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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.

An automated molecularly imprinted sorbent based assay (MIA) for the rapid and sensitive analysis of penicillin-type beta-lactam antibiotics (BLAs) has been developed and optimized. The polymers were prepared using penicillin G procaine salt as template (PENGp) and a stoichiometric quantity of a urea-based functional monomer to target the single oxyanionic species in the template molecule. Highly fluorescent competitors (emission quantum yields of 0.4-0.95), molecularly engineered to contain pyrene labels while keeping intact the 6-aminopenicillanic acid moiety for efficient recognition by the cross-linked polymers, have been tested as analyte analogues in the competitive assay. Pyrenemethylacetamido penicillanic acid (PAAP) was the tagged antibiotic providing for the highest selectivity when competing with PenG for the specific binding sites in the molecularly imprinted polymer (MIP). Upon desorption from the MIP, the emission signal generated by the PAAP was related to the antibiotic concentration in the sample. The 50% binding inhibition concentration of penicillin G standard curves was at 1.81 x 10(-6) M PENG, and the detection limit was 1.97 x 10(-7) M. The sensor showed a dynamic range (normalized signal in the 20 to 80% range) from 6.80 x 10(-7) to 7.21 x 10(-6) M (20-80% binding inhibition) PENG in acetonitrile:HEPES buffer 0.1 M at pH 7.5 (40:60, v/v) solutions. Competitive binding studies demonstrated various degrees of cross-reactivity with penicillin-type beta-lactam antibiotics such as ampicillin (71%), oxacillin (66%), penicillin V (56%), amoxicillin (13%), and nafcillin (46%) and a lower response to other isoxazolyl penicillins such as cloxacillin (27%) and dicloxacillin (16%). The total analysis time was 14 min per determination, and the MIP reactor could be reused for more than 150 cycles without significant loss of recognition. The automatic MIA has been successfully applied to the direct analysis of penicillin G in spiked urine samples with excellent recoveries (mean value 92%). Results displayed by comparative analysis of the optimized MIA with a chromatographic procedure for penicillin G showed excellent agreement between both methods.