1. Radio-labelled MIAs

2. Fluorescence-based MIAs

3. Enzyme-linked MIAs

4. Molecularly Imprinted Nanoparticle Assay (MINA)

5. References

And there's another problem--it never actually explains just what the method is. DGG (talk) 18:43, 11 November 2018 (UTC)}}

Molecularly imprinted polymers are used in a wide range of applications. They demonstrate greatest potential as alternative affinity reagents for use in diagnostic applications, due to their comparable (and in some regards superior) performance to antibodies. Many studies have therefore focused on the development of molecularly imprinted assays (MIAs) since the seminal work by Vlatakis et al. in 1993, where the term “molecularly imprinted [sorbet] assay” was first introduced. Initial work on ligand binding assays utilising MIPs in place of antibodies consisted of radio-labelled MIAs, however the field has now evolved to include numerous assay formats such as fluorescence MIAs, enzyme-linked MIAs, and molecularly imprinted nanoparticle assay (MINA).^[1]

Radio-labelled MIAs

Similarly to radioimmunoassay (RIA), radio-labelled molecularly imprinted assays rely upon binding to a radio-labelled analyte. This allows the use of analyte which is structurally identical to the template of interest, differing only through introduction of a radioactive isotope. It is a very sensitive technique, however issues concerning the commercial unavailability of isotopic-labelled tracers for many compounds of interest coupled with apprehensions over the handling and disposal of radionuclides made the development of assays based on other labelling and detection methods more preferential.

Fluorescence-based MIAs

In fluorescence-based MIAs, the fluorescent signal is typically generated through the addition of a fluorescent tag/group to the analyte of interest. Impressive results have been achieved with this MIA format, however fluorophore coupling to the analyte can potentially be detrimental to assay performance, as different binding behaviour may be observed when compared to the uncoupled analyte, impacting the sensitivity and selectivity of the assay. This problem has been resolved in a number of MIAs through substituting the fluorescently labelled analyte for fluorescent monomers/quantum dots incorporated into the MIPs themselves,^[2] or post-synthesis functionalisation of the MIPs with a fluorescent core-shell.^[3]

Enzyme-linked MIAs

The use of enzyme-labelling analytes was first implemented as early as 1968, and has since become the most popular method for labelling in immunoassays. This trend has translated over to MIAs also, as traditional problems of incompatibility with water and inaccessibility of binding sites with the use of enzymes with MIPs have been overcome. Enzyme-labels still suffer the same problems as fluorescent probes with regards to conjugation of the label to the analyte and the effect this consequently has on the recognition and binding of the labelled analogue; however, the commercial availability of many enzymes at low cost and general ease of conjugation offer significant advantages. Additionally, many enzyme labels undergo simple colorimetric/fluorimetric reactions during their application, requiring detection devices no more complex or expensive than a multichannel colorimetric/fluorimetric reader.

The aforementioned difficulty of binding site availability has led to adoption of in situ polymerization of imprinted films on the surface of 96-well plates as the most popular technique for development of biomimetic ELISA-like assays. By utilizing a film format, a large surface area can be achieved, whilst control of the film thickness assists in access to binding sites. The method has been used extensively for a variety of templates, with the developed assays being applicable to determination of their respective analytes in environmental water samples. The assay has been demonstrated for a large variety of templates, such as fumonisin B1,^[4] cocaine,^[5] gentamicin,^[6] vancomycin,^[7] and others.^[8][9]

Molecularly Imprinted Nanoparticle Assay (MINA)

A MINA is based on the same principle as a fluorescence immunoassay, where antibodies are substituted by MIPs and the signal is generated by MIPs themselves, due to fluorescent moieties incorporated into the polymer. The template of interest is covalently immobilised on the surface of iron oxide nanoparticles where it acts as a competitor to free analyte in the sample matrix. The assay is run in a 96-microtiter plate modified with magnetic inserts at the bottom of each well (see figure), and can be performed in either a competitive, displacement, or sandwich format.

This assay format offers the advantage of removing the requirement of a biological receptor/reporter, such as antibodies in the case of an immunoassay or enzymes in case of enzyme-labelled assays. This avoids common criticisms related to these assays, such as storage at controlled temperature and poor versatility of the components. In addition, compared to assays requiring several washing steps to separate the unbound label/template, MINA does not require any washing step due to the presence of the magnetic field which directly produces a physical separation in situ.

The assay format has the potential to become a commonly used technique for drug screening in forensic and clinical laboratories. MIPs are synthetic materials removing the requirement for use of animals in the production chain. This avoids some of the already acknowledged problems such as antibodies poor reproducibility due to differences in each animal’s immune system. This, plus the required storage at controlled temperatures (2-4⁰C) and the necessity for specialist personnel make immunoassays poorly suited for in situ detection or clinical screening, where highly specific facilities and trained staff are not always present. In these scenarios screening is also required within a limited period of time; MINA reduces the analysis time compared to that required for ELISA. The limits of detection reached in published assays for trypsin and biotin have been comparable to those of commercially available ELISA assays.^[10][11]

Iron-oxide nanoparticles functionalised with an analyte of interest are added to each well. When MIP nanoparticles are added excitation light passing through the middle of the well will excite the fluorescent moieties incorporated in the MIPs, generating the assay signal. If there is binding between MIPs and the analyte immobilised on iron oxide nanoparticles the resulting MIP-iron oxide nanoparticle complex will be attracted to the surface of the magnetic inserts, reducing the quantity of MIPs in solution and therefore decreasing the signal intensity.

A displacement assay is a method used to quantify free analyte in solution. It requires pre-incubation of MIP nanoparticles with analyte-functionalised iron-oxide nanoparticles. With the help of a magnet, the excess of unbound MIPs are removed and the media replaced. Different concentrations of free analyte are dispensed into each well of a 96-microtiter plate modified with magnetic inserts, ), and the solution of MIP/iron-oxide complex added. The magnetic field supplied by the magnetic inserts attracts the complexes removing the fluorescence generated by the bound MIP nanoparticles. MIP nanoparticles will dissociate from the iron-oxide nanoparticles and may either rebind to the immobilised analyte or bind to the free analyte in the sample solution, dependent upon how much is available to compete. MIP which binds to free analyte is therefore displaced and free to move in solution, increasing the fluorescent signal.

The signal generated by the fluorophore incorporated in the nanoMIPs matrix is detected by the microplate reader. Therefore the larger quantity of free analyte in solution, the higher fluorescence/signal detected. In this way it is possible to quantify a concentration dependent response.

A significant advantage of using a MINA displacement assay is the potential to dry a MIP/analyte-functionalised iron oxide nanoparticle solution to the surface of each well, with the ability to simply add a test sample and obtain a result with no further addition or separation steps necessary. The format is therefore very easy and straightforward for the end user, and due to this mix-and-read format allows results to be obtained very rapidly compared to a multistep ELISA assay.

Competition is an alternative method used to quantify free analyte in solution, which relies on similar principles to the displacement assay. The main difference is that nanoMIPs are incubated simultaneously with both functionalised iron-oxide nanoparticles and free analyte, as opposed to sequentially in the case of a displacement assay. Immobilised and free template are therefore both available to interact with nanoMIPs; if one is present at a higher concentration than the other, it is more likely to be bound to the nanoMIPs.

References

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