465959 Aptameric Peptide Mediated Capacitive Detection of Protein Kinase
Introduction- The phosphorylation of proteins regulates almost all aspects of cell life. Protein kinases are enzymes which catalyze the phosphorylation of target proteins. Unusual regulation of protein phosphorylation is associated with many diseases, including cancer. Therefore, accurate quantification of the protein kinases is crucial for the analysis of related abnormalities.
Recently, kinase activity assays based on mass spectroscopy, fluorescence microscopy, and quartz crystal microbalance have been reported for the analysis. However, miniaturization of these techniques for handheld device is quite not possible. μTAS technologies have been widely explored to develop miniaturized devices for portable use. Recent advances in microfabrication technique have facilitated the creation of microchips with electrochemical detection of different biomarkers. To detect protein kinases electrochemically, a variety of methods have been proposed. Major drawback in these reports is the use of multiple bio and nano particles for sensitive and selective detection. These steps make the analysis laborious and expensive.
For this reason, this work focuses on the development of a microchip for protein kinase A (PKA) detection with a minimalistic use of biomolecules. Electrolyte-insulator-semiconductor (EIS) devices as electronic transducers for label-free detection of biochemical reactions are very promising for the development of low-cost high-throughput sensors. The capacitive EIS device is the simplest field-effect bio-chemical sensor with a direct electrical readout. The change in the local pH at the electrolyte-insulator interface or the attachment of charged biomolecules to the insulator alters the surface potential, leading to a shift in flat-band voltage (Vfb).
Therefore, this work, uses an EIS capacitor structure to assay the protein kinase activity. A peptide based aptamer was immobilized on the insulator surface using a (3-Mercaptopropyl)trimethoxysilane (MPTES) and gold nanoparticle (AuNp) link. The EIS sensor detects the charge changes in AuNp/aptamer induced by the binding of PKA and aptamer. The concentration of enzyme is detected in terms of changes in the flat-band voltage of the EIS sensor.
Experimental- 50 nm of SiO2 was deposited by chemical vapor deposition (CVD) on p-type Si wafers. The wafers were then cut into small pieces of 8 x 8 mm in size. The Si-SiO2 wafers were cleaned using ultra-sonication in acetone and isopropyl alcohol for 20 min, each. After drying the wafers, 100 nm aluminium was deposited on the back of the Si wafers to serve as an ohmic back-contact, using a vacuum thermal evaporator.
The cleaned sensor surface was first silanized by dipping it in a freshly prepared 1% MPTES in ethanol for 24 hrs. The surface was then thoroughly cleaned using ethanol. For deposition of AuNPs on the sensor surface, the thiol functionalized surface was made to react with an AuNPs colloid for 12 hrs.
A rapid prototyping technique for microchip based on a polymer film and double sided tape was developed. A lower layer consists of an EIS sensor mounted on a PES film and attached to an Au electrode. The middle layer was fabricated from 3MTM double sided tape and contained the fluidic network and reservoir for the analysis. Finally, the upper layer consists of an Ag/AgCl thin-film reference electrode patterned on the PES film.
Results- Each modification step of the sensor surface was characterized by means of water contact-angle measurements with a 12 μl drop of ultrapure water. The hydrophilicity of the surface increased significantly to a contact angle of ~7.8° after the UV-O3 treatment from a contact angle of ~42° for a bare SiO2. The increase of surface hydrophilicity is due to the increase in hydroxyl groups. The sensor surface modified with MPTES regained the hydrophobicity (contact angle= ~37°). The change in the surface property of SiO2 confirms the functionalization of the sensor. A scanning electron microscopic analysis of the EIS surface was also performed to check the presence of AuNPs.
Binding of PKA enzyme with aptamer altered the surface charge, leading to the shift in Vfb. A linear shift in Vfb was obtained with the increasing concentration of PKA enzyme. Based on these results, we propose a flexible microchip with an EIS sensor for the label-free detection of PKA enzyme.
Conclusion- EIS based detection of PKA using a peptide aptamer is presented in this work. A label-free analysis without the use of any reporting molecule was performed. The proposed device can easily be modified to detect any other PK using specific aptamers. Polymer based chip and one-step detection makes this work more practical. This assay offers sensitive and reproducible quantification of PK.