Molecularly imprinted polymers as synthetic mimics of biorteceptors . 1 . General principles of molecular imprinting

The review is devoted to analysis of the publications in the area of synthesis of artificial mimics of biological receptors using the method of molecular imprinting. General principles of molecular imprinting as well as main types of polymers being used in molecular imprinting are described. The special attention is paid to the polymers-biomimics synthesized using the method of non-covalent molecular imprinting.

The abil ity of liv ing cells to ob tain in for ma tion from ex ter nal en vi ron ment is based on mo lec u lar rec og nition phe nom e non.Reg u la tion of vir tu ally all bio chem ical pro cesses in liv ing or gan isms is as so ci ated with mo lec u lar rec og ni tion based on complementarity of biomolecules: en zy matic ca tal y sis, intracellular transport, in ter ac tion of hor mones and other me di a tors with their re cep tors, and an ti gen-an ti body in ter ac tions.In other words, molecular recognition is a fundamental principle of life.
Unique se lec tiv ity of biomolecules pro vides their wide prac ti cal ap pli ca tions -for the de vel op ment elabo ra tion of an a lyt i cal meth ods and bio tech no log i cal pro cesses as well as in med i cal di ag nos tics.Nor mally nat u ral re cep tors in ter act with the cor re spond ing ligands with high af fin ity, how ever, un der non-phys i olog i cal con di tions they are very un sta ble.Un for tunately, all biomolecules are ex tremely sen si tive to changes of tem per a ture, pH, [the] pres ence of or ganic sol vents, tox ins, heavy met als, etc.The gen eral draw -backs of all biomolecules, lim it ing their wide prac ti cal ap pli ca tion, are com pli cated pro ce dures of their iso lation and pu ri fi ca tion as well as high cost.Pos si bil i ties of get ting nat u ral re cep tors in pre pa ra tory quan ti ties are often limited.Moreover, not all the molecules of interest have their natural receptors.
Syn the sis of ar ti fi cial re cep tors able to rec og nize and bind dif fer ent tar get mol e cules with high af fin ity and spec i fic ity is a top i cal prob lem of cur rent bio technol ogy, an a lyt i cal bio chem is try, and med i cine.Ide ally, these ma te ri als are to com bine the abil ity of bioreceptors to rec og nize cor re spond ing analytes selec tively with sta bil ity, easy synthetic procedure, and low cost.
Dur ing re cent years mo lec u larly-im printed polymers (MIPs or so-called poly mers-biomimics), mimick ing ac tive sites of an ti bod ies and bi o log i cal re ceptors, have at tracted sig nif i cant at ten tion [1].They can pro vide high se lec tiv ity [2][3][4], while the method of their syn the sis is quite sim ple.Nor mally, they dem on strate suf fi cient ther mal and me chan i cal sta bil ity, as well as sta bil ity in ag gres sive me dia [5].There fore, polymers-biomimics com bine high se lec tiv ity of biomolecules with the sta bil ity of synthetic polymers in harsh environments.
The method of mo lec u lar im print ing [1] is widely used for the syn the sis of poly mers-biomimics.It assumes for ma tion of highly cross-linked poly mers around so-called tem plate mol e cules.The syn the sis takes place due to co-poly mer iza tion of func tional and cross-link ing mono mers in the pres ence of tem plate mol e cule (the tem plate mol e cule is at the same time an analyte of in ter est).Ex trac tion of the tem plate mol ecules from the fully-formed poly meric net work re sults in for ma tion of cav i ties, which [are com ple men tary to the tem plate] in their size, shape, and spa tial ar rangement of func tional groups are com ple men tary to the tem plate.The mo lec u larly im printed poly mers syn thesized ac cord ing to this prin ci ple are able of fur ther selec tive bind ing the template molecules (Fig. 1).
The com plex be tween func tional mono mers and tem plates can be formed un der par tic i pa tion of both revers ible co va lent and noncovalent (hy dro gen, ionic, hy dro pho bic, van der Waals) in ter ac tions.The method of co va lent im print ing, pro posed by Wulff and co-authors [6][7][8][9][10][11][12][13], as sumes syn the sis of the tem plate mol ecule de riv a tive, ca pa ble of poly mer iza tion.Af ter the syn the sis of the co va lent mo lec u larly-im printed polymer, the tem plate mol e cule is to be re moved through cleav age of the co va lent bonds be tween the tem plate and func tional mono mers.Ap pli ca tion of this method has sig nif i cant lim i ta tions in se lec tion of po ten tial template mol e cules, while ki net ics of their bind ing by the poly mers is quite slow.How ever, a sig nif i cant ad vantage of this ap proach as com pared to the non-co va lent one is formation of more homogeneous (in terms of affinity) population of binding sites.
