Abstract
Herein we demonstrate molecularly imprinted polymers (MIP) as plastic antibodies for a microplate-based assay. As the most abundant plasma protein, human serum albumin (HSA) was selected as the target analyte model. Thin film MIP was synthesized by the surface molecular imprinting approach using HSA as the template. The optimized polymer consisted of acrylic acid (AA) and N-vinylpyrrolidone (VP) in a 2:3 (w/w) ratio, crosslinked with N,N′-(1,2-dihydroxyethylene) bisacrylamide (DHEBA) and then coated on the microplate well. The binding of MIP toward the bound HSA was achieved via the Bradford reaction. The assay revealed a dynamic detection range toward HSA standards in the clinically relevant 1–10 g/dL range, with a 0.01 g/dL detection limit. HSA-MIP showed minimal interference from other serum protein components: γ-globulin had 11% of the HSA response, α-globulin of high-density lipoprotein had 9%, and β-globulin of low-density lipoprotein had 7%. The analytical accuracy of the assay was 89–106% at the 95% confidence interval, with precision at 4–9%. The MIP-coated microplate was stored for 2 months at room temperature without losing its binding ability. The results suggest that the thin film plastic antibody system can be successfully applied to analytical/pseudoimmunological HSA determinations in clinical applications.
Highlights
A molecularly imprinted polymer (MIP) or plastic antibody is a designed polymer matrix containing selective recognition sites toward a given analyte
Acrylic acid (AA), magnesium chloride (MgCl2), sodium chloride (NaCl), sodium dodecyl sulfate (SDS), potassium chloride (KCl), calcium chloride (CaCl2), urea, dimethyl sulfoxide (DMSO), and acetic acid solution were obtained from Merck (Darmstadt, Germany). 4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES) was obtained from Alfa Aesar (Karlsruhe, Germany)
Since the amount of the blue form is proportional to the amount of human serum albumin (HSA) bound to MIP, one can determine the quantity of bound HSA directly by measuring the absorbance at 595 nm with a MultiskanTM microplate spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA)
Summary
A molecularly imprinted polymer (MIP) or plastic antibody is a designed polymer matrix containing selective recognition sites toward a given analyte. This poses substantial challenges in terms of polymer optimization and selectivity. There are many available dye-binding assays to monitor protein concentration (i.e., the Bradford method, the Lowry method, and the Biuret method); the Bradford reaction was chosen due to its wide use for protein assessment, leading to a broad dynamic range when quantifying a wide range of proteins. It is more sensitive and simpler, and it reacts faster than the Lowry and Biuret methods. Large batches can be analyzed by preparing MIP thin films in microplate wells before testing, shortening the analysis time
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