Table 1.

Examples of Protein Imprinting Research

refs	imprint platform	separation/ detection system	protein(s)	matrix	solvent	affinity/ detection	specificity (imprinting factor)b	comments
48, 49	amphoteric polymer for direct protein separation by HPLC	chromatographic phase	bovine serum albumin, lysozyme	methacrylic acid, N-[3-(dimethylamino) propyl]-methacrylamide	water + CaCO3 (porogen)	lysozyme imprint shows enhanced rebinding of initial template	slight cross-reactivity between two systems	acid wash to remove CaCO3 followed by 48 h pronase incubation and phosphate buffer wash to remove remnants of protein; lysozyme shown to enter but not interact with BSA imprint through chromatographic elution
50	recognition of a DNA protein using DNA as backbone of formed polymer	solution depletion measured by enzyme activity, via gel electrophoresis	EcoR1	DNA, psoralenterminated polyisopropyl-acrylamide	series of buffers during polymerization step	demon-started between active and denatured EcoR1	non-imprinted DNA blocks nearly all protein/DNA interactions	highly complex system with specifically synthesized monomer; requires specific DNA chain for protein recognition; possible uses for separation of DNA-binding proteins
51	combination of affinity separation and molecular imprinting for specific protein separation	solution depletion	trypsin chymotrypsin	acrylamide/N,N-ethylenebis(acrylamide) gel with additional polymerizable inhibitor	water/DMF	capacity of ~0.7 mg/g of polymer	2.92 for Trp imprinted/non-imprinted. 1.92 for Trp/CTrpon the Trp specific polymer	polymer dried and washed with acetone/chloroform; well-studied system used for polymerization incorporating a specific monomer; potential protein entrapment in gels as shown by Ou (52)
53–55	polyacrylamide gel based polymers for chromatographic separation of various proteins	chromatographic phase	hemoglobin, cytochrome c, RNase, human growth hormone, transferrin	acrylamide/N,N-ethylenebis(acrylamide)	aqueous buffers and water	various	various	well-studied system used for polymerization and demonstrated with several proteins; different wash steps performed for each protein; binding attributed to weak interactions; possible difficulties seen with cross-reactivity and protein entrapment
52, 56	polyacrylamide gel based polymers, designed with specific electrostatic groups	solution depletion	lysozyme	methacrylic acid, N,N-diethylamino ethyl methacrylate	aqueous Tris buffer	12.5–43.8% w/wa	1.34–3.38	gentle salt wash; good imprinting efficiency; nonspecific binding seen; ~27% of lysozyme trapped in system
57–59	peptide chain selectivity for an angiotensin II octapeptide; “epitope approach”	chromatographic phase	angiotensin II octapeptide; attempts to bind the whole corresponding protein	sodium acrylate/poly(ethylene glycol) diacrylate	water	0.4 µg/mL detection limit by HPLC for octapeptide	19.2–2.10 depending on environment	uses target epitope as an anchor for imprint, lowering cross reactivity due to template size; peptide “anchor” imprint recognized but failed to with the whole protein; small point of interaction not giving strong enough affinity between polymer and protein
60, 61	traditionally imprinted sol-gel monolith	solution depletion	urease, bovine serum albumin	3-amino-propyltriethoxysilane, tetraethylorthosilicate.	potassium phosphate buffer pH 7, 0.1 M	60–90% rebinding	preferential binding factor of 1.5 seen for both protein specific MIPs	~25% protein entrapment seen in crushed polymer after 160 h pronase digestion; repeated protocol using hemoglobin and myoglobin showed no selectivity
62	direct imprint of protein onto silane modifiedsilica particles	chromatographic phase	transferrin	borate-silane complex	potassium phosphate buffer pH 7, 0.1 M	not shown	relative retention of transferrin over BSA of 2.16	uses a specific functional monomer; gentle salt wash to remove protein; no evidence of template entrapment.
63, 64	macroporous chitosan beads with acrylamide	HPLC, solution depletion	hemoglobin	acrylamide	phosphate buffer pH 6.8, 10 mM	adsorption capacity ~12 mg/g	KD: 42.7 (Hb)1.41 (BSA)c	mechanically stable materials used in two forms of analysis; high selectivity demonstrated; time-consuming protocol to obtain equibrium due to mass transfer
