Molecular and bioelectronic functionalization

• Grant number: 13/10759-4
• Support Opportunities: Research Grants - Visiting Researcher Grant - International
• Start date: April 03, 2014
• End date: June 17, 2014
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal Investigator: Paulo Roberto Bueno
• Grantee: Paulo Roberto Bueno
• Visiting researcher: Jason Davis
• Visiting researcher institution: University of Oxford, England
• Host Institution: Instituto de Química (IQ). Universidade Estadual Paulista (UNESP). Campus de Araraquara. Araraquara , SP, Brazil

Abstract

Impedance sensors measure the electrical impedance of the electrode-solution interface usually by applying a small sinusoidal voltage at a certain (and controllably varied) frequency and measuring the resulting current. The ratio of the current/voltage gives the impedance, and this technique is called electrochemical impedance spectroscopy (EIS). If the target analyte is captured by an electrode-immobilised probe (such as an antibody, DNA or peptide aptamer), the impedance of the electrode-solution interface will change detectably. These assays can be demonstrably exceedingly sensitive (picomolar to femtomolar), calibratable and specific when carried out with appropriately prepared and responsive surfaces.1-3,4 Assays such as this can also be used to separately assess interfacial capacitance. Again, as receptor modified electrodes are exposed to specific targets this can be a very sensitive function of target binding. Capacitive sensors are usually based on the nonfaradaic (reagentless) scheme and are measured at a single frequency. Importantly, both assays are "label free", in that they do not require prior labeling of the target solution, and nondestructive (in terms of the underlying sensory surface). They can be, additionally, carried out in complex bodily fluid (through appropriate controls of surface chemistry to restrict nonspecific interfacial adsorption).5,6,7 Finally, they are potentially very low cost, portable and readily multiplexable. In multiplexing, one has access to shortened analysis time, decreased sample volume, improved analytical efficiency and reduced cost compared to standard, parallel, single analyte assays. Multiplexed methods also enable the identification of patterns associated with relative change across a number of proteins in a fluid (for example, in a range of Ig's - see below). Though they can be exceedingly sensitive, the standard methods of impedance and capacitance are,however, profoundly problematic and difficult to apply reproducibly in different media or with different receptor/target combinations. When one applies an oscillating voltage to an electrode under electrolyte, the detected current has a closely associated sinusoidal form. The ratio of applied voltage signal to detected current signal is specifically known as impedance (a complex function, Z*, composed of real, Z', and imaginary, Z'', components) and the current and voltage waveforms related by a phase difference, Õ. In typical assays, Z* or its components, Z' or Z'' are reported as a function of target protein concentration. For reasons which are rarely, if ever, clear, for many assays only one of these functions, if any, serves as a calibratable measure of target binding. Z' or Õ may, for example, act as sensitive probes of target binding at one specifically generated antibody interface but be an insensitive or unreliable reporter of binding with either difference surface chemistry or at a second antibody surface. By fitting the acquired Z* to an assumed "equivalent circuit", it is also possible to resolve capacitative and resistive terms that can potentially also report on target protein binding but, this process is also fraught with problems related to the assumption of an accurate equivalent circuit and subsequent fitting errors. In recent months, in a collaborative effort between the PI's group and the group of Professor Paulo Bueno in São Paulo, we have established a means of accessing an entire library of potentially sensory functions for any given interface; specifically, the relationship between the applied sinusoidal voltage and measured current can generate a large number of Immittance Functions (IF's), of which impedance and phase are just two. (AU)