated with polymer microspheres imprinted with 2,4-D, which were fixed in place by using poly(vinyl alcohol) as glue. In a competitive mode, the analyte-peroxidase conjugate was incubated with the free analyte in the microtiter plate, after which the bound fraction of the conjugate was quantified. After addition of the chemiluminescent substrates, light emission was measured in a high-throughput imaging format with a CCD camera. Calibration curves corresponding to analyte concentrations ranging from 0.01 to 100 microg/mL were obtained.

Characteristic and Synthetic Approach of Molecularly Imprinted Polymer

“…In this manner, near-monodispersed spherical beads can be prepared, and size and porosity can be fine-tuned thereby changing the polymerization conditions. This technique has been reported in MIP-based competition assays [71][72] and capillary electrochromatography [73][74], but only recently works have been published, in which it is clearly shown that precipitation polymerization can be a potentially fruitful technique for preparing chromatography-grade molecularly imprinted beads [75].…”

Does size matter? Study of performance of pseudo-ELISAs based on molecularly imprinted polymer nanoparticles prepared for analytes of different sizes

“…12 Glass beads were activated by boiling in NaOH for 10 min and washed with DI water and acetone. After drying, the beads were incubated overnight in a 2 % solution of APTMS in dry toluene and then washed with acetone and dried.…”

“…One of the major drawbacks to work with MIPs in ELISA is the absence of a reproducible and straightforward method for coating microplate wells with the material. In the literature, there are several papers describing the applications of MIPs in ELISA, [8][9][10][11][12][13][14][15] but only a few demonstrate direct application of MIPs in the assays for quantitative detection of the target analytes. [8][9]16 In one example, the surface of the microplate wells was modified with a homopolymer of 3-aminophenylboronic acid, which was imprinted with ephedrine 15 .…”

Capillary electrophoresis microchip coupled with on-line chemiluminescence detection

“…However, the high costs and large size of these instruments are incompatible with the concept of -TAS. Chemiluminescence (CL) is a highly sensitive detection method widely used in flow-injection analysis [12], immunoassays [13] and conventional capillary electrophoresis [14,15]. Due to its simple optical devices, wide linear range, and the presence of many well-characterized systems, CL is uniquely suited to on-line detection for -TAS.…”