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

Abstract

Abstract. The aim of this work is to evaluate whether the size of the analyte used as template for the synthesis of molecularly imprinted polymer nanoparticles (nanoMIPs) can affect their performance in pseudo-enzyme linked immunosorbent assays (pseudo-ELISAs). Successful demonstration of a nanoMIPs-based pseudo-ELISA for vancomycin (1449.3 g mol -1 ) was demonstrated earlier. In the present investigation, the following analytes were selected: horseradish peroxidase (HRP, 44 kDa), cytochrome C (Cyt C, 12 kDa) biotin (244.31 g mol -1 ) and melamine (126.12 g mol -1 ). NanoMIPs with a similar composition for all analytes were synthesised by persulfate-initiated polymerisation in water. In addition, core-shell nanoMIPs coated with polyethylene glycol (PEG) and imprinted for melamine were produced in organics and tested. The polymerisation of the nanoparticles was done using a solid-phase approach with the correspondent template immobilised on glass beads. The performance of the nanoMIPs used as replacement for antibodies in direct pseudo-ELISA (for the enzymes) and competitive pseudo-ELISA for the smaller analytes was investigated. For the competitive mode we rely on competition for the binding to the nanoparticles between free analyte and corresponding analyte-HRP conjugate. The results revealed that the best performances were obtained for nanoMIPs synthesised in aqueous media for the larger analytes. In addition, this approach was successful for biotin but completely failed for the smallest template melamine. This problem was solved using nanoMIP prepared by UV polymerisation in an organic media with a PEG shell. This study demonstrates that the preparation of nanoMIP by solid-phase approach can produce material with high affinity and potential to replace antibodies in ELISA tests for both large and small analytes. This makes this technology versatile and applicable to practically any target analyte and diagnostic field.

A novel thermal detection method based on molecularly imprinted nanoparticles as recognition elements

“…7,8 In addition, they can be considered as replacement for antibodies and have been used for the detection of several targets in ELISA-type assays. [9][10][11][12][13] The use of synthetic antibodies has a number of distinct advantages over their natural counterparts, including their cost-effectiveness, simple and scalable synthesis, and that no animals are required for the production process. 14,15 Due to their robust nature, nanoMIPs can be employed in harsh chemical environments such as extremes of pH, temperature and also in organic solvents.…”

A comparison of the performance of molecularly imprinted polymer nanoparticles for small molecule targets and antibodies in the ELISA format

“…The nanoparticles are isolated as an aqueous suspension which can be used as a direct replacement for antibodies. In a parallel study, the effect of the size (molar mass) of the templates used for imprinting in aqueous media was investigated16. It was found that nanoparticles prepared in this manner could be used in the biomimetic ELISA format to create effective assays for horseradish peroxidase (44 kDa), cytochrome C (12 kDa) and biotin (244.31 g mol −1 ) as well as vancomycin (1449.3 g mol −1 )7, however for the smallest template studied (melamine, 126.12 g mol −1 ), aqueous-based imprinting was not effective.…”

“…High affinity material is produced by virtue of an in-built affinity separation step, the template may be re-used, and the surface chemistry can be modified chemically without affecting the recognition properties of the MIPs56. The resultant particles are suitable for applications where antibodies are traditionally used, including sensors22232425 and assays71626. Imprinted nanoparticles have also been shown to selectively detect mammalian cell types by their expression of antigens2728.…”

“…Error bars represent ±1 standard deviation and are for experiments performed in triplicate. Data for biotin-MIPs (g) is also included in a report where the effect of template size on aqueous solid-phase imprinting was investigated16, (see above).…”

Surface plasmon resonance based sensor for the detection of glycopeptide antibiotics in milk using rationally designed nanoMIPs

“…NanoMIPs produced using a solid phase has been pioneered by our group 9 , 17 , 18 and the solid phase synthesis approach demonstrates superior advantages over the other MIP synthesis methods 17 – 21 : Low affinity polymers can be discarded during the manufacturing process by applying cold wash (1), high affinity nanoMIPs are collected by performing elution steps at high temperature (2), the template free receptors are obtained with this method, because nanoMIPs are removed from the template molecule rather than the template being removed from the MIP (3), the stability and size of the nanoMIPs are high owing to the selective washing and elution steps (4), a significant amount of MIPs can be obtained per synthesis that is adequate for long term experiments (5) 17 – 21 . These superior features have resulted in the successful applications of nanoMIPs in various fields such as pharmaceutical monitoring 9 , 13 , virus quantification 18 , 21 , endotoxin detection 22 , 23 , development of enzyme linked immonosorbent assays (ELISA) 24 , 25 , manufacturing of bioselective membrane filters 11 , and in vivo recognition of biomarkers 26 .…”