molecularly imprinted tailor-made functional polymer

Chromatography 2016, 37, 43-64

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Focusing Review

Molecularly Imprinted Tailor-Made Functional Polymer Receptors for Highly

Sensitive and Selective Separation and Detection of Target Molecules

Toshifumi TAKEUCHI*, Tomohiko HAYASHI, Shoko ICHIKAWA, Ayaka KAJI, Manami MASUI,

Hiroki MATSUMOTO, Reo SASAO

Graduate School of Engineering, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan

Abstract Molecularly imprinted polymers (MIPs) are functional materials capable of molecular recognition and other biorelevant functions and can be prepared with ease in a tailor-made fashion by copolymerization in the presence of a crosslinker and a template molecule (the target molecule or its analog) conjugated with a functional monomer(s). These robust and affordable synthetic polymer receptors are highly accepted as favorable alternatives to biomacromolecules such as antibodies and enzymes. This article provides a critical review of the present state of MIPs to establish perspectives on this technique via a survey of early to contemporary work, mainly conducted by the authors, covering new principles and methodology to generate MIPs. These include the design and synthesis of new functional monomers and crosslinkers to develop and/or introduce new functionalities, new polymerization methods to improve the imprint effect, and highly sensitive MIP-based assays and sensors. Keywords: Molecular imprinting; Synthetic polymer receptor; Post-imprinting modification; Bioinspired materials; Molecular recognition; Biomimetics

1. Introduction Protein-protein and protein-ligand interactions play

important roles in biological systems, where the corresponding binding proteins possess specific binding activities for target molecules. These biomolecules capable of molecular recognition are extensively employed in biotechnological and bioindustrial applications such as affinity media for bioseparation, immunoassays for biologically active compounds, and molecular recognition elements for biosensors. Recently, efforts have been made to extend their applications to biomedical fields, i.e., monoclonal antibody therapy, molecularly-targeted therapy, and immunotherapy.

Although the functions of biomacromolecules have been applied in a wide range of scientific fields and their application has been recognized as the global standard, the use of biomacromolecules is problematic due to their fragility, high cost and time-consuming production, and the difficulty of quality control in their bioproduction. In the face of these problems, substitute materials that can be

artificially prepared have been intensively studied. Molecular imprinting has been attracting attention as one of such artificial contenders for replacing biomacromolecules. Molecular imprinting is a template polymerization technique involving the copolymerization of a crosslinker(s) and a target molecule or its derivative (template molecule) covalently or non-covalently bound to a functional monomer(s). After removing the template molecule, imprinted cavities are left in the polymer matrix, which are complementary to the template molecule in shape and size and capable of the template molecule recognition [1-16].

We have been studying MIPs for more than 20 years and have developed many new functional monomers, affinity media, sensing materials, biofunctional materials, stimuli-responsive materials, and array chips, as well as greatly expanded MIP application. In this review, perspectives of MIP technology are given from our previous work and insights into further development of MIPs are discussed.

*Corresponding author: Toshifumi TAKEUCHI Received: 27 April 2016Tel: +81-78-803-6158; Fax: +81-78-803-6158 Accepted: 22 May 2016 E-mail: [email protected] J-STAGE Advance Published: 31 May 2016 DOI: 10.15583/jpchrom.2016.007


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2. MIPs for small molecules prepared using a single functional monomer 2.1. Acidic monomers: acrylic acid (AA), methacrylic acid (MAA) and 2-(trifluoromethyl)acrylic acid (TFMAA)

In early their stages, MIPs were applied to target- selective separation media, which were prepared using single functional molecules, and MAA was frequently used due to its wide applicability resulting from its ability to act as both a hydrogen donor and acceptor. MIPs for atrazine, a triazine herbicide, were prepared using MAA as the functional monomer [17-22] and ethylene glycol dimethacrylate (EGDMA) as the crosslinker, and applied as separation media for solid-phase extraction (SPE) [20,22] (Fig. 1). To prepare monolith-type MIPs, in-situ preparations inside a column and a capillary tube were also conducted, which allowed the MIPs to be ready for use immediately after preparation [18,23]. Since atrazine is toxic, structurally related analog dibutylmelamine was used as a dummy template molecule in place of atrazine [21], and the performance as SPE media was found to be comparable to MIPs prepared using atrazine [22]. In the case of similar triazine herbicide prometryn, TFMAA was more suitable as the functional monomer than MAA [24,25]. This was likely due to the difference in its basicity; prometryn is more basic than atrazine due to the substituent effect of its SCH3 and Cl groups, which may lead to more preferable interactions with strong hydrogen donor TFMAA, while atrazine possesses a more moderate basicity and can therefore interact with MAA as both a hydrogen donor and acceptor. Nicotine was also molecularly imprinted using MAA [26] and TFMAA [27,28]. In this case, TFMAA induced stronger affinity than MAA, probably due to the

fact that nicotine has one binding site and therefore a stronger hydrogen donor was more suitable for molecular imprinting than weaker hydrogen donor MAA.

MIPs for atrazine were applied as the molecular recognition element of an electrochemical sensor, where the electrochemical reduction of atrazine was conducted on a gold electrode [29]. The atrazine sensor was fabricated by directly polymerizing atrazine-MIPs composed of MAA and EGDMA onto the surface of the gold electrode. By introducing LiCl onto the MIP, atrazine was reduced below -800 mV vs Ag/AgCl reference electrode at pH 3. The cathodic current of atrazine depended on the concentration of atrazine in the range of 1-10 μM, and a selective response was displayed toward atrazine.

Bisphenol A (BPA), a suspected endocrine disruptor, was also molecularly imprinted using BPA dimethacrylate [30-32] as a covalent-type BPA-MAA template molecule (Fig. 2). After polymerization, the ester bonds were hydrolyzed to create binding cavities for BPA. Immobilized BPA on spherical porous silica beads was used as the template molecule and non-covalent MAA was employed as the functional monomer. After the formation of the polymer matrix in the pores of the silica beads, an NH4HF2 treatment was conducted to dissolve the silica matrix. Spherical MIP beads for BPA recognition were obtained as a result [33].

Biotin-MIPs were prepared using MAA in the presence

of biotin methyl ester [34]. Since the data from NMR titration suggested a one-to-one complex formation of biotin methyl ester with MAA, a possible complex structure was estimated by docking the most stable conformers by intermolecular Monte Carlo conformational search under the assumption of a one-to-one association (Fig. 3). A competitive binding assay for biotin methyl ester was demonstrated using the biotin-MIPs and biotin p-nitrophenyl ester as the competitor.

AA-based MIPs showed enantioselectivity and diastereoselectivity when chiral compounds were used as template molecules even though AA is achiral and normally provides no stereoselectivity. L-Phenylalanine anilide was

Fig. 1. Preparation of atrazine-MIPs using MAA and the procedure for solid-phase extraction.

Fig. 2. BPA-MIPs prepared using BPA dimethacrylate.


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used as the template molecule and the obtained monolith-type (continuous polymer rod) MIPs showed enantioselectivity, suggesting both that the imprinted cavity possessed two binding sites for both the amino group and the amide group of L-phenylalanine anilide and that the mobility of the phenyl group may have been reduced by the cavity [23] (Fig. 4). MAA-based [35] and TFMAA-based [36,37] MIPs for a cinchona alkaloid, cinchonidine, also showed diastereoselectivity. Interestingly, the fluorescence spectra of cinchonidine (λex: 330 nm) showed a blue shift in accordance with the increase in total concentration of cinchonidine with an emission maximum around 390 nm at lower concentrations and around 360 nm at higher concentrations when TFMAA-based MIPs were added [38]. Free cinchonidine without the addition of the MIP showed an emission maximum around 360 nm, suggesting that the observed fluorescence spectral shift may have been due to a balance of the spectra of free and bound cinchonidine, which indicates that the MIP can be applied to a shift reagent for quantitating the amount of cinchona alkaloid bound to the MIPs without bound/free separation. 2.2. Basic monomers: N,N-dimethylaminoethyl methacrylate (DMA) and 4-vinylpyridine (4-VPy)

DMA was used for the imprinting of acidic compounds. Plant hormone indole-3-acetic acid-MIPs were prepared using DMA, and these MIPs were applied as SPE media [39] and molecular recognition elements for a quartz crystal microbalance (QCM) sensor [40]. The effect of DMA was examined by comparing an MAA-based MIP on developed affinity, and it was found that MIPs prepared with DMA showed higher affinity than those prepared with MAA. An important point of this study was that the hydrophilicity of MIPs affected their affinity and selectivity, i.e. MIPs prepared with 2-hydroxyethyl methacrylate (HEMA) showed better recognition ability than those prepared without HEMA [41,42] (Fig. 5). The copolymerized HEMA

affected the hydrophilicity of the polymer matrices, and hydrophilic targets such as sialic acid and other water-soluble compounds could therefore more easily approach the polymer matrices, resulting in higher accessibility to the imprinted cavities.

4-VPy was used as a functional monomer capable of hydrogen bonding and π-π stacking in molecular imprinting for non-steroidal anti-inflammatory drug piroxicam [43], BPA [33], and sialic acid [44]. Since 4-VPy works as a ligand for metal ions, a diketon compound, dibenzoylmethane, was molecularly imprinted using a Co(II) complex of one dibenzoylmethane and two 4-VPy, where the formation of binding sites was controlled by metal ion coordination [45].

