Application of Molecular Imprinting for Development of Plasmonic Bio- and Chemosensors
Imprinting of molecular recognition sites in organic  or inorganic polymers [2, 3] has been the subject of extensive research efforts  (Fig. 5.1). There are two general methods to generate imprinted sites in polymer membranes. One approach  involves the polymerization of monomers that include a complementary function to the imprinted substrate such as H-bonds, electrostatic interactions, and 7r-donor-acceptor interactions. Polymerization of the monomer-substrate complex, followed by the removal of the substrate molecules acting as a template forthe polymerization, yields the imprinted sites in the polymer. The second approach involves the covalent attachment  or coordination  of the substrate to polymerizable monomer units, followed by the со polymerization of the functional monomers with other monomers, to yield rigidified polymer matrixes. Cleavage of the polymer-linked substrate units leads to the formation of the polymer with imprinted sites. Polymers with imprinted sites revealing structural  and chiral [9, 10] selectivities have been prepared. Molecularly imprinted polymers (MIPs) have been used as specific sensing interfaces , functional
Molecular Plasmonics: Theory and Applications Volodymyr I. Chegel and Andrii M. Lopatynskyi Copyright © 2021 Jenny Stanford Publishing Pte. Ltd.
ISBN 978-981-4800-65-5 (Hardcover), 978-0-429-29511-9 (eBook) www.jennystanford.com materials for chromatographic separations , and matrixes for selective and catalyzed chemical transformations . This artificial molecular recognition system has significant advantages such as mechanical/chemical stability, low cost, and ease of preparation, thus have increasingly attracted considerable attention [14,15].
Figure 5.1 Highly schematic representation of the molecular imprinting process. The formation of reversible interactions between the template and polymerizable functionality may involve one or more of the following interactions: (a) reversible covalent bond(s), (b) covalently attached polymerizable binding groups that are activated for non-covalent interaction by template cleavage, (c) electrostatic interactions, (d) hydrophobic or van der Waals interactions, or (e) coordination with a metal centre; each formed with complementary functional groups or structural elements of the template, (a-e) respectively. A subsequent polymerization in the presence of crosslinker(s), a crosslinking reaction, or other process results in the formation of an insoluble matrix (which itself can contribute to recognition through steric, van der Waals and even electrostatic interactions) in which the template sites reside. Template is then removed from the polymer through disruption of polymer—template interactions, and extraction from the matrix. The template, or analogues thereof, may then be selectively rebound by the polymer in the sites vacated by the template, the "imprints." Reproduced with permission from Ref. , Copyright 2014, John Wiley & Sons.
The use of imprinted polymers is particularly tempting for sensing applications since membranes with tailored recognition functions can be generated. The major difficulty encountered in the use of imprinted polymers as active components in sensor devices is, however, the coupling of the sensing membrane with an electronic transducer. The imprinted organic polymer is usually relatively thick, and the recognition sites lack direct electrical contact with the transducer. Indeed, most of the sensor devices based on imprinted polymers are either optical, and include chromogenic markers , or involve the microgravimetric analysis of the bound substrate using piezoelectric crystals (quartz crystal microbalance) . Only a few reports have addressed the use of surface plasmon resonance (SPR) to follow the association of substrates to imprinted polymers [18-20]. In these studies, however, the SPR spectra followed mass changes associated with the binding of the substrate to the polymer. These mass changes (for low molecular-weight substrates) are small, resulting in low signals and limited analytical performance of the respective sensors.
Addressing the limitations of conventional molecular imprinting techniques, several research groups have begun to explore alternative approaches for developing new imprinting methodologies. An effective approach is to control templates to locate at the surface of imprinted materials, typically exampled by surface imprinting, which is carried out by immobilizing template molecules at the surface of suitable substrates, forming thin imprinted films [21, 22]. Surface molecular imprinting is especially valuable as it solves the problems of limited mass transfer and template removal to some extent, as compared to the conventional molecular imprinting technique. Another attempt to address the limitations of the conventional MIPs is the development of molecularly imprinted nanomaterials . Nanostructured imprinted substrates have extremely high surface- to-volume ratio, so that most of the template molecules can be situated at the surface of the materials, resulting in a large amount of effective imprinted sites, a high binding capacity, and good site accessibility for the molecularly imprinted nanomaterials [24, 25].