More uni ver sal ap proach to the syn the sis of molec u larly-im printed poly mers was pro posed by Mosbach and co-au thors [14][15][16][17][18]. Ac cord ing to the non-co va lent ap proach, for ma tion of the com plex tem plate-func tional mono mer takes place due to non-co va lent in ter ac tions.This ap proach is more flexi ble as com pared to the co va lent one, since the choice of tem plates and func tional mono mers is vir tu ally unlim ited, while the tem plate mol e cules can be eas ily ex tracted from the poly mer by the cor re spond ing organic sol vent.It is widely rec og nized that the non-cova lent mo lec u larly-im printed poly mers con tain het ero ge neous (in terms of af fin ity to wards the tem plate mol e cule) pop u la tion of syn thetic bind ing sites, which is of ten com pared to pop u la tions of polyclonal an ti bod ies [19,20].
Vulfson et al. dem on strated a pos si bil ity of synthe sis of hy brid ma te ri als, where in ter ac tion of a template with func tional mono mers takes place due to both co va lent and non-co va lent in ter ac tions [21].
Since the ap proach based on non-co va lent imprint ing is more uni ver sal for the syn the sis of ar ti fi cial an a logues of biomolecules, the main at ten tion in the pres ent re view is fo cused on the mo lec u larly-imprinted poly mers, syn the sized ac cord ing to this princi ple.
To be suc cess fully used as a tem plate, a sub stance is to be sta ble un der poly mer iza tion con di tions (t=60-80°C, UV-ir ra di a tion), and it is not sup posed to con tain groups in hib it ing/able to take part in poly meriza tion.
Types of the poly mers used in mo lec u lar imprint ing.The struc ture of a poly meric ma trix is crucial in mo lec u lar im print ing, since it de ter mines se lec -tiv ity of syn thetic bind ing sites in re sult ing poly mers.The lat ter are to ful fill the fol low ing re quire ments [1]: 1) the polymer is to be highly cross-linked so that selective sites retain their shapes after removal of the template molecules, 2) cer tain flex i bil ity of poly mer chains, which for the first site con tra dicts high de gree of cross-link ing, is nec es sary for fast ki net ics un der [the] bind ing of tem plate mol e cules, 3) as many as pos si ble syn thetic bind ing sites are to be ac ces si ble for the in ter ac tions with template mol e cules, 4) the poly mer is to be me chan i cally sta ble, which is cru cial re quire ment in the case of its ap pli cation in harsh en vi ron ments (i.e. or ganic sol vents), HPLC or SPE un der in creased pres sures, in in dus trial re ac tors un der con stant stir ring, etc. 5) ther mal sta bil ity is es sen tial in the case of MIPs ap pli ca tion un der in creased tem per a tures, that are fa vor able for better ki net ics.
The main types of the poly mers that are be ing used in mo lec u lar im print ing are or ganic poly mers, compos ite ma te ri als con sist ing of thin lay ers of or ganic poly mers on/in in or ganic car ri ers, sil ica gels, and biopolymers.
Or ganic poly mers.Most of mo lec u larly-imprinted poly mers are macroporous acrylate or vi nyl poly mers.That is de ter mined by the wide spec trum and avail abil ity of the mono mers for their syn the sis.Macroporous or ganic poly mers are syn the sized by rad i cal co-poly mer iza tion of func tional and cross-link ing mono mers in the pres ence of tem plate mol e cules and in ert sol vents (porogens).Macroporous poly mers are formed in the case of a high con tent (up to 90-95%) of cross-linker in the initial mono mer com po si tion.Fur ther poly mer iza tion leads to phase sep a ra tion re sult ing in for ma tion of per ma nent pore struc ture.The most widely used functional mono mers for the syn the sis of mo lec u larly-imprinted poly mers are sum ma rized in Fig. 2.
