As summarized in several recent research articles1,2, researchers at De Montfort University (UK), in concert with Aptamer Group (UK), demonstrate how aptamers combined with nanoparticles can be used to create new hybrid molecularly imprinted polymers (MIPs) which provide improved binding and lower detection limits compared to traditional biosensing targets. The researchers’ aim is to provide a stable, highly sensitive recognition material for use in a variety of applications including, but not necessarily limited to, those needing very low detection limits, with the potential to serve as very sensitive disease markers. Improvements in reproducibility and reusability make MIPs an interesting alternative to traditional biomolecules. Combining them with an aptamer sequence provides additional benefits due to improved stability, protection from degradation, and improved affinity. These researchers used a Reichert2SPR to explore the improved binding properties using this new target surface compared to a traditional biomolecular system.

Molecular recognition is commonly carried out using antibodies due mainly to their specificity and sensitivity. However, antibodies have downsides which include high cost, limited shelf life and immunogenicity issues. An alternative is to use aptamers or small RNA or DNA strands as the recognition element. These are more affordable, have a better shelf life and do not exhibit immunogenicity, but suffer from risk of environmental degradation. Another suitable alternative would be MIPs. These have certain advantages including that they do not degrade, since they are not of biological origin. Also, many earlier problems with MIPs, like heterogeneity, have been solved due to improved understanding of polymer chemistry. Related to MIPs, there has been interest in recent years in obtaining smaller polymer structures. This has led to the combination of MIPs with nanoparticles, which exhibit good analytical performance, and has opened up new avenues for development.1,2

A critical step in the development outlined here is to use nanoparticles combined with aptamers, which create the recognition element in the MIP. The template is formed by polymerizing monomers around these recognition elements which are then removed, leaving the MIP behind.1,2

Experimental

BACKGROUND
Researchers were interested in comparing SPR results using MIPs with, and without aptamers, combined with immobilized nanoparticles.1,2 

Conditions

 
 Instrument:
Reichert2SPR
 Sensor Chip: Carboxymethyl Dextran
Reichert P/N 13206066
 Temperature:  25 C
 Flow Rate:  25 μL/min
 Target: hybrid aptamer MIP or nanoparticle MIP
 Analyte: trypsin1 or moxifloxacin2
Running Buffer PBS pH 7.4 and 0.01 % (v/v) Tween 20
 Association Time:  2 minutes
Dissociation Time  5 minutes
 Regeneration: 10 mM glycine-HCl, pH 2

Results

The target proteins were attached to a solid support around which a MIP nanoparticle is formed, either as-is, or bearing a specific aptamer sequence containing either carboxyvinyl, or acrylamide functionalized thymine. The solid support is readily released, due to its thermo-responsive properties, leaving behind an imprinted nanoparticle. Researchers then attached the MIPs by amine coupling to a dextran coated gold chip.

Two model systems were employed – trypsin1 and the antibiotic moxifloxacin2. Due to the significant size (and by association molecular weight) difference between the nanoMIP and target analyte, a significant localized SPR nanoMIP signal is present3. An improved signal to noise ratio (SNR) of the localized nano-MIP SPR signal is generated by post-processing of the data using a projection method3-5.

As a representative example, trypsin experiments involved studying both the desired binding and the specificity by using BSA and lysozyme as negative controls. It was found that incorporation of an aptamer into the scaffold of the MIP improved sensitivity.1 Examples of trypsin binding are shown here:



Figure 1: Representative SPR sensorgrams of molecular interactions of various nanoparticles immobilized on carboxymethyl dextran hydrogel coated Au chips, to solutions containing five concentrations of target protein (trypsin,). (A) trypsin binding to trypsin-imprinted aptaMIPs; (B) trypsin binding to plain (non- aptamer bearing) nanoMIPs.1

Summary

  • Using hybrid MIPs formed from a combination of aptamers and nanoparticles improved sensitivity of the target surfaces over either MIP or aptamer alone
  • An important consideration when deciding whether to use MIPs is their selectivity. The two model systems chosen provided good selectivity when tested with other molecules that could potentially fit the binding pocket that was created. In particular, selectivity was improved using aptamer modified scaffolds (hybrid aptaMIPs compared to nanoMIPs alone).
  • MIP nanoparticles incorporate effectively with the Reichert 2SPR sensor system.