|
To develop strategies for making sensors and actuators for biological processes |
| Principal Investigator : Pramod K Upadhyay
Co-Investigators Project Associates/Assistants Collaborators To
develop electrical property measurement based systems for monitoring
biological processes is the theme of research. The objectives include: (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 and (ii) to develop piezo
ceramic based static electricity generator as a tool to carry out
electroporation. The
prototype of the impediometric sensor was evaluated for the detection of
spores of Bacillus anthracis. A specific signal for spores and its
antibody reaction was clearly visible when the spores concentration was higher
than 1000/ml. This signal was also used to study the kinetics of binding. The
reproducibility of the results was found to be poor. It could be due to the
differences in the amount and nature of covalently immobilized antibodies over
the electrode surfaces. We
have used micro four probes to measure changes in the surface conductivity of
indium titanium oxide (ITO) coated glass electrode after antigen get bound to
the immobilized antibodies. These probes are gold plated silicon fingers
placed at 60µm spacing. When such micro probes are used to measure surface
conductivity a large fraction of the current flow near the surface resulting
in a surface sensitive measurement. We
have used ITO coated glass slides of 100 ohm resistance. These glass slides
were first silanated followed by a treatment with gluteraldehyde. One half of
the length of these functionalized ITO glass slides were placed in antibody
solution. After the immobilization of antibodies the entire electrodes were
dipped in a non specific protein (like BSA) to block any remaining functional
group. Finally the electrodes were dipped in a solution containing antigen.
Electrodes were then washed and air dried. An
indigenous socket of the micro four probe was made. It was mounted on the lens
of a microscope. ITO glass slides were placed on the bench of the microscope
and micro four probe was scanned over the electrode and resistance measured.
It has been shown that the binding of antigen over the immobilized antibody
make a measurable conductivity difference when measured using a micro four
probe. In
order to study the membrane electroporation by piezo ceramic based devices, a
few designs of such devices generating different static voltages were made.
These devices were used to immunize mice in combination with different designs
of electrodes and protocols. A
comparative study to immunize mice by different electroporation devices was
initiated. Animals were immunized with commercially available hepatitis B
surface antigen. It has been found that mice immunized using piezo-ceramic
based electroporation device and conventional electroporation protocol invoke
identical responses. The antibody titers of these responses were found to
slightly lower than conventional alum based immunization. Further
investigations of these responses are being taken up. The set of piezo ceramic devices were also tried to transfect COS cells with plasmid DNA encoding gfp. Conventional kits to transfect cells were compared with electroporation using a typical power supply based protocol and different piezo ceramic devices. We found that it is not possible to transfect adhered cells over conducting flat surfaces by electroporation. It could be due to the ‘low strength’ uniform electric field generated by flat surfaces. It was observed that transfection with DNA is possible in cell suspension by electroporation when placed under non uniform electric field which has points of ‘high field strength’. Comparable transfection efficiencies were observed by conventional kits and electroporation using a power supply. When a piezo ceramic based device was used in place of a power supply the transfection efficiency was around 10 times low. Different designs of electrodes and piezo ceramic devices are being tried to make transfection efficiencies comparable. Publications Original
peer-reviewed articles 1. Bhaskar S and Upadhyay P (2003) Design and evaluation of an aerosol infection chamber for small animals. Int J Pharm (in press). |