Direct replacement of antibodies with molecularly imprinted polymer nanoparticles in ELISA--development of a novel assay for vancomycin

Abstract

A simple and straightforward technique for coating microplate wells with molecularly imprinted polymer nanoparticles (nanoMIPs) to develop assays similar to the enzyme-linked immunosorbent assay (ELISA) is presented here for the first time. NanoMIPs were synthesized by a solid-phase approach with an immobilized vancomycin (template) and characterized using Biacore 3000, dynamic light scattering, and electron microscopy. Immobilization, blocking, and washing conditions were optimized in microplate format. The detection of vancomycin was achieved in competitive binding experiments with a horseradish peroxidase-vancomycin conjugate. The assay was capable of measuring vancomycin in buffer and in blood plasma within the range of 0.001-70 nM with a detection limit of 0.0025 nM (2.5 pM). The sensitivity of the assay was 3 orders of magnitude better than a previously described ELISA based on antibodies. In these experiments, nanoMIPs have shown high affinity and minimal interference from blood plasma components. Immobilized nanoMIPs were stored for 1 month at room temperature without any detrimental effects to their binding properties. The high affinity of nanoMIPs and the lack of a requirement for cold chain logistics make them an attractive alternative to traditional antibodies used in ELISA.

Figure 1

TEM image of nanoMIPs specific for vancomycin.

Figure 2

Testing the specificity of binding of the HRP-vancomycin conjugate to immobilized nanoMIPs. Binding of HRP-V to microplate wells coated with nanoMIPs, nanoNIPs or to bare wells, as revealed by color development with TMB reagent (100 μL ) for 10 min. Error bars represent ± 1 standard deviation for experiments performed in triplicate.

Figure 3

Optimization of blocking buffer. Aliquots (300 μL) of PBS containing different concentrations of a surfactant (Tween 20 or SDS) and BSA were incubated in microplate wells with immobilized nanoMIPs for 1 hour and the blank assay performed as explained in Material and Methods. Error bars represent ± 1 standard deviation for experiments performed in triplicate.

Figure 4

Calibration curve determined for the nanoMIP-based enzyme-linked competitive assay for vancomycin. Error bars represent ± 1 standard deviation and are for experiments performed in triplicate.

Figure 5

Calibration curves of the enzyme-linked nanoMIP-based competitive assay performed with vancomycin, amoxicillin, gentamicin and bleomycin. Error bars represent ± 1 standard deviation and are for experiments performed in triplicate.