Kubo et al. reported MIPs prepared using partial

structures and dummy molecules of target compounds and 4-VPy as the functional monomer. MIPs for an amnesic shellfish poison, domoic acid (DA) (Fig. 6), was prepared with mono-, di-, and tricarboxylic acids, and it was found that the highest selective recognition ability for DA was achieved when pentane-1,3,5-tricarboxylic acid (1,3,5-PeTA) was used as the template molecule [46]. Molecular imprinting of o-phthalic acid also showed high affinity and selective recognition of DA [47]. MIPs for 4-tert-butylphenol [48,49] and 2,6-bis-(trifluoromethyl) benzoic acid [50] were reported to possess selective retention of BPA and brominated/chlorinated BPA, respectively, suggesting that this selective retention is due to their shape recognition ability with respect to the alternative template molecules. The same dummy template molecules were applied in the preparation of MIPs selective

Fig. 3. The most stable complex of biotin methyl ester with MAA estimated by a Monte Carlo simulation.

L-phenylalanine anilideacrylic acidethylene glycol dimethacrylatecyclohexanol1-dodecanol2,2'-azobis(isobutyronitrile)

Fig. 4. Monolith-type (continuous polymer rod) MIPs possessing enantioselectivity toward L-phenylalanine anilide.

Fig. 5. Indole-3-acetic acid-MIPs prepared using DMA and HEMA.

Fig. 6. Chemical structure of domoic acid (DA).


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towards hydroxyl polychlorinated biphenyls [51]. MIPs for a hepatotoxin produced by cyanobacteria, microcystin (MC), were also prepared using partial and dummy structures of MC such as alkylbenzenes, 2-methoxypropylbenzene, phenylethyl methyl ether, 3-methoxy-2-methyl-4- phenylbutyric acid methyl ester, and N-carbobenzoxy- D-glutamic acid [52,53].

2.3. 4-Vinylphenylboronic acid (VPBA)

VPBA has frequently been used as a functional monomer for compounds possessing cis-diol structure, as VPBA easily forms reversible diester bonds under mild conditions. Saccharides often possess cis-diols in their structures, making VPBA a good candidate for molecular imprinting of saccharides. Sialic acid, a monosaccharide bearing a carboxyl group and commonly found in glycoproteins and gangliosides, was molecularly imprinted using VPBA [54]. As diester formation is pH-dependent, the binding affinity was changed by changing pH; optimum specificity toward sialic acid appeared to be at pH 8.1, and higher affinity with group selectivity for cis-diol containing sugars was achieved at a higher pH. VPBA was also employed to prepare MIPs for castasterone, a brassinosteroid in plant hormones bearing two cis-diols in its structure [55] (Fig. 7).

2.4. Functional monomers capable of multiple hydrogen bonding

MAA is capable of forming two hydrogen bonds due to it being both a hydrogen donor and acceptor, and in past studies when both hydrogen bonds were formed binding affinity and selectivity were enhanced, suggesting that functional monomers that can form multiple hydrogen bonds have the ability to provide strong affinity and high selectivity towards target molecules. 2,6-Bis(acrylamido) pyridine (BAP) was designed and synthesized such that it could mimic nucleotide bases pairs in DNA in forming multiple hydrogen bonds [56]. When cyclobarbital, a central nervous system depressant, was molecularly imprinted, the obtained MIP showed a distinct specificity for the template structure via the formation of one-to-two complexes of cyclobarbital and BAP residues (Fig. 8). In contrast, amobarbital bearing an isopentyl group on the 5th position on the malonylurea structure instead of a cyclohexenyl group showed only one half of the affinity toward cyclobarbital. Low affinity was also observed for 3-ethyl-3-methylglutarimide, which can form only a

one-to-one complex, and hexobarbital bearing an N-methyl group on the malonylurea structure that inhibited complex formation showed no affinity. These results revealed that the unsubstituted nitrogen atoms on the malonylurea structure were significant for binding, and that the two BAP residues precisely aligned according to the malonylurea structure. MIPs for a toxic glucose analogue alloxan were also prepared using BAP that were able to distinguished alloxan from structurally similar compounds thymine and theobromine [57].

BAP has a fluorescent property (λex: 270 nm, λem: 380 nm) and when cyclobarbital bound to the MIP, fluorescence intensity increased, possibly due to the increase in the rigidity of the BAP moieties by the formation of multiple hydrogen bonds. By using binding-responsive fluorescence enhancement, selective fluorescence detection of cyclobarbital was demonstrated with BAP-based MIPs [58]. Similarly, 2-acrylamidoquinoline, a fluorescent monomer capable of forming multiple hydrogen bonds and bearing a longer excitation wavelength than BAP (λex: 330 nm, λem: 376 nm) was reported [59] (Fig. 8) where highly sensitive and selective detection of cyclobarbital was achieved.

2.5. Stimuli-responsive monomers A novel stimuli-responsive functional monomer bearing

multiple hydrogen bonding diaminopyridine and photoresponsive azobenzene moieties was synthesized and applied to the preparation of photo-regulated MIPs for target molecule tri-4-carboxyphenyl-substituted porphyrin [60] (Fig. 9). The binding affinity of the imprinted cavities was regulated by UV irradiation, suggesting that azobenzene groups located inside the binding cavities worked as photosensitizers and that the trans-cis isomerization was able to regulate the affinity for the target compound. Repetitive binding of the target compound to

B

HO

O

O

O

O

B

Fig. 7. Chemical structure of castasterone VPBA diester.

Fig. 8. Chemical structures of 2,6-bis(acrylamido)pyridine (BAP) (a) and 2-acrylamidoquinoline (c), and a one-to-two complex of cyclobarbital with BAP (b).

NNH

NH

O O NHN

O

(a) (c)

(b)


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trans-MIPs and cis-MIPs was directly monitored by slab optical waveguide spectroscopy, and the photo-mediated regulation of binding affinity was successfully confirmed.

A photoresponsive benzospiropyran moiety was also utilized for preparing photoresponsive MIPs, where a benzospiropyran moiety containing a carboxy group was incorporated into the binding cavities that interacted with template molecule atrazine [61] (Fig. 10). Spectrophotometric analysis confirmed that the spiropyran moiety was photoresponsive even after polymerization, and photo-triggered switching of the selective binding activity of atrazine was observed; the activity was found to decrease dramatically after UV irradiation, suggesting that the spiropyran moiety in the binding cavities was transformed to its merocyanine form, resulting in unfavorable translocation of the carboxyl group for atrazine binding. Consequently, the reality of achieving reliable photoresponsive smart materials could emerge from the intensive investigations into MIPs prepared using deliberate stimuli-responsive functional monomers.

2.6. Other functional monomers

2-(Methacryloyloxy)ethyl phosphate (MEP), 3-acrylamido-N,N,N-trimethylpropan-2-aminium chloride (AMTC), 2,3,5,6-tetrafluoro-4-iodostyrene (TFIS), 2-sulfoethyl methacrylate (SEMA), and a polymerizable amino acid derivative, N-methacryloyl-L-valine t-butylamide (MVBA) have also been employed as functional monomer used for preparing MIPs. Steroid-MIPs were prepared using MEP [62], where the cholesterol-MIP showed a higher affinity for cholesterol than that for cholesterol derivatives such as cholesterol acetate and stigmasterol. The selectivity of the MIP was higher than that of MIPs prepared using TFMAA. Estradiol was also imprinted and gave similar results, demonstrating that MEP is suitable for preparing MIPs for cholesterol and related compounds.

In order to prepare MIPs for BPA in an aqueous solution, AMTC was adopted as a functional monomer that could interact non-covalently with negatively-charged BPA as a

substitute of water-insoluble BPA dimethacrylate [63] (Fig. 11). Under redox-polymerization in 50 mM NaOH, BPA formed an ion-pair with AMTC. The MIP was selective toward BPA in an aqueous solution while structurally related compounds were not recognized. Reduction of non-specific binding due to hydrophobic interactions was achieved by the addition of MeOH into the solvent for BPA binding. This technique provides a reliable way to prepare molecular recognition materials for hydrophobic compounds solubilized in aqueous media.

MIPs bearing halogen bonding-based binding sites were prepared using TFIS as the functional monomer [64] (Fig. 12). The binding cavities were generated by copolymerizing TFIS, styrene and divinylbenzene in the presence of a model template molecule, 4-dimethylaminopyridine. The MIPs preferentially adsorbed aminopyridine derivatives, suggesting that halogen bonding may have played a role in the selective recognition of analytes by the synthetic receptor.

SEMA bearing sulfonic acid was utilized for chiral

separation of indanyl substituted tetra-armed cyclens (TAC) octadentate Na+ complexes [65] (Fig. 13). Since their four side arms stand up and are bundled to form quadruplicated helical structures, they form Δ- or Λ-types enantiomers based on complex helicity. (S)-Indanyl substituted TAC sodium salt was used as the template molecule, and molecular imprinting was conducted using SEMA as a functional monomer capable of forming an ion-pair complex with the template molecule. Affinity for (S)-indanyl substituted TAC sodium salt was greater than the antipode, meaning that the MIP could discriminate helix

NN

O

N

NH

NH

C4H9

n-C4H9

O

n-

Fig. 9. Chemical structure of azobenzene-based photoresponsive functional monomer.