For ma tion of sta ble tem plate-func tional mono mer com plexes is cru cial in the tech nique of mo lec u lar imprint ing.Nor mally, for ma tion of sta ble com plexes with tem plate mol e cules in non-co va lent MIPs is achieved un der ex cess of func tional mono mers in the ini tial mono mer mix ture.This shifts the equi lib rium to for ma tion of the com plexes.It is widely known that due to exo ther mic na ture of the poly mer iza tion re action as well as pos si ble struc tural changes of both template and func tional mono mers, the struc ture of a part of these com plexes will be changed or de stroyed.The num ber of "de fect" sites can de crease in the case of for ma tion of strong com plexes be tween tem plates and func tional mono mers.There fore, se lec tion of functional mono mer is of great im por tance, since it in fluences di rectly the af fin ity and se lec tiv ity of a mo lec ularly-im printed poly mer.Most of sci en tists chose func tional mono mer taking into ac count just gen eral con sid er ations [90][91][92][93].Some pa pers de scribe ap pli ca tion of com bi na torial ap proach to MIP syn the sis and func tional mono mer se lec tion [94][95][96].The au thors syn the sized MIP librar ies, where the type of func tional mono mer and a ra tio tem plate:func tional mono mer were var ied in the ini tial mono mer mix ture.That was fol lowed by [the] screen ing of the re sult ing MIPs as for their abil ity to rec og nize tem plate mol e cules se lec tively.Us ing this ap proach, com po si tions of the poly mers able to recog nize se lec tively triazine her bi cide terbutylazine [95], phenytoin, and nifedipine [92] were op ti mized.De spite the fact that ev ery MIP from the li brary was syn the sized in small quan tity, the ap proach it self is labo ri ous and time-con sum ing, which sig nif i cantly limits its ap pli ca tion.
Sig nif i cantly more ef fec tive ap proach to op ti miza tion of MIP com po si tions (se lec tion of func tional mono mers able to form strong com plexes with template mol e cules) was pro posed by the other au thors [38,40,[97][98][99].The method pro vides a pos si bil ity of fast pre lim i nary screen ing [of] a large num ber of poten tial func tional mono mers, while [the] re sults of the screen ing of a vir tual li brary of func tional mono mers cor re late with [the] ex per i men tal data on bind ing template mol e cules with com pu ta tional MIPs.More over, [an] anal y sis of [the] com pu ta tional mod el ing data as for for ma tion of com plexes tem plate-func tional mono mer gives a pos si bil ity to syn the size ma te ri als ca pa ble of both highly-se lec tive rec og ni tion of in divid ual sub stances and rec og ni tion of groups of substances with sim i lar struc tures [39,40].Up to now, this ap proach is the most prom is ing, since sig nif icantly de creases time losses for op ti mi za tion of MIP com po si tion.At the same time, it gives in for ma tion as for pos si ble struc ture of tem plate-se lec tive bind ing sites in poly mers-biomimics.
The ef fect of mo lec u lar im print ing is based on rigid fix a tion of tem plate-func tional mono mer complexes in a poly meric net work, which pro vides a desired spa tial ar range ment of func tional groups of the mono mers and, as a re sult, of a whole syn thetic binding site.As it was men tioned, that is achieved by ad dition of 90-95% of bi/three-func tional cross-link ers in the ini tial mono mer com po si tion.The most widely used cross-link ers in non-co va lent mo lec u lar im printing are ethyleneglycol dimethacrylate, trimethylolpropane trimethacrylate, n-divinylbenzene, N,N'-bisacrylamide (Fig. 3).
The nu mer ous in ves ti ga tions dem on strate that ethyleneglycol dimethacrylate is the cheap est cross-linker, re sult ing in syn the sis of MIPs with op timal prop er ties.It pro vides high se lec tiv ity un der sepa ra tion of en an tio mers and struc tural an a logues [100][101][102], while chro mato graphic col umns based on these poly mers don't loose their se lec tiv ity un der constant use at 80°C and pres sure 6-10 MPa for months [103].There fore, most of re search groups use ethyleneglycol dimethacrylate-based poly mers for the pur poses of se lec tive bind ing [104][105][106][107][108]. In re cent years syn the sis of MIPs based on polyphenol [109], polyaminophenylboronic acid [110], co-poly mer of poly(phenylenediamine) with an i line [111], polyuretanes [63], and ox i dized polypyrroles [112,113] was re ported.
