To validate the technique, we have compared the plasmonic-based impedance spectroscopy with the simultaneously measured conventional electrochemical impedance spectroscopy and surface plasmon resonance (SPR) detection. for simultaneous SPR and surface impedance analysis of molecular binding processes. Keywords:SPR, Surface Plasmon Resonance, Electrochemical Impedance Spectroscopy, Plasmonic-based impedance spectroscopy, P-EIS == Introduction == Electrochemical impedance spectroscopy (EIS) is usually a powerful electrochemical technique for studying numerous surface processes and properties.1,2It measures electrical current response to a potential applied to an electrode, which is extremely sensitive to molecular adsorption taking place around the electrode surface. EIS has also been widely used as a label-free detection method to study numerous molecular binding processes, such as antigen-antibody3-7, protein-DNA conversation8-11, and DNA hybridization12-16, and to monitor numerous cellular processes. Surface plasmon resonance (SPR) is usually another label-free detection technique for real-time study of molecular interactions.17-25Unlike EIS, SPR is an optical method that detects and images changes in refractive index near a metal surface (sensor chip).26-29However, SPR signal is prone to interference due to changes in bulk refractive index, and SPR signal decreases with the mass of molecules, making small molecule detection challenging. Besides electrochemical and optical based techniques, piezoelectric sensor (e.g. Quartz Crystal Microbalance)30-32is another main category of label free method for molecular binding detection. Immunoassay is based on the Capecitabine (Xeloda) highly specific molecular acknowledgement between antigen and antibody.33,34Immunoglobulin G (IgG) antibody is one of the most common and important proteins in the immune system Capecitabine (Xeloda) and clinical application, which is also widely used in the immunoassay. One IgG molecule with a molecular excess weight of ~150 kDa has two identical antigen binding Capecitabine (Xeloda) sites, to form antigen/antibody complexes.35 Recently we have shown that it is possible to perform electrochemical measurements, such as cyclic voltammetry, based on a plasmonic detection. In other words, an electrochemical current can be decided and imaged optically, which is in contrast to the conventional electrochemistry that relies on electrical measurement.36-40We have also shown that electrochemical impedance can be obtained Capecitabine (Xeloda) based on the plasmonic detection.36Here we show that it is possible to obtain impedance spectrum by measuring impedance vs. frequency over a wide frequency range (from to 0.01 Hz to 10 kHz) with the plasmonic-based electrochemical impedance spectroscopy (P-EIS). We validate P-EIS by comparing it with the simultaneously recorded electrically measured EIS, and with theoretical modeling. Furthermore, as the new technique combined the features of both SPR and EIM, P-EIS has more potential in the area of molecular binding studies, which is exhibited by studying human IgG and anti-human IgG conversation as a model system. The kinetics and affinity results from P-EIS are compared with the simultaneously recorded SPR. == Experimental Section == == Materials == 6-mercaptohexanoic acid, 3-mercapto-1-propanol, NaCl, KCl, Na2HPO4, KH2PO4, NaF, sodium acetate, glucose,N-hydroxysuccinimide (NHS),N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC), ethanolamine, IgG from human serum (IgG), anti-human IgG (Fab specific) antibody (anti-IgG), albumin from bovine serum (BSA) were purchased from Sigma-Aldrich (St. Louis, MO). 1x phosphate buffered saline (1x PBS, 10 mM Na2HPO4-KH2PO4,137 mM NaCl, 2.7 mM KCl, pH 7.4), 10 mM sodium acetate (pH 5.0) were prepared as stock answer. == Plasmonic-based Impedance Measurements == BI-2000 SPR instrument with an EC-dual-flow analysis module (Biosensing Instrument Inc.) was used in the experiments. The SPR sensor chip was a BK7 glass cover slide coated with 2 nm chromium followed by 47 nm gold by thermal evaporator at high vacuum (3 106Torr). The Au sensor chip was used as working electrode (WE) and a glassy carbon electrode or Pt wire served as counter electrode (CE). Its potential was controlled with respect to Ag/AgCl reference electrode (RE) by a potentiostat (EG&G Model 283 Potentiostat/Galvanostat, Princeton Applied Research). Potential modulations with different frequencies and amplitudes were applied to the chip using an external function generator (Model DS345, Stanford Research Systems). The SPR responses Rabbit Polyclonal to MMP23 (Cleaved-Tyr79) to the modulations were measured by a lock-in amplifier (Model SR830 DSP, Stanford Research Systems). The amplitude and phase outputs from your lock-in amplifier, as well as the corresponding potential, current, SPR signals were collected with the sampling rate of 10,000 points per second by Data Acquisition (NI USB-6251, National Instruments) using a program written in MATLAB. == Chip preparation == The Au sensor Capecitabine (Xeloda) chip was rinsed with water and ethanol, and then annealed with hydrogen flame to further clean the surface before each SPR and plasmonic-based impedance measurement. The chip was altered by immersing it in 1 mM 1:1 6-mercaptohexanoic acid and 3-mercapto-1-propanol combination answer in ethanol for 24 h. IgG was subsequently immobilized around the sensor chip using the standard amine-coupling chemistry using the flow-through system of BI-2000 instrument with 1x PBS as running buffer at a circulation rate of 10 L/min. The.