65, 66	thins films formed around proteins coated with disaccharide layers	competitive adsorption using radiolabel target protein	bovine serum albumin, immunoglobulin G, fibrinogen, lysozyme, RNase	disaccharide-coated hexa-fluoropropylene (C3F6), on fixed support	phosphate buffered saline	not directly measured	5–26 depending on substrate	demonstrated for a variety of templates; however complexity limits large scale applications; protein recognition only shown in competitive assays
67	acrylate beads by inverse-phase suspension polymerization	solution depletion	bovine serum albumin	acrylamide/N,N-ethylenebis(acrylamide), methacrylic acid	phosphate buffer pH 3.7	not shown	defined imprinting effect compared to non-imprinted beads; specificity between BSA and ovalbumin demonstrated	simple protocol with clear optimization; cross reactivity data shown for 1 protein; stability issues of gels not discussed; protocol requires demonstration in chromatographic conditions
68–71	modification of silica beads with acrylate-based polymer	batch binding/enzyme activity test/ QCM	glucose oxidase, lysozyme	N,N-1,2 dihydroxy-ethylenebis(acrylamide), N,N’-methylenebisacrylamide, acrylamide, acrylic acid	range of phosphate buffers	0.557 mg/g of polymer, for GO imprint. 0.8 mg/mL by QCM for lysozyme	not shown for GO system. demonstrated between Hb and Lzy on Lzy system	simple protocol, demonstrated in two formats and for two proteins; gentle salt wash used for template removal; lack of cross reactivity data; nonspecific binding seen for both proteins with QCM
72–74	supported polymers grafted to wells of polystyrene microplate; grafted layers to glass surfaces (microcalorimetry); grafted layers onto gold QCM crystal	solution depletion, QCM, microcalorimetry	horseradish peroxidase, hemoglobin, microperoxidase, lactoperoxidase, lysozyme, cytochrome c	3-aminophenylboronic acid	water	various for different polymers	various between different polymers and templates	simple method, using only one polymer component; rebinding subject to environmental conditions; further demonstration with QC by Rick and Chou showing “dual imprint”
75	modified silica surfaces	solution depletion	hemogloblin	3-amino-propyl-trimethoxysilane, trimethoxypropyl silane	MOPS pH 7, 10mM	not directly measured	demonstrated between hemoglobin and a range of competing proteins	complex protocol for formation of highly selective imprints; template not completely removed from material; indirect detection
	direct detection of protein using film formed on electrode	pulsed amperometric detection	bovine leukaemia virus glycoprotein	polypyrrole	KCI 100 mM	10 ng/mL detection shown	not shown	direct detection; regeneration problems, matrix not suited for all proteins; high nonspecificity seen
77	specific monomer used to coat silica particles	stationary chromatography phase	ribonuclease A, lysozyme	metal chelating monomer, N-(4-vinyl)-benzyl iminodiacetic acid	70/30 water/DMF rebinding in 70/30 HEPES buffer/DMF	capacity factor of 5.79 compared to 2.68 on ref	2.35 between ribonuclease A and lysozyme	good recognition of target template; however, this technique is specific for His- bearing proteins; requires presence of Cu2+ ions
78	micro-contact approach with thin films (protein stamping)	competitive binding by ELISA	C-reactive protein, lysozyme, human serum albumin	O-(4-nitrophenylphosphoryl)-choline/PEG400 dimethacrylate	water + CaCO3 1 mM	3.78 ng/cm2 for CRP 2.78 µg/cm2 for HSA	0.08 ng/cm2 for HAS 0.27 µg/cm2 for HSA	low cross reactivity, demonstrated for two templates; microscale application; stability and reusability issues due to fragility of material
79–82	imprinted Langmuir monolayer formed at the air/water interface, measured in situ or transferred to hydrophobic support	SPR, QCM	ferritin	methyl stearate, dioctadecyldimethyl-ammonium bromide, poly(ethylene glycol) bearing phospholipids in various ratios	water	demonstrated between varying ratios of monolayers components	6 on PEG:SME:DOMA 6:3:1 layers by QCM, 3.8 on 20:9:1 layer (molar ratios)	generic protocol, with imprinting coming though complementary patterning of monolayers; stability issues with formed layers; specificity not demonstrated

^aProtein absorbed/total protein initial × 100.

^bImprinted activity/nonimprinted activity.

^cDistribution coefficient K_D = C_p/C_s where C_p = concentration of protein in beads (mg/g) and C_s = concentration of protein in solution (mg/mL).