Or ganic poly mers on/in in or ganic car ri ers.Thin (5-10 nm) lay ers of macroporous poly mers hav ing the struc ture sim i lar to those de scribed in the sec tion "Organic poly mers" can be syn the sized on the sur face of macroporous sil ica-gels.That is achieved by co va lent at tach ment of methacrylate groups to the sil ica surface through re ac tion with 3-(trimethoxysilyl)propylmethacrylate.That is followed by ini ti a tion of the rad i cal poly mer iza tion of mono mers tra di tion ally used in mo lec u lar im print ing on the sur face of sil ica-gels.Us ing this ap proach one can ob tain poly meric adsorbents, which don't swell in or ganic sol vents and aque ous so lu tions.These ma te ri -als are mainly used for chro mato graphic sep a ra tion of en an tio mers [17,114,115] and close struc tural an alogues [29], as well as in solid-phase ex trac tion [116].
Since sur face mod i fi ca tion of in or ganic ma te ri als is of great im por tance from the point of view of sen sor tech nol ogy, that is of spe cial in ter est.A num ber of papers re port sur face mod i fi ca tion of glass, gold, and tin di ox ide [98,[117][118][119][120][121][122][123][124][125][126].The pa per [117] de scribes mod i fi ca tion of glass sur faces with self-as sem bled MIP lay ers.Monolayers of trichloro-n-octadecylsilane were formed on glass surfaces in the pres ence of a de ter gent-mod i fied dye as a tem plate mol e cule.Ex trac tion of the tem plate mol ecules re sulted in ap pear ance of cav i ties, formed by poly-con densed sil ane mol e cules.The MIP monolayers were ca pa ble of pre dom i nant ad sorp tion of the tem plate as com pared to its struc tural an alogues.Un for tu nately, the rec og ni tion pro cess was too slow, since mass trans fer took place through the layer of long-chain alkylsilanes.Amphiphylity of mol e cules is of great im por tance in this ap proach.The re-ad sorp tion pro cess was in ves ti gated elec tro chem ically [119], us ing Raman spec tros copy [118], and ellipsometry [120].Tin di ox ide as well as gold can be also mod i fied us ing this method [121,122].
In ter est ing data on mod i fi ca tion of gold sur faces by mo lec u larly-im printed poly mers are pre sented in [123].The au thors de scribed for ma tion of hy dro pho bic self-as sem bled mo lec u larly-im printed monolayers of hexadecanthiol and de vel oped a sen sor sys tem for choles terol de tec tion on their ba sis.The de vel oped amperometric sen sor sys tem was found to be se lec tive to cho les terol as com pared to its close struc tural an alogues (cholic and deoxycholic ac ids), while ki net ics of the sen sor re sponses was quite fast (the time of the sen sor re sponse com prised 5 min only).Un for tu nately, the sen sor dem on strated poor stor age sta bil ity and lost 60% of its ini tial sen si tiv ity af ter 10 days of stor age.That was a limitation for its wide practical application in medical diagnostics.
Sig nif i cantly much more ef fec tive ap proach based on mod i fi ca tion of golden elec trodes by thin lay ers of mo lec u larly-im printed poly mers us ing the method of graft ing poly mer iza tion was de vel oped re cently [98,[124][125][126].The highly cross-linked struc ture of mo lec ularly-im printed poly mers pro vided highly-se lec tive rec og ni tion of analytes (triazine her bi cides), while sensor re sponses re mained sta ble for a long time (approximately 1 year).
Im printed sil ica gels.Ap pli ca tion of sil ica-gels in mo lec u lar im print ing was pi o neered by Dickey, the first who syn the sized sub strate-se lec tive adsorbents ca pa ble of se lec tive rec og ni tion of dyes [127][128][129].The im printed ma te rial with the in creased af fin ity to the tem plate mol e cule was ob tained by pre cip i ta tion of silica-gels in the pres ence of methyl or ange.Sil ica gels were also im printed by other tem plate mol e cules and used for sep a ra tion of en an tio mers [130][131][132], pes ticides [133,134], and drugs [135].The ma te ri als synthe sized in this way were not sta ble and quickly lost their se lec tiv ity.There fore, this di rec tion was not further pro ceeded.More over, a sig nif i cant dis ad van tage of this ap proach is im pos si bil ity of se lec tive sil ica gel syn the sis to wards wa ter-in sol u ble sub stances, while Partikeev's method of synthesis of silica gels from organogels was found to be ineffective [133].
Mo lec u larly-im printed com pos ite in or ganic ma teri als.This ap proach is a con tin u a tion of Dikey's pa pers [127][128].It is based on syn the sis [of] highly-cross-linked polysiloxanes on the sur face of silica gel par ti cles.The polycondensation re ac tion takes place in the pres ence of sil anes hav ing func tional groups able to in ter act with tem plate mol e cules.This ap proach is sim i lar to the above-men tioned one, where polymerizable dou ble-bonds are re placed with sil ane groups ca pa ble of polycondensation.A num ber of works on ap pli ca tion of cross-linked polysiloxanes for se lec tive rec og ni tion of dyes [136], glycoproteins [114], and NAD [137] were published.