OHO

ON

N+

O

-O

O

O

HN

N O O

NH

N

O O

Fig. 10. Chemical structure of benzospiropyran-based photoresponsive functional monomer.

Fig. 11. Electrostatic interaction between BPA and 3-acrylamido-N,N,N-trimethylpropan-2- aminium chloride (AMTC) in aqueous solution.

Fig. 12. Halogen bonding-based MIPs prepared using 2,3,5,6-tetrafluoro-4-iodostyrene (TFIS).


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enantiomeric structures of TAC Na+ complexes. MVBA was synthesized for use in molecular imprinting

of peptides, where this monomer was expected to form multiple hydrogen bonds with peptide derivatives [66]. The obtained MIPs exhibited high stereoselectivity for the template molecule Boc-D-phenylalaninyl-L-alanine p-nitroanilide (Boc-D-Phe-L-Ala pNA) and its diastereomer Boc-L-Phe-L-Ala pNA, and amino acid sequence selectivity was also observed, i.e. Boc-D-Phe-L-Ala pNA was more highly bound to the MIPs than Boc-L-Ala-D-Phe pNA. 3. MIPs for small molecules prepared simultaneously using different functional monomers

Efforts have been made to use different functional monomers simultaneously for enhancing the affinity and selectivity of MIPs. It was natural to employ plural functional monomers bearing appropriate functional groups as the interactive sites on target molecules. For examples, sialic acid was successfully imprinted using N,N,N-trimethylaminoethyl methacrylate chloride (TMAEMA) in addition to VPBA [44,67], where TMAEMA was selected because of apparently better affinity for the target molecule than commonly used 4-VPy [44] (Fig. 14). These MIPs were successfully employed as sensing elements for QCM sensors [68] and surface plasmon resonance (SPR) sensors [69]. It should be noted that the sialic acid-MIPs also recognized monosialotetrahexosylganglioside (GM1) which contain one sialic acid moiety, meaning that MIPs fabricated using a partial structure of target molecules can also recognize the whole structure. Allyl amine has also been employed as a functional monomer instead of TMAEMA [70].

In another study, antitumor agent 5-fluorouracil (5-FU) was used as the target molecule and 5-FU-MIPs were synthesized using BAP and/or TFMAA as functional monomers [71]. The 5-FU-MIP showed higher affinity for 5-FU than for 5-FU derivatives, and by using both BAP and TFMAA simultaneously, the affinity and selectivity for 5-FU were improved, possibly due to the interaction between TFMAA and F on 5-FU.

Molecular imprinting for (+)-catechin (CA) recognition was demonstrated using 7-hydroxyflavanon (7-HF) as a structurally similar dummy-template molecule [72] (Fig. 15). 7-HF was methacrylated and monomethacrylamidyl β-cyclodextrin was used as the functional monomer. After polymerization, 7-HF was removed by alkaline hydrolysis, yielding CA binding cavities containing both a MAA residue and a β-cyclodextrin residue, which were able to work cooperatively for CA binding. The binding property of these MIPs was confirmed by fluorescent measurements (λex: 280 nm, λem: 620 nm) taken of the CA remaining in the supernatant after interaction with the MIPs.

Catalytic MIPs for atrazine were prepared using a combination of MAA and SEMA that could bind atrazine and convert it to the less toxic compound atraton, where Cl at the 6th position of the triazine structure was substituted with a methoxy group in a methanol-containing solvent [73]. Competitive inhibition of this atrazine methanolysis occurred with the addition of a structurally related binder, ametryn, which is inactive for methanolysis, suggesting that the catalytic reaction proceeded within the atrazine binding cavity generated by the molecular imprinting process.

4. Combinatorial molecular imprinting A diverse range of MIPs can be prepared by combining

functional monomers and crosslinkers in the presence of various template molecules. However, monomer recipes for preparing MIPs have generally been decided using trial and error, making it rather difficult and time consuming to obtain optimally functionalized MIPs. In order to solve this problem, we have developed a strategy called Combinatorial Molecular Imprinting, i.e. a semi-automated technique for preparing small-sized MIP libraries and screening for high affinity and selectivity toward the template molecule. MIPs were prepared directly on the bottom surface of each glass vial by a programmable

Fig. 13. Interaction between SEMA and the indanyl-substituted tetra- armed cyclen forming octadentate complexes with Na+.

Fig. 14. Sialic acid-MIPs prepared using N,N,N- trimethylaminoethyl methacrylate chloride (TMAEMA), VPBA and HEMA.

Fig. 15. MIPs for (+)-catechin (CA) prepared using 7- hydroxyflavanon (7-HF) as the template molecule coupled with MAA and monomethacrylamidyl β-cyclodextrin.


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auto-liquid handling system, followed by an examination of the binding activity in the same vials automatically [74] (Fig. 16). As model target molecules, two triazine herbicides atrazine and ametryn were employed and MIP libraries were prepared using two functional monomers, MAA and TFMAA, with EGDMA as the crosslinker. Examining the MIP libraries, it appeared that, depending upon the functional monomer used, the imprinting efficiency is different for each triazine herbicide; MAA is preferred for the atrazine receptor preparation and TFMAA for the ametryn receptor preparation. From these results, this high throughput combinatorial molecular imprinting technique was proved to be a powerful tool for finding optimal conditions for preparing MIPs with high affinity for their target molecules.

Catalytic MIPs for atrazine described before [73] were

also examined by this combinatorial approach for the selection of functional monomers to construct the atrazine decomposition sites [75]. Combined use of MAA and SEMA provided atrazine-decomposing polymers, in which atrazine was converted to the non-toxic compound atraton and the imprinting effects enhanced the catalytic activity.

A (-)-cinchonidine-MIP library was prepared on a 96-well microtiter plate using liquid-handling equipment that was programmed to prepare various breeds of the MIP [76]. The resulting polymers immobilized on the bottom surface of the wells were screened to determine their binding capacity by fluorescent measurement using a microplate reader (λex: 360 nm, λem: 465 nm), clearly displaying the high reliability and high-throughput performance of microtiter plate-based combinatorial molecular imprinting.

A method of experimental design with multivariate

analysis (partial least squares regression method) was applied to the combinatorial approach and was found to be effective toward obtaining optimal conditions with reduced library size [43] (Fig. 17). Combinatorial libraries of MIPs for non-steroidal anti-inflammatory drug piroxicam were prepared automatically and screened for high affinity with regard to six experimental factors that have a significant impact on MIP synthesis: the amount of 4-VPy (functional monomer); the amount of either EGDMA, trimethylolpropane trimethacrylate (TRIM), divinylbenzene, or BPA dimethacrylate (crosslinker); the amount of piroxicam (template molecule); the volume of acetonitrile (porogen); the amount of 2,2'-azobis(isobutyronitrile) (AIBN, initiator); and the type of initiation (photo-initiation or thermal initiation). MIP libraries were prepared by varying the six factors according to a two-level fractional factorial design of resolution VI (26-1) which was considered for each 4-VPy/cross-linker combination. The proposed procedure is time and cost effective and can be used as a general tool for preparing MIPs for different target molecules. It also provides new insight into the influence and interaction of the main factors that affect the MIP performance and could thereby facilitate improvements in MIP fabrication and design process.

5. Metalloporphyrins as functional monomers for signaling MIPs

Metalloporphyrins are a group of metal-coordinated heterocyclic macrocycles comprised of four pyrrole subunits, which can form metal complexes with various ligands. Because metalloporphyrins possess flat and rigid structures with a coordinate bonding interactive site and unique spectroscopic properties, they should be good candidates as functional monomers in molecular imprinting, especially for application in MIP-based sensing materials. The first report of MIPs prepared with polymerizable Zn(II) porphyrin was published in 1998, where the methacrylated metalloporphyrin was used in the molecular imprinting of a nucleobase derivative, 9-ethyladenine (9-EA) [77]. When the concentration of 9-EA was increased, the visible absorbance spectra of the polymer dispersed in chloroform

53Fig. 16. Combinatorial molecular imprinting using a programmable auto-liquid handling system.

53

Fig. 17. Surface plot by a method of experimental design with multivariate analysis for a library of piroxicam-MIPs prepared using 4-VPy as the functional monomer and EGDMA as the crosslinker.


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showed a red shift, and the degree of the shift was dependent upon the concentration change. This readable molecular recognition phenomenon suggests the potential application of the MIPs as optical sensor materials.

An investigation on the combined use of methacrylated Zn(II) porphyrin and MAA was conducted [78] (Fig. 18) where the metalloporphyrin moiety and the MAA residue were complexed with a nitrogen on the purine structure and the amino group at the 6th position on adenine, respectively, and it was found that selectivity was enhanced when the both functional monomers were simultaneously copolymerized compared to that of MIPs prepared using only the Zn(II) porphyrin monomer or MAA. Fluorescence quenching (λex: 423 nm, λem: 605 nm) was observed when 9-EA was added, confirming that the metalloporphyrin moiety was engaged in the selective rebinding of 9EA. These results allowed for the conclusion that metalloporphyrin-based molecular imprinting was effective for constructing ligand complementary binding sites in the imprinted cavities in which additional functional monomers can be cooperatively arranged for multiple interactions capable of signaling second messages as fluorescence.

