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.

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

“…The detection limits of the pseudo-ELISA tests performed with nanoMIPs prepared for templates of different sizes, including the pseudo-ELISA for vancomycin carried out in a previous work, 17 are summarised in Table 1. 17 The table shows that the size of the templates, whether these are large proteins or small molecules such as biotin and melamine, has little effect on the sensitivity of pseudo ELISA based on nanoMIPs. In case of melamine, due to its small size, a modification of the protocol by grafting nanoparticles with PEG shell is necessary to improve assay performance.…”

“…17 The size of nanoparticles prepared in water was analysed by DLS and measured as 250 ± 30.1 nm, 170 ± 21.8 nm, 148 ± 7.3 nm, 133.4 ± 19.6 nm for HRP, Cyt C, biotin and melamine respectively. The value indicates that in general larger templates produce larger nanoMIPs.…”

“…Both assays were performed using the same conditions as in the assay developed for vancomycin. 17 These included the same blocking and washing steps, as well as adjusting the concentration of the stock solution of the two conjugates (for the competitive mode) to 2.5 mg mL -1 (before diluting them 800 times for the competition reaction) .…”

“…Recently we have described the use of nanoMIPs, produced by solid-phase approach in a microplate-based assay for accurate measurements of vancomycin (1449.3 g mol -1 ) with a limit of detection of 2.5 pM. 17 In the present work, we have decided to investigate the effect of the size of the template on the performance of MIP nanoparticles in ELISA. For this, we have selected a variety of target analytes with different size: horseradish peroxidase (HRP, 44 kDa), cytochrome C (Cyt C, 12kDa), biotin (244.31 g mol -1 ) and melamine (126.12 g mol -1 ) and we have used them as templates to produce nanoMIPs with the same synthetic protocol (Figure 1).…”

Molecularly Imprinted Polymer Waveguides for Direct Optical Detection of Low‐Molecular‐Weight Analytes

“…[ 17,18 ] In optical sensor applications, nanoMIP monolayers were attached to the sensor surface by means of physisorption, [ 19 ] affi nity reactions, [ 20 ] or covalent binding [ 7 ] for the capture of target analyte.…”