An important practical chemosensing field, which has significant prospects for improvement by application of MIPs, is the selective and highly sensitive detection of explosives, which attracts substantial efforts in homeland security research [26-30]. Different analytical procedures for the detection of explosives were reported, and these include optical [31-33], electrochemical [34-36], surface acoustic wave [37-39], or competitive immunoassay  methods. Recently, imprinted Au nanoparticle (Au NP) composites as ultrasensitive and selective matrixes for the electrochemical or SPR analyses of the trinitrotoluene (TNT) or RDX explosives [41-43] were introduced. The key element of this approach involves the generation ofmolecularly imprinted sites in Au NP matrixes. In contrast to the well-established principles of preparation of molecularly imprinted matrixes in organic or inorganic polymers (e.g., Ti02, Si02) [44-46], the synthesis of molecularly imprinted matrixes of metal nanoparticles, for example, Au NPs, demonstrates significant advantages emerging from the nanoscale dimensions of the building blocks of the composite-sensing interface. According to this principle, Au NPs modified with electropolymerizable thioaniline units are electropolymerized onto Au-coated surfaces in the presence of a molecular template that acts as a structural analogue for the respective substrate analyte and exhibits affinity interactions with the thio- aniline-modifying groups, or with the electrogenerated bis(aniline) bridging units. The electropolymerization of the functionalized NPs onto a thioaniline-modified surface, and the subsequent removal of the template molecules from the resulting bis(aniline)-crosslinked Au NP matrixes, resulted in the formation of molecularly imprinted contours that bind the analyte by complementary affinity interactions. For example, by the electropolymerization of the functionalized NPs in the presence of picric acid or Kemp's acid, imprinted Au NP composites for the ultrasensitive detection of TNT or RDX were, respectively, demonstrated. The binding of the substrates to the imprinted sites was monitored by SPR spectroscopy.
This chapter presents the results of specific studies related to the development ofSPRbio-and chemosensors based on the exploitation of molecular imprinting with two different approaches for SPR signal enhancement. The first study applies imprinted polymers that undergo a swelling process upon binding of the substrates, resulting in a substantial change in the refractive index of the polymer-sensing interface. This enables the improved application of SPR spectroscopy to follow the association of substrates to the imprinted polymers. As a result, the possibility to detect nicotinamide adenine dinucleotide cofactors NAD(P)+ and NAD(P)H using the imprinted polymer films by means of SPR spectroscopy was demonstrated. Additionally, these functionalized membranes have been used to analyze biocatalytic transformations involving NAD+-dependent enzyme lactate dehydrogenase. The second study is based on the application of molecularly imprinted composite polymer-Au NP matrix for the detection of ultralow concentrations of pentaerythritol tetranitrate
(PETN), nitroglycerin (NG), and ethylene glycol dinitrate (EGDN) explosives.
Application of Macromolecules Conformational Changes as a Signal Parameter for Studying the Biospecific Reactions Using SPR and Molecular Imprinting
Investigation of Enzymatic Reactions Involving NAD(P)+ and NAD(P)H
Investigation of enzymatic reactions involving p-nicotinamide adenine dinucleotide (NAD+) and p-nicotinamide adenine dinucleotide phosphate (NADP+), as well as their reduced forms NAD(P)H (see Fig. 5.2), occupies one of the leading places in biochemistry and bioelectrochemistry [47, 48]. Due to the control of the content of these cofactors, it becomes possible to significantly expand the range of enzymes that can be used for the development of intelligent biosensors and monitoring ofbiocatalytic transformations involving these cofactors. However, the role of the NADH/NAD+ and NADPH/NADP+ pairs is not limited to the promotion of enzymatic reactions. In many cases, it is much more convenient to determine the amount of the reduced (or oxidized) form of the cofactor than any product of the reaction. Often, the quantitative analysis of one of the forms of these cofactors is carried out electrochemically. However, the amperometric analysis of NAD(P)+ reveals irreversible electrochemical reduction accompanied by high overpotentials  resulting in a non-enzymatically active dimer . The NAD(P)+ cofactors were reduced electrocatalytically or bioelectrocatalytically in the presence of Rh complexes , or in the presence of NAD(P)+- dependent enzymes (e.g., ferredoxin-NADP+ reductase, lipoamide dehydrogenase, formate dehydrogenase) . Direct, non-mediated electrochemical reduction of NAD(P)+ was also achieved at modified electrodes, for example, in the presence of an L-histidine-modified Ag electrode . Similarly, the direct oxidation of NAD(P)H is electrochemically irreversible and involves high overpotentials
 . The electrocatalyzed two-electron oxidation of NAD(P)H was extensively studied in the presence of different electrocatalysts
 , for example o-quinones , phenazine, phenoxazine, and phenothiazine derivatives . There is no method, however, to selectively analyze NAD+ in the presence of NADP+ or alternatively to selectively analyze NADH and NADPH. The proposed alternative non-electrochemical method for the determination of NAD(P)+ and NAD(P)H  is based on the principle of molecular imprinting, which in recent years has begun to be used for the identification and quantification of various substances.
Figure 5.2 Structures of NAD(P)* and NAD(P)H cofactors. Reprinted from Ref. , Copyright 2004, with permission from Elsevier.
Boronic acid ligands bind strongly and reversibly vicinal diols (Eq. 5.1). This property has been employed to develop optical sensors for sugars . Boronic acid acrylamide copolymers were employed as active matrixes for the sensing of glucose  or nucleotides . The boronic acid ligand is often used to imprint molecular recognition sites in polymers for specific binding  and separation  of sugars. The selective analysis of the NAD+, NADP+, NADH, and NADPH cofactors in acrylamide-acrylamidophenylboronic acid copolymer membranes associated with ISFET devices was reported . In complementary microgravimetric quartz crystal microbalance experiments, it was found that the association of the NAD(P)+ or the NAD(P)H cofactors to the imprinted polymer matrixes linked to the Au-quartz crystals involved the uptake of water and the swelling of the polymer films.