A molecularly imprinted film was fabricated, in the presence of epitope-peptides, onto a quartz crystal microbalance (QCM) chip. These five peptides are known linear or conformational epitopes of the anthrax protective antigen PA(83). Imprinting resulted in an epitope-cavity with affinity for the corresponding template. With the use of a basic monomer, the binding-effect was further enhanced increasing the affinity to nanomolar levels. The affinities of the peptide to their corresponding molecularly induced polymers (MIPs) were more closely related to the molecular weight of the analyte than to the number of residues. All epitope-cavities differentiated their epitope region on the protective antigen PA(83) as well as the corresponding furin cleavage fragments PA(63) and PA(20). The QCM chip differential response to the protective antigen fragment was observed in the picomolar range, thus demonstrating a method to manipulate protein on the surface with defined orientation.

A quantitative method using artificial antibody to detect creatine kinases was developed. Linear epitope sequences were selected based on an artificial-epitope mapping strategy. Nine different MIPs corresponding to the selected peptides were then fabricated on QCM chips. The subtle conformational changes were also recognized by these chips.

Serum is a readily available source for noninvasive studies in clinical research, but it contains abundant proteins such as albumin and immunoglobulin G that can hinder the presence of low-abundant proteins as well as decrease sample loading capacity of analytical methods. Therefore, depletion of these two proteins is required to observe low-abundance serum proteins. Molecularly imprinted polymers are template-induced artificial antibodies with the ability to recognize and selectively bind the target molecule. In this study, artificial albumin and immunoglobulin G antibodies were developed by using two epitopes of human serum albumin and immunoglobulin G as templates. Acrylic acid, acrylamide, and N-acryl tyramine were the corresponding monomers; N,N'-ethylene bisacrylamide served as a cross-linker, and cellulosic fibers were used as a supporting matrix. The adsorption capacity of these artificial antibodies was 15.2 mg, 10 mg, and 15 μL per gram for human serum albumin, immunoglobulin G, and human serum, respectively. The dissociation constant (Kd ) of these artificial antibodies toward the human serum albumin and immunoglobulin G was 1 μM and 0.6 μM, respectively. The biomimetic properties of these artificial antibodies, coupled with their economical and rapid production, high specificity and their reusability, make them attractive for protein separation and analysis.

A complementary peptide separation and purification system, using a novel, artificial, affinity-type extraction material, is described. Cellulose paper was first coated with a 3-methacryloxypropyltrimethoxysilane (MPS) layer, followed by imprinting with peptides, to form an approximately 2-µm layer of film (molecularly imprinted polymer film; MIPF) on both sides and resulted in an increased affinity toward the corresponding template as well as to matched polypeptides. MIPF-cellulose paper (MIFC) was immersed in 2% peptide solution (acetonitrile/water) for the sorption of targeted-peptides at room temperature for an hour in a 96-well microtiter plate. Desorption of targeted-peptides from the MIPF-and NIPF (non-imprinted polymeric film)-coated filter papers was nearly complete (~99%) in two desorptions of 10 min using 5% acetic acid in water. Clean extracts of targeted-peptides were obtained demonstrating the suitability of MIPF-coated filter papers for the extraction or analysis of biological samples. To compare their extraction characteristics under various conditions, analyses were carried out by an enzyme-linked immunosorbent assay (ELISA) microplate reader as peptide-detecting/peptide-capturing microarrays. In load ability studies using acetonitrile/water, peptide masses up to 100 µg could be applied onto a piece of MIPF coated filter paper (0.7 cm in diameter). ArticleTai et al. Scheme II Operation of MIFC for the extraction of peptide Fig. 2. Scatchard plot for the binding of the Phe-Phe-Phe-Phe to MIFC. Data points are the average of triplicates.

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Evaulation of the severity of jaundice in the neonate may be determined by measuring the reserve binding capacity of serum proteins for free bilirubin. Determination of protein-bound bilirubin has been labor intensive, necessitating multiple runs on gel-permeation chromatography columns or, more recently, enzyme assays or fluorescence quenching analysis. We present a method for quantitation of free bilirubin and of bilirubin-binding capacity of serum by liquid chromatography. A gel-permeation column binds free bilirubin while allowing passage and quantitation of protein-bound bilirubin. Subsequent injection of a desorbing agent releases the adsorbed bilirubin from the column, permitting quantitation of free bilirubin. Bound and free serum bilirubin may be determined directly in less than 15 min using 10 microL of serum. The binding of bilirubin to neonatal serum is seen to be quite different from the binding to adult serum. Ion-exchange chromatography of adult and neonatal serum samples shows that their protein profiles are radically different. This difference probably accounts for the binding characteristics.