|
To
develop strategies for making sensors and actuators for biological processes |
| Principal Investigator : Parmod K Upadhyay
Co-Investigators Project
Associates/Assistants The
theme of research is to develop electrical property measurement based systems
for monitoring biological processes. The objectives of the project are (i) to
develop piezo ceramic based static electricity generator as a tool to carry
out electroporation and (ii) to study the dielectric properties during
biological processes like complexation, aggregation, solublization etc. and to
develop devices and sensors based on such studies. An
exceptionally simple and effective DNA detection methodology based on latex
microparticles and gold nanoparticle probes has been developed. Separate
oligonucleotide sequences were linked to latex microparticles and gold
nanoparticles. In the presence of target strands that are complementry to both
the oligonucleotides, these probes undergo hybridization. This hybridization
results in linking of gold nanoparticles to the latex microparticles. Finally,
these gold nanoparticles are made visible to the naked eye after silver
enhancement. We have selected oligonucleotides sequences from IS6110 insertion
to develop a DNA based diagnostic test for tuberculosis. The sensitivity limit
of this technique was found to be 2-10 ng of target DNA. If we assume 10pg DNA
per cell, the sensitivity is approximately 200-1000 cells. Immunization
by electroporation We
have used a commercial preparation of hepatitis B surface antigen to immunize
mice by electroporation. A previously standardized power supply and a piezo
ceramic device have been used to apply electric pulses. Last year we observed
the problem of lower levels and variations of antibody titre. These issues
have been addressed. In the hetrostructure, skin-antigen formulation -
electrodes - electric device, we focused on antigen formulation and electrodes
to improve the response. In
designing an electrodes for electroporation important considerations are the
surface area of individual electrode and the distance between the two
electrodes. The ease of operation is a major concern. We have made a number of
designs to achieve desired electric field strength and current density. We
have found that electrodes based on ‘self closing tweezers’ design are the
most convenient. In
order to increase the amount of antigen delivered through skin we have
formulated a permeation enhancer, dimethylsulfoxide (DMSO) along with antigen
and systematically modified the electric pulse sequence and timing. Although,
DMSO is not the best permeation enhancer available due to is toxicity, it can
be used for short ‘skin contact’ time. Among various combinations, a
sequence of events like apply electrodes bearing antigen with DMSO on
previously shaved skin, wait for 3 mins, apply 10 electric pulses sepatated by
10 secs, wait for 1 min and remove electrodes resulted in best immune response
in terms of antibody titre. The exact quantification of antigen delivered by
various protocols is yet to be done. We
have explored the role of adjuvant in immunization by electroporation. It is
not possible to deliver alum by electroporation along with antigen due to its
large particle size. Lipopolysaccharides (LPS) are strong adjuvants to
generate humoral response. These molecules have moderate molecular weight
(around 30KD) and are ‘skin friendly’ to permeate inside the skin. The
adjuvant activity of LPS is related to lipid A, which is the toxic moiety of
the LPS molecule. We detoxified the lipid A of LPS by alkylation with n-amino
butyric acid. This alkylation of LPS has been confirmed by spectroscopic
studies. The toxicity of detoxified LPS was checked on mouse macrophage cell
line by comparing percentage of dead cells at different concentrations. At 0.3µg/ml
detoxified LPS was found to cause 16% cell death compared to 34% cell death
when LPS was used of same concentration. Detoxified LPS concentration less
than 0.1µg/ml was found to be ‘non toxic’. We are also trying to use this
alkylated LPS to make a water soluble conjugate of taxol. In
early investigations, mice were immunized intra muscularly by hepatitis B
surface antigen along with detoxified LPS. Mice which were immunized with this
adjuvant showed significantly higher antibody titre and high recall response,
compared to animals in which alum was used as an adjuvant. We have started
immunizing animals by electroporation in which detoxified LPS is incorporated
along with antigen and soon we expect to get the results.
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 Pharmaceutics 255:43-48 (*in press
last year, since published). 2. Agarwal KS, Tyagi S, Kumar A, Khanna R, Puliyel JM and Upadhyay P (2003) Bone densities in mothers of children with vitamin D deficiency due to atmospheric pollution. J Tissue Res 3:57-59. |