Protein-responsive polymers for point-of-care detection of cardiac biomarker

This work describes a novel use for the polymeric film, poly(o-aminophenol) (PAP) that was made responsive to a specific protein. This was achieved through templated electropolymerization of aminophenol (AP) in the presence of protein. The procedure involved adsorbing protein on the electrode surface and thereafter electroploymerizing the aminophenol. Proteins embedded at the outer surface of the polymeric film were digested by proteinase K and then washed away thereby creating vacant sites. The capacity of the template film to specifically rebind protein was tested with myoglobin (Myo), a cardiac biomarker for ischemia. The films acted as biomimetic artificial antibodies and were produced on a gold (Au) screen printed electrode (SPE), as a step towards disposable sensors to enable point-of-care applications. Raman spectroscopy was used to follow the surface modification of the Au-SPE. The ability of the material to rebind Myo was measured by electrochemical techniques, namely electrochemical impedance spectroscopy (EIS) and square wave voltammetry (SWV). The devices displayed linear responses to Myo in EIS and SWV assays down to 4.0 and 3.5 μg/mL, respectively, with detection limits of 1.5 and 0.8 μg/mL. Good selectivity was observed in the presence of troponin T (TnT) and creatine kinase (CKMB) in SWV assays, and accurate results were obtained in applications to spiked serum. The sensor described in this work is a potential tool for screening Myo in point-of-care due to the simplicity of fabrication, disposability, short time response, low cost, good sensitivity and selectivity.

Electrochemical biosensor based on biomimetic material for myoglobin detection

A novel reusable molecularly imprinted polymer (MIP) assembled on a polymeric layer of carboxylated poly(vinyl chloride) (PVCsingle bondCOOH) for myoglobin (Myo) detection was developed. This polymer was casted on the gold working area of a screen printed electrode (Au-SPE), creating a novel disposable device relying on plastic antibodies. Electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and Fourier transform infrared spectroscopy (FTIR) studies confirmed the surface modification. The MIP/Au-SPE devices displayed a linear behaviour in EIS from 0.852 to 4.26 μg mL−1, of positive slope 6.50 ± 1.48 (kΩ mL μg−1). The limit of detection was 2.25 μg mL−1. Square wave voltammetric (SWV) assays were made in parallel and showed linear responses between 1.1 and 2.98 μg mL−1. A current decrease was observed against Myo concentration, producing average slopes of −0.28 ± 0.038 μA mL μg−1. MIP/Au-SPE also showed good results in terms of selectivity. The error% found for each interfering species were 7% for troponin T (TnT), 11% for bovine serum albumin (BSA) and 2% for creatine kinase MB (CKMB), respectively. Overall, the technical modification over the Au-SPE was found a suitable approach for screening Myo in biological fluids.

Smart Plastic Antibody Material (SPAM) tailored on disposable screen printed electrodes for protein recognition: application to Myoglobin detection

This work introduces two major changes to the conventional protocol for designing plastic antibodies: (i) the imprinted sites were created with charged monomers while the surrounding environment was tailored using neutral material; and (ii) the protein was removed from its imprinted site by means of a protease, aiming at preserving the polymeric network of the plastic antibody. To our knowledge, these approaches were never presented before and the resulting material was named here as smart plastic antibody material (SPAM). As proof of concept, SPAM was tailored on top of disposable gold-screen printed electrodes (Au-SPE), following a bottom-up approach, for targeting myoglobin (Myo) in a point-of-care context. The existence of imprinted sites was checked by comparing a SPAM modified surface to a negative control, consisting of similar material where the template was omitted from the procedure and called non-imprinted materials (NIMs). All stages of the creation of the SPAM and NIM on the Au layer were followed by both electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). AFM imaging was also performed to characterize the topography of the surface. There are two major reasons supporting the fact that plastic antibodies were effectively designed by the above approach: (i) they were visualized for the first time by AFM, being present only in the SPAM network; and (ii) only the SPAM material was able to rebind to the target protein and produce a linear electrical response against EIS and square wave voltammetry (SWV) assays, with NIMs showing a similar-to-random behavior. The SPAM/Au-SPE devices displayed linear responses to Myo in EIS and SWV assays down to 3.5 μg/mL and 0.58 μg/mL, respectively, with detection limits of 1.5 and 0.28 μg/mL. SPAM materials also showed negligible interference from troponin T (TnT), bovine serum albumin (BSA) and urea under SWV assays, showing promising results for point-of-care applications when applied to spiked biological fluids.

Artificial antibodies for troponin T by its imprinting on the surface of multiwalled carbon nanotubes: Its use as sensory surfaces

A novel artificial antibody for troponin T (TnT) was synthesized by molecular imprint (MI) on the surface of multiwalled carbon nanotubes (MWCNT). This was done by attaching TnT to the MWCNT surface, and filling the vacant spaces by polymerizing under mild conditions acrylamide (monomer) in N,N′-methylenebisacrylamide (cross-linker) and ammonium persulphate (initiator). After removing the template, the obtained biomaterial was able to rebind TnT and discriminate it among other interfering species. Stereochemical recognition of TnT was confirmed by the non-rebinding ability displayed by non-imprinted (NI) materials, obtained by imprinting without a template. SEM and FTIR analysis confirmed the surface modification of the MWCNT. The ability of this biomaterial to rebind TnT was confirmed by including it as electroactive compound in a PVC/plasticizer mixture coating a wire of silver, gold or titanium. Anionic slopes of 50 mV decade−1 were obtained for the gold wire coated with MI-based membranes dipped in HEPES buffer of pH 7. The limit of detection was 0.16 μg mL−1. Neither the NI-MWCNT nor the MWCNT showed the ability to recognize the template. Good selectivity was observed against creatinine, sucrose, fructose, myoglobin, sodium glutamate, thiamine and urea. The sensor was tested successfully on serum samples. It is expected that this work opens new horizons on the design of new artificial antibodies for complex protein structures.