A cinchona alkaloid, (-)-cinchonidine, was also applied to

the Zn(II) porphyrin/MAA-based MIP system [79]. These MIPs showed diastereoselective fluorescence quenching (λex: 404 nm, λem: 604 nm) according to the binding of (-)-cinchonidine, and the quenching was significant in the low-concentration range, suggesting that the high-affinity binding sites contained the porphyrin residue. The correlation of the relative fluorescence intensity against the logarithm of (-)-cinchonidine concentrations showed a linear relationship, suggesting that these MIPs could act as fluorescence sensors that selectively respond to the binding events of the template molecule.

A non-fluorescent methacryloyl Fe(III) porphyrin was also utilized for preparing MIPs for (-)-cinchonidine with MAA [80]. The obtained MIPs showed a signal response proportional to the chiral binding events of cinchonidine, where a linear relationship was observed between change in

the absorbance at 572 nm derived from the Fe(III) porphyrin moiety in the MIPs and the logarithm of cinchonidine concentrations. As can be seen, metalloporphyrin/MAA-based MIPs are applicable to selective sensing materials, and furthermore, various metalloporphyrins can be applied as functional monomers for preparing MIPs, enabling us to prepare functional MIPs for a wide range of target compounds capable of coordination with such metalloporphyrins.

6. MIPs for proteins

Attempts have been made to prepare MIPs for proteins (Fig. 19) because they could conceivably play an important role as substitutions for natural receptors in practical use. Among these synthetic contenders, molecular imprinting has emerged as a promising technique for the preparation of synthetic protein recognition materials.

MIPs selective toward lysozyme were prepared on SPR

sensor chips by radical copolymerization with AA as the functional monomer, 2-methacryloyloxyethyl phosphorylcholine (MPC) as a comonomer for protecting against the non-specific binding of other proteins, and a hydrophilic crosslinker, N,N’-methylenebisacrylamide (MBAA). SPR sensor chips were acrylated with N,N’-bis(acryloyl)cystamine, followed by the preparation of MIP thin films on the acrylated SPR chip surfaces [81]. For protein imprinting, a balance of electrostatic interactions and hydrophobic interactions is important, so the effect of salt concentration during both the preparation and the evaluation steps was examined for lysozyme imprinting. Here, MIPs were prepared using AA as the functional monomer and MBAA as the crosslinker in NaCl solutions of 0 to 40 mM concentration, and an SPR sensor was used for the evaluation of binding activity using 10 mM HEPES buffer (pH 7.4) containing NaCl at concentrations of 0 to 40 mM as the running buffer. The selectivity factor (the amount of lysozyme bound divided by that of cytochrome c) was improved with increasing NaCl concentration during polymerization. This suggests that the presence of NaCl during the polymerization reduced weak electrostatic

Fig. 18. 9-Ethyl adenine (9-EA)-MIPs prepared using methacrylated zinc(II) porphyrin and MAA.

Fig. 19. Schematic illustration of MIPs for proteins.


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interaction between lysozyme and AA, resulting in the assembling of AA residues in a conformation favorable for strong lysozyme binding. In contrast, with respect to binding evaluation conditions, 20 mM NaCl resulted in the highest selectivity factor, implying that the balance of electrostatic interactions and hydrophobic interactions was important in order to optimize specific binding for proteins due to the effect of polymer matrices in addition to the interaction between the protein and functional monomers. From this study, it became apparent that highly specific protein recognition in MIPs could be realized by optimization of salt concentration in addition to molar ratios of functional monomers and proteins.

Lysozyme-MIPs were prepared on silica beads by copolymerizing AA, acrylamide (AAm), and MBAA [82]. The molar ratio of AA to lysozyme affected the selectivity, and the optimal ratio appeared to be 5 to 1. No binding specificity toward lysozyme was observed when there was no AA present, and binding specificity decreased when the amount of AA was increased beyond this ratio.

Array-based techniques have been in great demand for use in protein profiling. Molecular recognition by differential receptor arrays could provide an enhancement in selectivity, and in 2007, protein MIP array-based analysis was reported for the first time [83]. Six different protein-MIPs prepared using three template proteins, cytochrome c, ribonuclease A, and lactalbumin, with acidic (AA) or basic (DMA) functional monomers resulted in unique fingerprints not only for the corresponding template protein but also for other proteins, allowing for the profiling of proteins for identification and classification via a multivariate analyses: principal component analysis (PCA) for the binding data of the six MIPs and the corresponding two non-imprinted polymers prepared without the addition of the target proteins (Fig. 20).

Protein-MIPs capable of specific transduction of protein

binding events into fluorescent signal change were designed and synthesized using dansyl ethylenediamine-conjugated

O-acryloyl L-hydroxyproline (Hyp-En-Dans) [84] (Fig. 21). Human serum albumin (HSA) was used as a model target protein and HSA-MIPs ware prepared on glass substrates. Specific fluorescence change (λex: 390 nm, λem: 490 nm) was observed for HSA binding on the imprinted thin film, while weaker response was observed for other proteins such as bovine serum albumin, chymotrypsin, lysozyme, and avidin. In contrast with SPR measurement results, the non-specific binding caused by the polymer matrix and/or fluorescent monomer residues randomly located outside of specific binding sites did not contribute to the observed fluorescence change. These results revealed that the proposed protein-imprinting technique using Hyp-En-Dans could provide a highly selective protein-sensing platform in which only specific binding events would be detected by fluorescent measurements.

Inorganic materials have been frequently used in the field

of nanotechnology, and the products are stable with rigid structures making them ideal for use as molecular recognition materials. Organic/inorganic hybrid thin films for protein recognition were prepared by molecular imprinting coupled with the liquid phase deposition (LPD) which is a soft-solution process for preparing metal oxide thin films from aqueous solutions at room temperature. In this process, metal oxide thin films can be deposited onto various kinds of immersed substrates using the chemical equilibrium reaction between a metal fluoro-complex and a metal oxide in an aqueous solution. The formation of metal oxides in solution may have proceeded via the following ligand-exchange (hydrolysis) equilibrium reactions. Reaction (1) was shifted to the right by adding boric acid, which can react with F- ions to form more stable, complex ions (Reaction 2). F- ions were consumed and the ligand exchange reaction was accelerated. As a result, thin films were slowly deposited homogeneously on the substrate; this process requires no special equipment.

MFx

(x-2n)- + nH2O → MOn + xF- + 2nH+ (1) H3BO3 + 4HF → BF4

- + H3O+ + 2H2O (2) Pepsin was used as a model protein and TiO2 was

deposited on gold substrates in the presence of pepsin/poly-L-lysine (PL) complexes [85] (Fig. 22). The complexes remained in the templated film after the deposition, and the PL-based binding sites for pepsin were constructed after pepsin was removed from the film.

Fig. 20. Principal component analysis (PCA) score plots showing the discrimination of four trials of five different proteins based upon bound amounts on AA-based and DMA-based MIPs. Cyt: cytochrome c; Rib: ribonuclease A; Lac: α-lactalbumin; Alb: albumin; Myo: myoglobin. Alb and Myo are non-templated proteins.

Fig. 21. Chemical structure of dansyl ethylenediamine- conjugated O-acryloyl L-hydroxyproline (Hyp-En-Dans).


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Specific binding of pepsin was confirmed by measuring SPR signals of the deposited films, while TiO2 films prepared without PL did not show any selectivity. The procedure for preparing the films was simple to perform, and the process for hybridization of the thin films with nanometer-order thickness was easily controlled by changing the LPD reaction time period. Using LPD for preparing MIPs is among the most appropriate methods to prepare hybrid materials with protein recognition ability, as it proceeds under mild conditions in an aqueous environment.

Peptide fragments originating from the target protein were used for constructing selective binding sites within imprinted cavities. Cytochrome c-MIPs bearing peptide fragment-based binding sites were prepared by copolymerization of acrylated cytochrome c with 6-monoacryloyl-trehalose (MAT) and 6,6'-diacryloyl-trehalose (DAT) as a novel hydrophilic comonomer and crosslinker, respectively, followed by enzymatic decomposition of the grafted protein in the polymer matrix by pepsin, resulting in the creation of peptide fragment-based protein-binding sites [86] (Fig. 23). Rebinding was quantitatively evaluated using MALDI TOF MS with polyethylene glycol (PEG) as the internal standard, and the selective binding ability of the MIPs was confirmed, where a greater binding ability was observed when more acryloyl moieties were conjugated with cytochrome c. The MIPs prepared using the acrylated protein showed greater selectivity than those prepared using the corresponding non-modified protein.

