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To develop strategies for making sensors and actuators for biological processes |
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Principal Investigator : Pramod K Upadhyay Co-Investigators Research Assistant The project involves development of electrical
property measurement based systems for monitoring biological processes. The
specific objectives are i) to study the dielectric properties during the Ag:Ab
complexation and to develop a biosensing device based on such a study for a
model system and to further extend this approach to make useful biosensors,
ii) to study the dielectric spectra of a suspension of cells and to develop
sensors to estimate the concentration of cells, their morphology, viability
etc. and iii) to develop piezo ceramic based static electricity generator as a
tool to carry out electroporation. The current activities are focused on making an
impediometric sensor for Bacillus anthraci. To begin with, polyclonal
antibodies were used to develop an ELISA based test for Bacillus anthraci. The
lower detection limit of this ELISA based test is 103-104 spores/ml of
Bacillus anthraci. The test involves coating the micro well plates with
purified antibodies; incubation with test solution and revealing the bound
spores with HRPO labeled antibodies. The test was also modified to test
AB-Bacillus anthraci by coating spores on microplates. Anodically oxidized aluminum and titanium electrodes
were used to make the impediometric sensor for Bacillus anthraci. Electrodes
made from aluminum and titanium were evaluated for their reproducibility and
drift. It was found that the impedance of Al/Al2O3 electrode drift
significantly in non-reproducible manner. The drift associated with Ti/TiO2
electrode was reproducible making it a usable electrode for impediometric
sensors. Polyclonal antibodies were immobilised on Ti/TiO2
electrode by adsorption and covalent bonding. Relatively higher degree of
uniformity was obtained with covalently bound antibodies made by the
silanization of oxide layer of Ti electrode. For subsequent studies,
electrodes made by polyclonal antibodies attached to Ti/TiO2 through
3-aminopropyl trietoxysilane-glutaraldehyde were used. A flow cell was designed to analyze the samples.
Quantitative reproducible change in impedance was not observed when spores of
Bacillus anthraci were injected into the flow cell. It has been argued that at
higher electrolyte concentration the diffuse ion cloud outside the immobilized
antibody layer will be compressed resulting in an increased capacitance. This
reduces the precision of the measurement. Electrodes are being evaluated at
lower ion strength of the buffers now. We have investigated how the voltage pulses generated
by triggering a piezo ceramic-based static electricity generator can cause
electroporation of the skin. Piezo ceramic-based devices have a ceramic disc
inside. Application of compressive force on ceramic disk produces voltage
across its ends. In a typical domestic gas igniter a force of around 2000 N is
applied on the piezo ceramic disk at each trigger and a voltage of around 18
KV is generated and a charge of 10-6 C is delivered within 10-7 s. A
domestic piezo gas igniter was modified to deliver charge pulses. An insulated
wire was joined by silver loaded conducting epoxy to the central rod of the
gas igniter and the other connection was taken from the outer jacket. Ten
triggers, each separated by a second, were applied from the gas igniter for
immunization. An in-house designed power supply was used to deliver 100 V
square wave pulses of 0.1 ms duration. Ten such pulses each separated by a
second were applied. The cups containing the antigen solution were left in
place for one hour. Some of the mice were immunized with chicken albumin
by electroporation using a power supply and some by a gas igniter. Twenty
micro-liter antigen solution (10 µg/ml chicken albumin in PBS) was placed in
two hemispherical cups (2 mm inner diameter and 50 µl capacity) stuck to the
skin by an adhesive. Platinum wire electrodes were inserted into the antigen
solution, but did not touch the skin surface. The change in impedance of the
skin of a mice after applying high voltage electrical pulses from a power
supply and that with a gas igniter were found to be similar. In both the
groups identical immune responses were generated. This new device design has
made electroporation as handy as a syringe with a needle. Publications Original peer-reviewed articles 1. Upadhyay P and Bhaskar S (2000) Real time monitoring of lymphocyte proliferation by an impedance method. J Immunol Methods 244:133-137. 2. Upadhyay P (2001) Electroporation of the skin to deliver antigen by using a piezo ceramic gas igniter. Int J Pharma (in press). |