Bioimprinting.The method of mo lec u lar im printing is ap pli ca ble not only for syn thetic poly mers, but also for biopolymers [1,138].So-called bioimprinting was de vel oped for pro duc ing en zy matic ac tiv ity/substrate se lec tiv ity/change of en zy matic ac tiv ity in proteins [139,140], for pro duc ing enantioselectivity [141], as well as for re ten tion of pro tein re cep tor proper ties in or ganic sol vents [141,142].Usu ally, partly denaturated pro tein un der goes in ter ac tion with a template mol e cule, while its struc ture is sta bi lized by bi-func tional cross-link ers.Us ing this ap proach en zymatic ac tiv ity can be pro duced in pro teins (al bu min, concavanin A), en zy matic ac tiv ity of ribonuclease, glucoseoxidase, urease, a-am y lase can be changed, while their stability can be increased [143][144][145].
An other ap proach to bioimprinting pro teins is based on their abil ity to re tain "struc tural mem ory" after the trans fer from aque ous to an hy drous en vi ronment.In the case of pre cip i ta tion or liophilization of a the pro tein from aque ous en vi ron ment in the pres ence of a tem plate mol e cule, the lat ter can fur ther dem onstrate pre dict able changes of its prop er ties in or ganic sol vents [146][147][148][149][150][151][152][153][154][155][156][157], and (un der con di tion of ad di tional sta bi li za tion) in aque ous so lu tions [158].Syn thetic recep tor sites can be also formed us ing the method of molec u lar im print ing in cross-linked car bo hy dratesstarch [159] and am y lase [160,161].Us ing this approach, poly mers ca pa ble of se lec tive rec og ni tion of methylene blue [159], and glucose [160,161] were obtained.
All the meth ods de scribed in the pres ent re view give a pos si bil ity to ob tain syn thetic mim ics of bi o log ical re cep tors with dif fer ent se lec tiv ity, since all of them un der cer tain con di tions pro vide for ma tion of complexes be tween tem plates and func tional groups on the sur face of poly mers as well as ac ces si bil ity of the se lective sites due to formation of porous structure.
In au thor's opin ion, mo lec u larly im printed polymers due to their se lec tiv ity un der rec og ni tion of template mol e cules and sta bil ity in harsh en vi ron ments are much more prom is ing ma te rial as to their prac ti cal appli ca tion in cur rent an a lyt i cal chem is try and bio technol ogy (in clud ing sen sor tech nol ogy) as com pared to than tra di tional biomolecules.One of the at trac tive fea tures of these ma te ri als is a pos si bil ity of syn the sis of in ex pen sive ar ti fi cial re cep tors to vir tu ally un lim ited num ber of sub stances.Syn the sis of MIPs to wards substances which have no nat u ral re cep tors or syn the sis of nat u ral re cep tors to which is prob lem atic (low-mo lec ular weight com pounds, myco toxins, bac te rial tox ins, etc.) is of spe cial in ter est.From this point of view ap plica tion of the meth ods of com pu ta tional mod el ing, provid ing fast op ti mi za tion of ar ti fi cial re cep tors' com posi tion able to bind ef fec tively tar get analytes is of special in ter est.Highly cross-linked or ganic poly mers with sta ble physicochemical char ac ter is tics ob tained in the form of both poly meric par ti cles and thin films seem to be the most prom is ing to be used in cur rent bio tech nol ogy.The anal y sis of lit er a ture data dem onstrates ef fec tive ness of ap pli ca tion of polymers-biomimics for both fundamental investigations of molecular recognition processes and practical applications.
Fi nan cial sup port from Na tional Acad emy of Sciences of Ukraine (pro grams "Sen sor sys tems for med ical-eco log i cal and industri al pur poses" and "Novel med i cal-bi o log i cal prob lems and human environment")

Fig. 1 .
Fig. 1.The main principle of molecular imprinting.a.A template molecule (3) forms a complex with functional monomers (1) in a solution³.b.Polymerization in the presence of a cross-linker (2).c.Extraction of the template and formation of synthetic binding sites complementary to the template in their shape and arrangement of functional groups.