7. Immobilized proteins as template molecules

Since thermodynamic motion of free protein molecules can interfere with the imprinting process, using immobilized target proteins as template molecules for MIPs allows the proteins to be more easily and effectively imprinted. In this context, protein-MIP thin films were prepared using a crystallized protein as a template molecule

in HEPES buffer (pH 7.4) containing AA, MPC, MBAA, and PEG as a precipitant [87] (Fig. 24). Compared with the conventional solution-based MIPs prepared using free lysozyme, the crystallized protein-based MIPs showed higher selectivity for lysozyme than cytochrome c, trypsin, and chymotrypsin, suggesting that the imprinting for the orderly orientation of the motifs of the surface of crystallized lysozyme provides higher specificity toward lysozyme. The use of crystals has a merit of the better stability than dissolved proteins, and can be easy to apply dry processes for mass-production of protein recognition chips including array chips. Furthermore, crystallized protein-based MIPs may facilitate protein crystallization, therefore, the MIPs could be useful in the field of protein chemistry and biophysics.

Recently, MIP arrays were prepared via a novel

methodology known as transcription-type molecular imprinting [88,89], where patterned dots composed of biotinylated nanoparticles were first immobilized on a glass substrate followed by the immobilization of versatile biotinylated proteins via avidin-biotin interactions, yielding a multiple protein-immobilized stamp as a mold that could

64Fig. 22. TiO2-based pepsin-MIPs prepared by liquid phase deposition (LPD) using poly-L-lysine as the component of binding sites in pepsin-imprinted cavities.

81

Fig. 23. Chemical structures of 6-monoacryloyl trehalose (MAT) and 6,6'-diacryloyl trehalose (DAT) (a), and schematic illustration of cytochrome c-imprinted polymers bearing peptide-fragment binding sites prepared using acrylated cytochrome c followed by the enzymatic digestion to yield peptide-fragment binding sites (b).

Fig. 24. Preparation of MIPs for crystallized proteins.


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be transcribed (Fig. 25). MIPs were prepared between the stamp and a methacrylated glass substrate, and after the stamp was peeled off, MIP dots were revealed on the substrate according to the positions of the immobilized proteins on the stamp. Cytochrome c-MIPs, ribonuclease A-MIPs, lysozyme-MIPs, and myoglobin-MIPs were prepared, and competitive binding assays were conducted using the fluorescein-labeled proteins (λex: 490 nm, λem: 520 nm) as corresponding competitors. This proposed platform involving easily handled nanoparticle-based protein stamps for the preparation of MIP arrays can provide a new type of pattern recognition-based protein chip, which can be adopted as a highly stable, low cost, and tailor-made substitute for the use of conventional protein arrays in various research and industrial fields in the life sciences.

8. Preparation of MIPs using microfluidics Microfluidic techniques have been used for producing

highly uniform droplets by the shearing force of a continuous phase using a microchannel. As a new preparation method for MIP microspheres, a Y-junction microchannel was employed. There are two advantages for the preparation of MIPs using microfluidic devices: (1) the obtained polymers are spherical and monodispersed, and particle diameter can be controlled by flow speed, width and depth of path, and the mixed ratio of the organic phase and the aqueous phase; and (2) it is possible to reduce both cost and the influence on the environment, as the required amount of materials for the preparation can be minimized.

Atrazine-imprinted microspheres were prepared using a Y-junction microfluidic device of 153.1 μm width and 80.0 μm depth [90]. With a continuous phase containing 1.0 wt % poly(vinyl alcohol) (50 μL min-1) and a dispersed phase of a mesitylene solution containing atrazine, MAA, EGDMA, and 2,2-azobis(2,4-dimethylvaleronitrile) (8 μL min-1), monodisperse droplets were produced, followed by polymerization with UV light to yield spherical MIPs for atrazine recognition (50 μm in size).

BPA-MIP particles were also prepared using a similar microfluidic device with a width of 630 μm and a depth of 330 μm [91]. A dispersed phase of a dichloromethane solution containing styrene, divinylbenzene, and 2,2'-azobis(2,4-dimethylvaleronitrile) was pumped at 10 μL min-1, and a continuous phase containing 5.0 wt % poly(vinyl alcohol) was pumped at various flow rates between 300 and 2200 μL min-1. The size of the droplets could be changed by controlling the flow rate, and under the fixed flow rate of the dispersed phase of 10 μL min-1, droplets between ca. 90 and 250 μm could be prepared by changing the flow rate of the continuous phase. MIP microspheres 90 μm in size prepared at a continuous phase flow rate of 2200 μL min-1 were employed as an SPE medium. When dichloromethane was used as the adsorption solvent, BPA was strongly bound to the BPA-MIP particles by hydrogen bond formation, and after switching the solvent to methanol as the removal solvent, BPA was eluted quantitatively due to the weakening of the hydrogen bonding, confirming that the BPA-MIP particles could be applied to affinity-type SPE for BPA.

Protein-MIP microgels were also prepared using

monodispersed submillimeter-sized water in oil (W/O) droplets produced with a microchannel 500 μm wide and 100 μm deep [92] (Fig. 26). Mineral oil was used as the oil phase at a flow rate of 150 μL min-1, and an aqueous phase containing HSA as the template molecule, pyrrolidyl acrylate as the functional monomer, AAm and HEMA as comonomers, MBAA as the crosslinker, Irgacure 2959 (1-[4-(2-hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-

Fig. 25. Transcription-type molecular imprinting. Fabrication of patterned multiple protein-immobilized dot stamps for transcribed MIPs (a) and protocol for preparation of MIP arrays (b).

Fig. 26. Synthesis of sub-millimeter-sized MIP particles selective for human serum albumin (HSA) using inverse suspension polymerization with a water-in-oil emulsion prepared using a Y-junction microfluidic device.


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propane-1-one) as the water-soluble photoinitiator, and Span 85 as the surfactant dissolved in 10 mM phosphate buffer (pH 7.4) was used at a flow rate of 3 μL min-1. The prepared W/O droplets were polymerized by inverse suspension polymerization with photoirradiation at 365 nm, yielding 320 μm-sized HSA-MIP microgels. The microgels exhibited high affinity and selective bonding toward HSA. This was the first report of monodispersed submillimeter-sized microgels synthesized by inverse suspension polymerization in W/O droplets prepared using a microchannel.

9. MIPs prepared by controlled/living radical polymerization

One of the most important issues for MIPs is homogeneity of the polymer matrices, resulting in the formation of homogeneous binding cavities. Controlled/living radical polymerization seems to be a good solution and such efforts have been made using this technique to obtain homogeneously crosslinked polymers and elaborate target recognition cavities in MIPs with size- and shape-selectivity. For example, MIPs for BPA were prepared by reverse atom transfer radical polymerization (reverse ATRP) [93]. As a new template molecule, BPA di(4-vinyl benzoate) was synthesized and copolymerized with styrene and divinylbenzene. Compared with conventional radical polymerization-based BPA-MIPs, the selectivity of the ATRP-based MIPs appeared to be enhanced.

Controlled/living radical polymerization has been applied to MIPs for proteins. Ribonuclease A-MIPs were prepared by ATRP of AA, AAm, and MBAA in the presence of ribonuclease A [94]. The binding activity of the MIPs was evaluated by SPR response of MIP thin layers prepared on the gold-coated substrates. The MIPs prepared by ATRP showed a much higher binding affinity and capacity toward ribonuclease A than the MIPs prepared by conventional free radical polymerization. The selectivity was confirmed by the binding of reference proteins such as cytochrome c, myoglobin, and α-lactalbumin to MIP films at an optimal thickness of 15-30 nm.

A target protein immobilized using its specific ligand was employed as a template molecule, where the ligand was anchored on a gold substrate and served to both orient the immobilized target protein for the precise formation of homogeneous binding cavities and act as a binding site with high affinity and selectivity on the obtained MIP thin films after releasing the immobilized protein (Fig. 27). Glutathione-S-transferase-π (GST-π) and glutathione (GSH) were employed as a model protein-ligand pair, and surface initiated-ATRP using an activator generated by electron transfer (SI-AGET ATRP) of AAm, MBAA, and HEMA or N-[tris(hydroxymethyl)methyl]acrylamide (THMA) was

conducted on a gold substrate bearing a mixed self-assembled monolayer comprised of anchored GSH and bromoisobutyryl groups (initiators for ATRP) [95]. The selectivity was confirmed by comparing the binding of HSA and fibrinogen, where the MIP film thickness affected the selectivity, and it was found that a thickness of approximately 15 nm yielded the highest selectivity. More hydrophilic THMA provided higher selectivity in the presence of NaCl, giving more selective binding of GST-π. These findings show that the proposed bottom-up synthetic route using protein-ligand interaction has potential for facilitating the fabrication of highly specific MIPs for proteins.

10. Nano and sub-micrometer-sized MIPs Since MIP-nanoparticles (MIP-NPs) possess a large

surface area to volume ratio, high dispersibility, accessibility and reactivity can be expected. As the molecular recognition element of a slab-type optical waveguide-based microfluidic SPR measurement system for BPA, MIP-NPs were prepared and immobilized on the surface of consecutive parallel gold and silver deposition bands in the line of plasmon flow, allowing two individual SPR signals to be independently obtained as a result of the difference in resonant reflection spectra of these metals [96] (Fig. 28). BPA sensing was conducted in a competitive binding manner with BPA-gold nanoparticles (BPA-Au-NPs).

