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Multiplexed protein and kinase activity detection assays in complex media using impedance/capacitance

Final Report Summary - MPKADAIC (Multiplexed protein and kinase activity detection assays in complex media using impedance/capacitance)


The main objective of this project is to develop electrical assays capable of sensing multiple proteins in complex biological media such as human serum, using highly sensitive electrochemical techniques (impedance/capacitance). As a major obstacle preventing researchers from assaying proteins in biological samples accurately is the nonspecific adsorption or bio-fouling, we have focused our research topics on the development of different bio-sensing systems that can effectively resist nonspecific adsorption. With the support of this Marie Curie Fellowship, we have developed a few protein assay systems that can be performed in human serum without significant nonspecific adsorption problem. In short, we have successively achieved the goal of this research project in the past 2 years, and the main research topics have been studied and successively finished are summarized as follow.

1) Protein assay systems based on polyethylene glycol (PEG) thiol self-assembled on gold electrodes.

We have tried to immobilize different antibodies (instead of proposed peptide aptamers, as currently there are few peptide aptamers available) such as insulin, C-reactive protein (CRP) and alpha-synuclein antibodies on gold electrode surfaces. The PEG moiety containing thiol molecules HS-C11-(EG)3-OCH2-COOH were firstly self-assembled on the gold electrodes, and then different antibodies were attached to the electrode surface through the formation of amide bonds between the amine groups of the antibodies and the carboxyl groups of the PEG-thiol, via the assistance of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimde (NHS). With the immobilization of antibodies on the gold electrodes, these biologically functionalized interfaces can selectively bind their corresponding target antigens/proteins, which can cause significant electrical impedance change of these electrode interfaces and be monitored using Faradaic electrochemical impedance spectroscopy (EIS). Therefore, different protein biosensors for insulin, CRP and alpha-synuclein can be developed.

All these protein assays exhibited good selectivity owing to the selective binding of proteins towards their unique corresponding antibodies. Those protein assays can be easily performed using Faradaic EIS in phosphate buffer saline (PBS) with the added common redox probe ferry cyanide. Proteins present in the solution can selectively bind to the immobilized specific abtibodies and block the electron transfer of the redox probe to the electrode, resulting a significant changes of the EIS. As the changes in EIS has a linear relationship with the log concentration of the protein, it can be used for the quantitative detection of proteins. EIS assays for proteins insulin, CRP and alpha-synuclein have been developed, with detection limits of 1.2 pM, 176 pM and 55 pM, respectively. As the PEG moiety is highly hydrophilic, and it is neutral in charge, it can prevent the adsorption of proteins to the electrode surface when a PEG-thiol self-assembled monolayer is formed. All the biosensors based on the self-assembled PEG-thiol can effectively reduce the nonspecific adsorption of other proteins in human sera.

2) Insulin assay in human serum based on antifouling zwitterionic polymers.

Zwitterionic polymer is a kind of new antifouling polymers, which normally is highly hydrophilic and neutral in total charge, and can strongly resist nonspecific protein adsorption. Based on the nonspecific response elimination effect of the zwitterionic polymer poly(carboxybetaine methacrylate) (PCBMA), we have developed a highly sensitive and selective electrical insulin biosensor based on the chemisorbed zwittorionic polymer PCBMA support and a novel reagentless sensing technique based on phase monitoring electrochemical impedance spectroscopy. The polymer adlayer is exceptionally effective in both reducing background response and maintaining receptive antibody binding efficacy, while the non-Faradaic analysis avoids potential interference from background electro-active molecules. Applied to the detection of even a low molecular weight protein (here, insulin), a linear range from 0.1 to 200 pM and an unprecedented femtomolar detection limit are possible in undiluted blood serum.

3) Multiplexed protein detection based on novel PEG modified microelectrode arrays.

Simultaneous measurement of multiple proteins in the same complex biological media, such as human serum is of great importance to ensure efficiency and reliability of point-of-care disease diagnosis, while a major obstacle to such multiplexed protein assays in biological fluids is the nonspecific adsorption of proteins or bio-fouling. We have developed a novel bioassay system for two different disease markers, insulin and CRP, based on cross-linked polyethylene glycol (PEG) modified microelectrode arrays. These functional PEG films not only render the electrode array excellent antifouling property but also provide biocompatible substrates for biomolecule immobilization. Microelectrodes in the same array with their surfaces attached with different antibodies are highly specific to their own targets, without suffering from cross interference and biofouling of serum. We have also applied this assay system for the detection of insulin and CRP simultaneously in real human serum samples, and the impedance based quantification agrees well with an NHS standard chemiluminescence assay, and the correlation of the two assays are mainly within 20% for both proteins (note that agreement between commonly used methods in clinical use can show up to 200% variation in results through differences in assay sensitivity and specificity).

We believe multiplexed bioassays such as these, capable of detecting different proteins in the same biological sample in a label free and non-fouling manner, to be of great value for point-of-care diagnosis, especially for those complex diseases that require multiple protein biomarkers to be measured so as to ensure diagnostic reliability.