MOLECULARLY IMPRINTED POLYMERS FOR RECOGNITION OF TOBACCO VIRUSES

Postgraduate

ABSTRACT

The goal of this research is to elucidate the mechanism of virus recognition in molecularly imprinted polymers (MIPs) using already utilized techniques. The clinical relevance of this study relates to the development of a virus imprinted MIP, which would apply to the identification, classification, and removal of viruses. The separation of viruses and virus-like particles from various media represents an enormous challenge to the fields of medicine, healthcare, and biotechnology.

Since virus MIPs must function in aqueous environments, our approach employs a more flexible non-covalent imprinting method which starts from a readily available polymer and utilizes an aqueous environment for both MIP synthesis and testing. Crosslinked polymers imprinted against Tobacco mosaic virus (TMV) via non-covalent interactions were synthesized using poly (allylamine hydrochloride) (PAA), epichlorohydrin (EPI), and TMV. The TMV imprinted polymer exhibited an increase affinity to the target virus compared to the control polymer and demonstrated a preferential affinity (imprinting factor of 2.1), based on shape, to the target virus compared to a non-target virus, Tobacco necrosis virus (TNV). In contrast, there was no significant increase in binding of the control polymer to either target or non-target virus.

Once it was determined that virus imprinted polymers can be successfully synthesized having preferential binding to a targeted virus, the synthesis procedure was optimized to obtain better binding characteristics to the targeted virus. Efforts were made to avoid polymer-template aggregation in the MIP prepolymerization mixture, and determine a proper wash solution by the ability to remove the templated virus from the crosslinked polymer. TMV imprinted hydrogels were synthesized using an optimized procedure and binding test performed on these MIPs to determine binding capacity, and more importantly, imprinting factor. The highest imprinting factor of 2.3 resulted from the MIP composed of 35 % PAA at pH 7, 15 %, ethylene glycol diglycidyl ether (EGDE), and 0.4 mg/mL TMV. The TMV imprinted hydrogels exhibited a lower binding capacity to TNV than when exposed to TMV. These results show that using optimized procedures, TMV MIPs with better shape selectivity can be achieved.


Significance

Molecular imprinting is a technique that creates synthetic materials containing highly specific receptor sites that have an affinity for a target molecule. Threedimensional cavities are created within a polymeric matrix that is complementary to the size, shape, and functional group orientation of the target molecule. The size and shape of the cavity allow the target molecule or similar molecules to occupy the cavity space, while the functional group orientation within the cavity will bind in specific locations complementary to only the target molecule and not to similar molecules. The result is molecular imprinted polymers (MIPs) that can mimic the recognition and binding capabilities of natural biomolecules like antibodies and enzymes. MIPs have several advantages over biomolecules, such as synthesis, stability, and reusability. MIPs can be seen applied in a wide range of technologies such as catalysis, separation and purification, drug delivery, and detection [1, 2, 3, 4, 5, 6, 7]. There remains an important need across many applications for materials that display selective and high affinity binding of biological analytes.

MIPs synthesized in this work would be applied to the removal of viruses. This is currently a very difficult task, but the need is widespread in diverse sectors such as human and animal health, crop protection, biopharmaceuticals, and biological warfare. For example, biopharmaceutical products need to be virus-free. These MIPs, when placed into a packed column and used as a purification stage, will act as virus-specific sponges and selectively capture the targeted virus while allowing other non-targeted molecules to pass through. Research in virus MIP have been done in the past [5, 6]. However, their applications are very different than the virus MIP system used in this work. Such MIP systems consist of two-dimensional surface imprinting of viruses on a crosslinked polymer surface that is attached to a sensor. When the virus template is imprinted and removed, these sensors are able to bind small amounts of the target virus on the polymer surface. Twodimensional surface imprinting is designed to detect the presence of small amounts of viruses whereas three-dimensional imprinting can be used to bind and extract large amounts of virus from a given solution. For example, two-dimensional imprinting can be used to detect the presence of hepatitis virus in blood, whereas three-dimensional imprinting can be used to extract hepatitis virus from the blood. Currently no work has been published on the synthesis of MIPs for large virus extraction.


Background

The concept of molecular imprinting was started by Linus Pauling in the 1940’s, in an attempt to explain antibody formation [8]. In his theory, an antigen serves as a template around which an antibody would bind to form a mold. Although it was proven later that his theory was incorrect, he did start the concept of what is now called molecular imprinting. The first studies of molecular imprinting were performed by a student of Pauling’s, Frank Dickey [9, 10]. Attempting to prove the Pauling’s theory, Dickey successfully synthesized silica gels made in the presence of either methyl orange, ethyl orange, propyl orange, or butyl orange, and exhibited selective binding of the MIPs to their respective targeted dye molecule. Since this initial discovery, there has been a variety of work aimed at the development and refinement of molecular imprinting procedures.