A highly selective and sensitive nanosensing system for

BPA based on the supraparticles composed of MIP-NPs and

Fig. 27. Preparation of MIP thin films for glutathione-s- transferase-π (GST-π) by surface-initiated controlled/living radical polymerization (SI-AGET ATRP).

MIP-NPs immobilized on a slab optical waveguide

SPR detection

BPA-AuNPs

HO OH

9

O

NH

S AuHO OH=

Free BPA

Fig. 28. Slab-type optical waveguide-based SPR sensing for BPA using MIP-NPs immobilized on a slab optical waveguide coexistent with BPA-immobilized Au nanoparticles (BPA-Au-NPs).


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BPA-Au-NPs was designed [97] (Fig. 29). The MIP-NPs functioned as molecular recognition materials and BPA-Au-NPs functioned as signal transduction materials for binding events, inducing spectral changes of localized surface plasmon resonance (LSPR) of the Au-NPs in the visible region. This novel supraparticles-based nanosensing system enabled specific sensing towards target molecules to be achieved simply, rapidly and inexpensively using only a UV-Vis spectrophotometer. Moreover, an affinity constant was obtained with this sensing system that was 12,000 times larger than that previously reported [30], and the limit of detection of sub-nM appeared to be comparable to the corresponding enzyme-linked immunosorbent assays.

Core-shell MIP-NPs bearing specific binding cavities for

cortisol was prepared by two-step emulsifier-free emulsion polymerizations of cortisol-21-monomethacrylate as the template molecule, itaconic acid as an additional functional monomer, styrene as the comonomer and divinylbenzene as the crosslinker [98,99] (Fig. 30). In using the MIP-NPs for cortisol detection, a fluorescence polarization-based sensing nano-platform was established for the competitive binding assay of cortisol using S-dansylaminoethyl-4-mercapto- cortisol (dansyl-cortisol) as the competitor, where the binding events of cortisol were transduced into the fluorescence anisotropy change of dansyl-cortisol from the bound-state to the free-state on the basis of the concentration-dependent competitive replacement of dansyl-cortisol by cortisol in the MIP-NPs (λex: 365 nm, λem: 450 nm). When MIP-NPs were prepared without

itaconic acid, the response decreased, suggesting that the itaconic acid and cortisol-21-monomethacrylate-derived MAA residues were working cooperatively. Highly sensitive cortisol detection was achieved by the proposed molecularly imprinted nanocavity-based fluorescence polarization assay for cortisol sensing with dansyl-cortisol, and the apparent limit of detection was estimated to be ca. 80 nM.

MIPs based on inorganic NPs were also reported, where a ribonuclease A-imprinted TiO2 layer was prepared on the surface of carboxylated quantum dots (QDots) by LPD [100] (Fig. 31). Fluorescence spectra of the TiO2-based MIP-QDots were measured in the presence of various concentrations of ribonuclease A at pH 7.0 (λex: 350 nm, λem: 530 nm), and fluorescence intensity was found to decrease with increased concentration of ribonuclease A, while the fluorescence of the non-imprinted QDots were varied only slightly, revealing that the ribonuclease A binding cavities were successfully constructed by the proposed LPD-based molecularly imprinting process, and the readout of specific ribonuclease A binding events could be achieved by measuring fluorescence change.

11. Post-imprinting modifications In order to achieve more sensitive and specific binding

with MIPs, a novel approach known as post-imprinting modifications (PIM) was developed, which was inspired by the mechanism of protein biosynthesis found in nature. Biological functions are acquired by proteins via post-translational modifications after biosynthesis in ribosomes, and sometimes additional modifications are provided by forming adducts with non-protein prosthetic groups and cofactors, allowing the proteins to develop a diverse range of biofunctions. Such post-biosynthetic processing through a chemical-modification strategy is also applicable when fabricating MIPs, where common binding scaffolds are first constructed by molecular imprinting using cleavable functional monomers and/or modifiable fu nc t io na l mo no mer s fo r sub seq ue nt sp ec i f i c post-polymerization modifications within the imprinted cavity. This can provide MIPs with the following

MIP-NPs

Free-BPA

OH

OH

OHO

HOOOHHO

OHO

HOO

ONH

OHHO

SAu

9

Red shift Blue shift

BPA-Au-NPs

ONH

OHHO

SAu

9

99

Fig. 29. Supraparticles formed with MIP-NPs and BPA- immobilized Au nanoparticles (BPA-Au-NPs).

Fig. 30. Chemical structures of itaconic acid, cortisol-21- monomethacrylate, and S-dansylaminoethyl-4-mercapto-cortisol (Dansyl-cortisol).

Fig. 31. Ribonuclease A-MIPs prepared by liquid phase deposition (LPD) of TiO2 on quantum dots (Qdots) for fluorescence sensing.


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modifications: 1) the addition of new functional groups, 2) transformation and rearrangement of intrinsic functional groups into more favorable ones, and 3) complexation with different macromolecular and/or inorganic substances via scaffold matrices (Fig. 32).

11.1. Transformation of binding sites within the molecularly imprinted cavity

The first PIM was reported in 2002, where site-directed conversion of recognition groups in imprinted cavities was conducted by posteriori-chemical modifications [101] (Fig. 33). Template molecule allyl phenyl disulfide was copolymerized with divinylbenzene as the crosslinker followed by reductive cleavage of the disulfide bond with NaBH4 to remove the thiophenol moiety, yielding binding cavities containing thiol groups complementary in size and shape to the phenyl ring. The obtained MIPs selectively recognized phenol rather than thiophenol. The higher affinity for phenol was confirmed by ab initio calculation, where the stabilization energies of hydrogen bonding of phenol, thiophenol, and other compounds towards methyl mercaptan, which was used in place of the thiol group at the imprinted cavity, were calculated with GAUSSIAN 98W [102] based on Hartree-Fock/6-31G(d). Hydrogen bonding energy ΔEHB can be expressed by the equation ΔEHB = E(A:B) - [E(A:β) + E(α:B)], where E(A:B) is the total energy optimized for the supermolecule A:B, E(A:β) is the

total energy of molecule A with additional basis sets β put on the position of molecule B, and E(α:B) is the total energy for molecule B with additional basis sets α put on the position of molecule A. This equation represents the stabilization energy between the two molecules at the geometries optimized for the complex A:B in which the basis set superposition error (BSSE), which often substantially affects the calculated stabilization energies, was corrected by means of the counterpoise method [103]. As expected, the hydrogen bonding strength of phenol to methyl mercaptan (-2.67 kcal mol-1) was higher than that of thiophenol (-0.82 kcal mol-1), supporting the higher retention of phenol [104].

The thiol groups in the imprinted cavities were then

oxidized with H2O2/acetic acid to transform the thiol groups into sulfo groups, allowing amines to be strongly bound via a single electrostatic interaction between the amino group and the sulfo group in the binding cavity [101]. It should be noted that conventional molecular imprinting is always conducted using functional monomers that possess the desirable functional groups to be introduced into the binding cavities. However, with this methodology functional groups are often grafted not only within the imprinted cavity but also randomly outside of the imprinted

Fig. 32. Schematic illustration of molecular imprinting using modifiable and cleavable monomers followed by post-imprinting modifications (PIMs). 1st PIM: addition of new functions; 2nd PIM: transformation and rearrangement of functional groups.

Fig. 33. Post-oxidative conversion of a thiol residue to sulfonic acid in the binding sites of MIPs.

Fig. 34. Dopamine-MIPs prepared using a designed template molecule consisting of a dopamine-like structure connected with an allyl moiety via disulfide bond formation and with a 4-vinylphenyl group via cyclic boronic diester formation followed by post-imprinting oxidation.


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cavity throughout the polymer matrix. In contrast, using PIM strategy the sulfo groups could be site-directedly generated only within the imprinted cavities, resulting in the decrease of non-specific binding.

Dopamine imprinting via PIM was conducted using a newly designed template molecule bearing a dopamine-like structure equipped with an allyl moiety via a disulfide bond on the amine side and a 4-vinylphenyl group via a cyclic boronic diester bond on the catechol structure, which allowed for PIM after polymerization was carried out [105] (Fig. 34). After copolymerization with styrene and divinylbenzene, the cyclic diester bonds were hydrolyzed and the disulfide bond was reduced to remove the template molecule moiety from the polymer matrix, followed by PIM oxidation to transform the thiol residues into sulfo groups. The imprinted polymer adsorbed dopamine selectively in an aqueous solution via these two-point interaction binding cavities, i.e. the formation of a cyclic boronic diester with the catechol moiety and an electrostatic interaction of the sulfonic acid residue with the amino group of dopamine. The affinity for dopamine was 400 times greater than that of the MIP before oxidation, and the oxidated MIP showed selective binding of dopamine over analogues such as tyramine, catechol, 3,4-dihydroxyphenylacetic acid, homovanillic acid, epinephrine, and norepinephrine, confirming that the binding activity and selectivity of the MIP was significantly improved by employing the strategy of PIM.

