A technique allowing high-throughput synthesis and evaluation of molecularly imprinted polymer sorbents at a reduced scale (mini-MIPs) was developed and used for the optimization of MIPs for use in pure aqueous environments. The technique incorporated a 4-port liquid-handling robot for the rapid dispensing of monomers, templates, solvents and initiator into the reaction vessels of a 96-well plate. A library of 80 polymers, each ca. 50 mg, could thus be prepared in 24 h. The MIP rebinding capacity and selectivity could be rapidly assessed in the batch mode by quantifying nonbound fractions in parallel using a UV monochromator plate reader. This allowed a complete evaluation of the binding characteristics of an 80 polymer library in approximately 1 week. With the objective of optimizing a polymer imprinted with the local anaesthetic Bupivacaine for use in pure aqueous systems, a polymer library was prepared by varying the original poly(MAA-co-EDMA) MIP composition. The variable factors were the added amount of the hydrophilic comonomer, 2-hydroxyethyl methacrylate (HEMA), the cross-linking ratio, and the porogen. This optimization resulted in polymers showing high imprinting factors (IF = K(MIP)/K(NIP)) in water as a result, mainly, of reduced binding to the nonimprinted polymer. Normal scale batches of these materials showed strong retention of the template and low nonspecific binding when assessed as chromatographic stationary phases using pure phosphate buffer, pH 7.4, as mobile phase, by equilibrium batch rebinding experiments and as sorbents for extractions of the analyte from blood plasma samples.

Treatment of Alzheimer's disease (AD) is plagued by a lack of practical and reliable methods allowing early diagnosis of the disease. We here demonstrate that robust receptors prepared by molecular imprinting successfully address current limitations of biologically derived receptors in displaying affinity for hydrophobic peptide biomarkers for AD under denaturing conditions. C-terminal epitope-imprinted polymers showing enhanced binding affinity for Aβ1-42 were first identified from a 96-polymer combinatorial library. This information was then used to synthesize molecularly imprinted polymers for both of the β-amyloid (Aβ) isoforms and a corresponding nonimprinted polymer. A solid-phase extraction method was developed to be compatible with sample loading under conditions of complete protein denaturation. This resulted in a method capable of quantitatively and selectively enriching a shorter C-terminal peptide corresponding to the sequences Aβ33-40 and Aβ33-42 as well as the full-length sequence Aβ1-40 and Aβ1-42 from a 4 M guanidinum chloride solution. Application of the method to serum allowed selective, high-recovery extraction of both biomarkers at spiking levels marginally higher than clinically relevant concentrations found in cerebrospinal fluid.

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A novel approach addressing the classical deficiencies of molecularly imprinted polymers (MIPs), that is, low binding capacity and nonuniform binding sites, is reported. The thin walled beads were produced in two steps by first grafting thin MIP films, under controlled (RAFT) or noncontrolled conditions, from porous silica beads following previously reported procedures. The resulting composites were compared in terms of film thickness, the grafted layer homogeneity, the effect of different support morphologies, and for their ability to recognize the template in chromatographic or static binding tests. Thus, using L-Phenylalanine anilide (L-PA) as template to imprint poly(MAA-co-EDMA) in such a way led to nanometer thick films where the resulting composite were able to selectively retain the template in relation to the thickness of the grafted film. In the second step, removing the silica supports from the above composites by etching, led to nanometer thin walled beads with structure, morphology and recognition properties strongly depending on grafting chemistry (RAFT or non-RAFT), monomer dilution and on the film thickness of the original composite. Thus whereas the thicker walled materials retained their mesoporous morphology and displayed enhanced enantioselectivity, load capacity, and higher surface areas compared to their composite precursors, the thin walled beads showed lower surface areas indicating network collapse. The thin walled beads prepared under dilute conditions in absence of RAFT displayed a perfectly uniform binding site distribution and a saturation capacity exceeding that of a conventional monolithic MIP. The beads prepared by RAFT control showed a further enhanced saturation capacity significantly exceeding that of the reference material. Finally, the reduced hydrophobic character of the thin walled materials indicated the existence of two separate pore systems with different pore wettabilities.