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PROFORMA – I
PROFORMA FOR SUBMISSION OF PROJECT PROPOSALS ON
RESEARCH AND DEVELOPMENT, PROGRAMME SUPPORT
(To be filled by the applicant)
PART I: GENERAL INFORMATION
1. Name of the Institute/University/Organisation submitting the Project Proposal:
PSG Institute of Advanced Studies,
Nanotechnology Research Facility,
Post Box No. 1609, Avinashi Road, Peelamedu,
Coimbatore-641 004
2. State: Tamil Nadu 3. Status of the Institute: Private Research Institute
4. Name and designation of the Executive Authority of the Institute/University
forwarding the application:
Dr. P. Radhakrishnan,
Director, PSG Institute of Advanced Studies
5. Project Title:
Development of gold nanoparticle based electrochemical immunosensor for
snake venom detection using chicken egg yolk antisnake venom antibodies
(IgY).
6. Category of the Project (Please tick): R&D / Programme Support
7. Specific Area (Please see Annexure - II):
2.8 Medical Sciences: vaccines and diagnosis
8. Duration: 3 Years
9. Total Cost (Rs): Rs 28,17, 500.00
10. Is the project Single Institutional or Multiple-Institutional (S/M)?: M
11. If the project is multi-institutional, please furnish the following:
Name of Project Coordinator: Dr.R.Selvakumar
Affiliation: Nanotech Research Facility, PSG Institute of Advanced Studies,
Coimbatore-641004
Address: PSG Institute of Advanced Studies, Post Box No. 1609, Avinashi
Road, Peelamedu, Coimbatore-641 004
12. Scope of application indicating anticipated product and processes
In India, approximately 15000 people die every year due to snake bite and many
cases remain unreported (Meenatchisundaram and Michael, 2009). Bites by non-poisonous
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snakes are common and in these cases only mild to moderate local reactions are
observed. However, the signs, symptoms and prognosis in poisonous snake bite cases
depend upon the species and the type and quantity of venom injected. The venom of
cobras, kraits and coral snakes affect mainly the nervous system producing paralysis. The
venom of viper and pit viper act mainly on blood and body tissues, whereas venoms of
sea snakes are myotoxic. At present, after a snake bite, the physicians have to wait for
symptoms caused by envenoming due to lack of specific detection kit and forced to give
polyvalent polyclonal antivenom. The use of polyvalent polyclonal antivenom is not
recommended by WHO due to its hypersensitivity reaction. Hence in International
conference on snakes, venoms and snakebite (2008), Kerala, India, it was concluded that
diagnosis of snakebite is essential in treating envenomed patients. The increasing number
of deaths due to snake bite is due to lack of immediate medical treatment and detection of
the type of snake venom. Usually patients are administered with polyvalent snake venom
antibodies as a life saving medicine. Such polyvalent snake venoms will lead to the
development of many hypersensitive reactions in our body system. Thus immediate
detection of snake venom can be used to treat patient with particular monovalent
antibody. In most snake bite incidence, the patient retains no information on the type of
snake which has bitten. Hence development of biosensor to detect the possible toxin
present in the bitten site could help to treat patients with specific anti-snake venom
antibody instead of giving him a polyclonal antibody. Such treatment could enhance the
survival rate of patients and easy recovery. The out come of the proposed project will
help upto detect the quantity and type of venom in the blood/ tissue samples collected
from the patient. The proposal is aimed to detect highly sensitive electrochemical
biosensor for snake venom diction using nanobiotechnology method.
Project Summary
Snake bite remains a public health problem in many countries. It is estimated that
the incidence of snake envenomation could exceed 5 million per year (Chippaux, 1998). In
Asia, it has been estimated that a million snakebites occur each year, of which
approximately 50% are envenomed, resulting in 1,00,000 annual deaths. The snake venom
comprises of complex pool of proteins, organic and inorganic compounds. Among these
compounds, some enzymes like acetylcholine esterases, Adpase, phospholipase,
metalloprotease and serineproteinase leads to the toxicity of the venom. Among these
enzymes the metalloprotease and serineproteinase are haemostatic active components of
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venom and differs from one species to another with widely varying toxicological features.