Catalytic MIPs were also prepared via PIM. As described in the previous section, SEMA-based MIPs processed atrazine into non-toxic atraton [73,75]; however, because of the use of free SEMA, undesirable binding cavities were also generated, which resulted in decreased selectivity for atrazine. Therefore, PIM was conducted using designed template molecules composed of an atrazine structural

component coupled with allyl mercaptan or 4-vinyl thiophenol via a disulfide bond at the 6th position instead of the original Cl group [106] (Fig. 35). MAA, styrene, and divinylbenzene were used as the functional monomer, comonomer, and crosslinker, respectively. After polymerization, the disulfide bond was reduced to remove the atrazine moiety from the polymer matrix, followed by PIM oxidation of the resulting thiol groups to form sulfo groups that could work as catalytic sites, resulting in both MAA residues and sulfo groups existing in an atrazine-imprinted cavity. The resulting MIPs displayed the selective binding of triazine herbicides and specific catalytic activity for methanolysis at the 6th position of atrazine with Michaelis–Menten kinetics-like behavior over other types of pesticides.

Another oxidative PIM procedure was designed where a dummy template molecule for BPA, 4,4'-diaminodiphenylmethane was used coupled with two styrenes via Shiff base formation [107] (Fig. 36). After polymerization with styrene and divinylbenzene, the Schiff-bases were cleaved by a weak acid treatment, followed by PIM oxidation of the residual aldehyde to form carboxylic acid residues. These designed binding sites with affinity toward BPA were created only inside the binding cavity. These PIM-based MIPs showed much higher selectivity than conventional MIPs prepared using BPA dimethacrylate [30-32].

11.2. Reconstitution and exchange of binding sites within the molecularly imprinted cavity

Conjugated protein mimics bearing cofactor-binding sites were prepared by metalloporphyrin-based covalent molecular imprinting of D-tyrosine anilide [108] (Fig. 37). Methacryloyl D-tyrosine anilide (D-TyrAN) was used as the template molecule and tris(4-methacryloyloxyphenyl)- substituted Zn(II) porphyrin (Zn-TMPP) was used as the functional monomer. After copolymerization with styrene and divinylbenzene, D-TyrAN and Zn-TMPP moieties were removed from the obtained polymer by hydrolysis with KOH, resulting in an apo-type scaffold. When tripyridyl- substituted Zn(II) porphyrin (Zn-TPyP) was added as a

Fig. 35. Atrazine-transforming catalytic MIPs prepared using a designed template molecule consisting of an atrazine-like structure connected with an allyl moiety via a disulfide bond, followed by post-imprinting oxidation.

Fig. 36. MIPs for BPA prepared using the Schiff base-type dummy template followed by post-imprinting oxidation.


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cofactor to re-construct the binding cavity (holo-type imprinted cavity), D-TyrAN could be bound to the holo-type cavity via interactions of the residual MAA within the cavity and the Zn(II) ion on the porphyrin plane with the phenolic hydroxy group by hydrogen bonding and the primary amine by coordinate bonding, resulting in enantioselective binding activity where the apo-type scaffold did not show any enantioselectivity. This may have been due to the synergetic activity of the MAA residue and Zn-TPyP, meaning that precise re-constitution of the binding cavities was achieved by recalling the structure of the template molecule even after replacement of the original porphyrin by the structurally related cofactor.

PIMs made possible the construction of prosthetic group-coupled tunable binding cavities for BPA by using a novel template molecule containing a BPA structural

component covalently conjugated with two allyl(4-carboxyphenyl)disulfides via ester bonds (BPA-D) [109] (Fig. 38). After copolymerization with styrene and divinylbenzene, the BPA di(4-mercaptobenzoate) moieties were removed by reductive cleavage of the disulfide bonds, resulting in an apo-type scaffold bearing two thiol residues. As the PIM, two 4-mercaptobenzoic acids were introduced into the apo-type scaffold as prosthetic groups via disulfide exchange reaction with 4,4’-dithiodibenzoic acid, resulting in holo-type cavities with two carboxylic acid residues available for binding with BPA. The attaching and detaching of the prosthetic group could be repeatedly conducted, which indicated that the apo-type scaffold was highly durable and reusable. When pyridyl prosthetic groups were introduced instead of 4-mercaptobenzoic acid by modifying with 4,4’-dithiodipyridine, BPA recognition ability was maintained with improved selectivity, confirming that the binding activity was tunable by PIMs. A non-covalent binding cofactor was also examined for tuning BPA binding affinity [108]. The thiol group in the apo-type scaffold was oxidized to form a sulfo group, and then 1,2-ethylenediamine was added to form sulfonate salt from sulfo groups in the cavity, resulting in the improvement of the binding properties of the MIPs due to the introduction of a space-filling interactive cofactor with affinity toward BPA. Testing the control of binding properties in the MIPs by introduction/removal of prosthetic groups/cofactors clearly showed the strong potential of PIM techniques for mimicking natural proteins.

More complicated conjugated protein mimics were prepared using a new molecular imprinting strategy combined with circumspect PIMs which involved the independent conjugation of the two different prosthetic groups at pre-determined positions within the apo-type

Fig. 37. Non-covalent cofactor (tripyridyl-substituted Zn(II) porphyrin)-coupled MIPs for D-tyrosine anilide prepared using tris(4-methacryloyloxyphenyl)-substituted Zn(II) porphyrin as the functional monomer and methacryloyl D-tyrosine anilide as the template molecule.

Polymerization

Reduction

Oxidation

Coupling with DTBA Coupling with DTPy

Coupling with the cofactor

Fig. 38. Molecularly imprinted tunable binding sites based on conjugated prosthetic groups and ion-paired cofactors. An apo-type scaffold (Apo-SH) was prepared using BPA-D as the template molecule. Prosthetic groups such as 4-mercaptobenzoic acid and 4-mercaptopyridene were conjugated using 4,4’-dithiodibenzoic acid (DTBA) and 4,4’-dithiodipyridine, respectively. Ethylene diamine was used as an ion-paired cofactor toward the oxidative apo-type scaffold (Apo-SO3H).


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scaffold. Beta-lactam antibiotic was employed as a model target molecule, and a designed template molecule that contained the basic structure of cephalexin and two different prosthetic groups attached via disulfide bonding and Schiff base formation was prepared [110] (Fig. 39). After copolymerization with crosslinker triethyleneglycol dimethacrylate, the template molecule was removed together with the prosthetic groups, yielding the apo-type scaffold (PRECURSOR). When 4-formyl benzoic acid (BA) and 4,4’-dithiodianiline (AN) were used as the prosthetic groups, the complete binding cavity HOLO(BA)(AN) was obtained and showed specific binding for ampicillin, an analog of cephalexin, whereas the imprinted cavities with one prosthetic group, either HOLO(BA) or HOLO(AN), in which only one binding site was available showed much less binding activity for ampicillin, confirming that the re-construction and tuning of the binding cavities was achieved by PIMs.

Fluorescent MIPs were also constructed by attaching a fluorescent dye, 5-formylsalicylic acid (FSA), instead of 4-formyl benzoic acid, yielding HOLO(FSA)(AN), and fluorescence detection of ampicillin was successfully carried out with this MIP (λex: 365 nm, λem: 446 nm) (Fig. 40). Furthermore, a photo-responsive property was introduced into the MIPs by a PIM involving the conjugation of 4-nitrosobenzoic acid with the amino group in the apo-type scaffold as an irreversibly attached prosthetic group to form a photo-tunable binding domain based on an azobenzene moiety.

As can be seen, through the independent conjugation of the two different prosthetic groups at pre-determined positions within the apo-type scaffold, the apo cavity was transformed into a functionalized holo cavity possessing a

diverse range of introduced functionality including site-specific addition, transformation, rearrangement, and other functional alterations, depending on the reaction and prosthetic groups used with the PIMs. In addition, on/off switching of functions was achieved by tautomeric interaction between apo-/holo-type structures.

11.3. Site-specific introduction of reporter molecules for sensing

Regarding MIPs for proteins, PIMs exhibit tremendous power for the introduction of site-specific fluorescent dyes into the imprinted cavity, yielding signaling MIPs that respond to the binding events of target proteins. A functional monomer 4-[2-(N-methacrylamido) ethylaminomethyl]benzoic acid (MABA) was designed specifically for PIMs that contained three functional groups for protein imprinting: a polymerizable methacryloyl group, a secondary amino group for fluorescent dye conjugation by PIM, and a benzoic acid moiety capable of interacting with amino groups on target proteins by electrostatic interaction [111] (Fig. 41). Lysozyme-imprinted thin films were prepared using MABA. After the removal of lysozyme, fluorescein isothiocyanate (FITC) was introduced into the secondary amino group on MABA residues in the imprinted thin film as a fluorescent reporter dye. Fluorescence

HN

ONH

NO

S

O SS

HN

NH

ON

HN

O

O HN

ONH

NO

S

O SS

HN

NH

ON

HN

O

O

SSNH

ONH2

HN

O

Template molecule

SS H+

PRECURSOR

HOLO(BA)

OHSS

NH

ON

HN

O

O

HOLO(BA)(AN)

OHSS

H2N

NH

ON

HN

O

O

HOLO(NONE)

SSNH

ON

HN

O

HOLO(AN)

SSH2N

NH

ON

HN

O

Polymerization

O

OOH

O

OOH

OO

S S

NH2

H2N

S S

NH2

H2N

78

Fig. 39. MIPs for β-lactam antibiotics bearing molecularly imprinted reconstructible and transformable regions in the apo-type scaffold (PRECURSOR) that are assembled using space-filling prosthetic groups such as 4-formylbenzoic acid (BA) and 4-mercaptoaniline (AN).

SSNH

ONH2

HN

O

SHOHO O

OH

OHO

MBA FSA NOH

HN

OOH

OSS

NH

OOHO

PRECURSOR

HOLO(FSA)(MBA)

Fig. 40. Fluorescent MIPs for β-lactam antibiotics constructed by the post-imprinting conjugation of fluorophore 5-formylsalicylic acid (FSA) and 4-mercaptobenzoic acid (MBA).


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intensity increased with added lysozyme, and the relative change in fluorescence intensity (λex: 490 nm, λem: 520 nm) was greater than that of the non-imprinted polymer film prepared without lysozyme. These results indicate that the imprinted cavity was created via the imprinting process, and that the fluorescent dye was successfully introduced into the lysozyme-imprinted cavity. Upon comparing SPR response for the binding of lysozyme, the apparent selectivity for the fluorescence system was found to be high, implying that the introduction of fluorescent dye by PIM provides a means to prepare fluorescent MIPs in which only specific binding events are transduced into changes in fluorescence due to specific labeling of the functional monomer residues within the imprinted binding cavity.

It is well known that non-covalent imprinting-based

binding cavities tend to have heterogeneous affinities and erroneous functional monomer residues outside of the binding cavities as well as unextracted buried template proteins, resulting in the inaccurate labeling of fluorophores via PIM and therefore high fluorescent background noise. In order to solve this problem, a new capping treatment to void the low affinity binding cavities was conducted by PIM for MABA-based lysozyme-MIPs [112] (Fig. 42), where the PIM was carried out in two steps: (1) the protection of high affinity binding cavities by adding a low concentration of lysozyme, followed by a capping treatment with p-isothiocyanatophenyl α-D-mannopyranoside (MITC) on unprotected low affinity sites, and (2) the site-directed introduction of FITC into the high affinity binding cavities after the removal of lysozyme. The capping treatment was found to improve the sensitivity of specific transduction of the binding events of lysozyme as fluorescent change (λex: 485 nm, λem: 510 nm), facilitating further development of sensitive and selective MIPs with signal transduction abilities as artificial molecular recognition elements in chemical sensors.

A cleavable functional monomer containing a disulfide

link and a maleimide group was designed and synthesized for PIM, yielding protein-MIPs with specific fluorescent signaling binding cavities [113] (Fig. 43). Cytochrome c containing a free thiol group was used as a model template protein and the cleavable functional monomer was covalently conjugated with cytochrome c. After copolymerization of the conjugated cytochrome c with AAm and MBAA, cytochrome c was removed by reduction of the disulfide bond in the cleavable functional monomer, leaving only free thiol groups within the imprinted cavity. This was followed by PIM by which a thiol-reactive fluorophore DBD-F (λex: 410 nm, λem: 537 nm) was site-directedly introduced into the imprinted cavity, resulting in specific transduction of the binding events into fluorescence spectral change. In contrast to MIPs prepared using a non-covalent binding-type cleavable monomer, the incidental fluorescence background noise was suppressed during cytochrome c binding due to the fact that the reporter molecule reactive site was present only inside the MIP cavity. As is apparent from this study, protein imprinting using cleavable monomers is a powerful tool for the production and development of specific signal transducing MIPs for detecting protein binding events.

Most proteins have no free cysteine in their structures, so carboxylic acid-type cleavable monomer ({[2-(2- methacrylamido)-ethyldithio]ethylcarbamoyl}methoxy) acetic acid (MDTA) was also prepared, and protein- imprinted cavities with exchangeable domains that could be used for PIMs were constructed for lysozyme-MIPs [114] (Fig. 44). Copolymerization was carried out with AAm, MBAA, and MDTA in the presence of lysozyme, where MDTA interacted with lysozyme and assemble close to lysozyme in the resulting polymer. After reduction to remove lysozyme, the exposed thiol groups within the

Fig. 41. Fluorescent MIPs for proteins prepared using 4-[2-(N-methacrylamido)ethylaminomethyl]benzoic acid (MABA) as the modifiable functional monomer followed by post-imprinting fluorophore introduction.

HN

OHO

HN

OHO

NOH

O

NOH

O

FITC

MITC

Uncapped MIP Protection of the reactive sites within the Lyso-binding cavity

Hindrance of reactivity of the amine on MBAB unprotected

Introduction of FITC to the reactive sites within the Lyso-binding cavity

FITC reactive sites

HN

OHO

HN

OHO

HN

HHHN

LysoHN

OHO

NOH

O

Capped site

HN

OHO

NOH

O

MITC-Capped MIP

(a)

(b)

Fig. 42. Fluorescent MIPs for proteins prepared by the site-directed two-step PIM. 1st PIM: Protection of high affinity binding cavities by adding a low concentration of the template protein, followed by a capping treatment with p-isothiocyanatophenyl α-D-mannopyranoside (MITC); 2nd PIM: Site-directed introduction of a fluorophore into the high affinity binding cavities


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imprinted cavities were modified with aminoethylpyridyldisulfide to transform them into aminoethyl groups that could work as active sites for amine-reactive fluorophores. FITC was then coupled with the aminoethyl groups yielding site-specific FITC-modified imprinted cavities for the detection of lysozyme (λex: 485 nm, λem: 520 nm). Because the in-cavity fluorescent labeling was achieved via formation of a disulfide link, it was easy to remove, exchange, and/or replace these amine-reactive fluorophores. This facilitated the screening of fluorophores to select for the one displaying the highest readout of binding events, the replacement of fluorophores when photo-bleaching occurred, and the introduction of other functionalities into the binding cavities.

12. Conclusion Molecular imprinting has improved greatly over the past

couple of decades and has become a truly innovative field of study. It has evolved from an interesting idea into a practical and powerful tool for creating synthetic recognition materials whose sensitivity, selectivity and functionality rival and even exceed those of natural

recognition elements. Every year new and exciting advances are being made, and in coming years we expect to see MIPs become ever more prominent in the eyes of the scientific community as their diversity and capabilities are steadily explored and their application continues to expand into new fields of science.

Acknowledgements

The Takeuchi Lab work on MIPs began in Prof. Isao Karube’s Lab at the University of Tokyo with Dr. Jun Matsui in 1991, and obviously none of our success with MIPs would have been achieved without this opportunity. Therefore, I would like to express my deep appreciation for Prof. Karube (currently the president of Tokyo University of Technology, Japan) and Prof. Matsui (currently a professor at Konan University, Japan). Many dedicated efforts have been made by university/company collaborators, former and current staff members, post-doctoral fellows, and students of the University of Tokyo, Hiroshima City University, and Kobe University, Japan. Prof. Klaus Mosbach (Lund University, Sweden), Prof. Günter Wulff (Heinrich Heine-Universitat Duseldorf, Germany), and Prof. Kenneth J. Shea (University of California, Irvine, USA) always encouraged me to go forward in this field, and I am grateful for their valuable support. Finally, I would like to thank Prof. Takenori Tanimura, Dr. Shigeo Yamazaki, and the late Dr. Rikizo Horikawa for giving me the opportunity to pursue a Ph.D. at Toyama Medical and Pharmaceutical University, Japan. The Takeuchi Lab work presented here was supported by MEXT (KAKENHI), JSPS (KAKENHI, PRESTO, A-STEP, SENTAN-JST), NEDO (Industry/academia collaboration program, Innovation promotion program), Hyogo Science and Technology Association, CASIO Science Promotion Foundation, Kawanishi Memorial Shin Meiwa Education Foundation, Nakatani Foundation, Ciba-Geigy Foundation, Kato Memorial Bioscience Foundation, Kowa Life Science Foundation, Japan Securities Scholarship Foundation, Towa

SS

Co-polymerizationwith cross-linker

Template removal

FL dye Introduction

NH

OS S

HN O

ONH

ON

O

O4 N

H

OS S

HN O

ONH

ON

O

O4

Cleavable monomer

Target protein

Cleavable monomer-protein conjugate (Template)

SH S

Fig. 43. MIPs for proteins prepared using the template protein covalently conjugated with cleavable functional monomers followed by site-specific post-imprinting introduction of fluorescent reporter molecules (FL-dye).

Fig. 44. Molecularly imprinted protein recognition cavities bearing exchangeable binding sites for PIMs prepared using a cleavable functional monomer ({[2-(2-methacrylamido)- ethyldithio]ethylcarbamoyl}methoxy)acetic acid (MDTA) capable of non-covalent interaction with the target protein during the imprinting process.


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Foundation for Food Science & Research, Urakami Foundation for Food and Food Culture Promotion, and other research funds from private companies. This focusing review is written in commemoration of the 2015 Chromatographic Sciences Award, and I am deeply grateful to the award committee and all members of the Society for Chromatographic Sciences (T. Takeuchi). References [1] Matsui, J.; Takeuchi, T. In Molecularly Imprinted

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