Polyvalent antivenom antibodies from pre-immunized horse sera are the only scope for the
doctors to neutralize venoms which have varying composition and type of haemostatic
active compounds. Avian yolk immunoglobulin (IgY) based antivenom had been first
suggested by Thally and Carrol (1990) to be less expensive, safer and more robust than
horse antivenoms. Many countries around the world claim great success in trials of IgY
antivenom for veterinary use. Dr.Michael and group developed similar IgY based
antibodies for various common poisonous snake in India (Meenatchisundaram et al.,
2009).
In this context, we are aiming to develop a gold nanoparticle based
electrochemical sensor for the detection of type of snake venom from the bitten site of the
patient. The proposed study will exploit the gold nanoparticles as electro active labels due
to its high stability, easy preparation, biological compatibility and excellent conductivity.
The gold nanoparticles will be tagged with biotinylated IgY antivenom (developed by
Dr.A.Michael, Co-PI) and coated onto glassy carbon electrode. A sandwich ELISA
method will be used for the reaction with venom in the sample. The electrochemical
signals produced will be analyzed using electrochemical impedance spectroscopy and
results will be interpreted. This study will help us to develop a specific electrochemical
biosensor for detection of low quantity venom in the blood/tissue samples and its type.
PART II: PARTICULARS OF INVESTIGATORS
Principal Investigator 1:
14 (a). Name: Dr.R.Selvakumar
Date of Birth: 24/05/1981 Sex (M/F): M
Designation: Assistant Professor
Department: Nanobiotechnology
Institute/University: PSG Institute of Advanced Studies
Address: Post Box No. 1609, Avinashi Road, Peelamedu, Coimbatore, Tamil
Nadu, PIN: 641 004
Telephone: +91-422-4344000 Fax: +91-422 -257 3833
E-mail: [email protected], [email protected]
Number of research projects being handled at present: two
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Principal Investigator 2:
14 (b). Name: Dr.A.Michael
Date of Birth: Sex (M/F): M
Designation: Associate Professor and Head
Department: Department of Microbiology
Institute/University: PSG College of Arts and Science
Address: Civil aerodrome post, Coimbatore, Tamil Nadu, PIN: 641 014
Telephone:0422-574620 (Res)
E-mail: [email protected] Number of research projects being handled at present: one
Co-Investigator: 1
15. Name: Dr P. Rani
Date of Birth: 10.04.1965 Sex (M/F): F
Designation: Associate Professor
Department: Dept of Biotechnology
Institute/University: PSG College of Technology
Address: Avinashi Road, Peelamedu, Coimbatore, Tamil Nadu, PIN: 641 004
Telephone: +91-422-4344000 Fax: +91-422 -257 3833
E-mail: [email protected], [email protected]
Number of Research projects being handled at present: 2
Note: Use separate page, if more investigators are involved
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PART III: TECHNICAL DETAILS OF PROJECT
16. Introduction (not to exceed 2 pages or 1000 words)
16.1 Origin of the proposal
Snakes are a fascinating part of nature. Around 215 different species of snakes are
found in India, out of which 80 are reported to be poisonous. The common poisonous
snakes found in India are Cobras, Kraits, Vipers, Coral snakes and Sea snakes. In a
tropical country like India the morbidity and mortality rates due to snake bite are of a
considerable magnitude. About 15,000 people die of snake bites every year in India.
Bites by non-poisonous snakes are common and in these cases only mild to moderate
local reactions are observed. The signs, symptoms and prognosis in poisonous snake bite
cases depend upon the species and the type and quantity of venom injected. The specific
treatment of snake venom poisoning is the administration of anti-venom. Antivenoms
used to treat poisonous bites and stings are usually derived from horse sera, consequently,
they contain horse immunoglobulins which frequently cause complement mediated side
effects, and other proteins that can cause serum sickness and, occasionally, anaphylatic
shock. Considering these complications in the horse sera based antivenom, the chicken
anti-venom offers many advantages over conventional mammalian anti-venom. The eggs
from immunized chickens provide a continuous daily source of antibody and this
convenient approach offers greater compatibility with animal protection regulations.
Laboratories in developing countries like ours would only need relatively simple
techniques and equipments to produce a safe product.
The group lead by Dr.Michael at PSG college of Arts and Science have
successfully developed antivenom in chicken egg yolk antibodies (IgY) in chicken and
have purified it. These antivenoms are made from yolk of eggs laid by pre-immunized
chicken (Meenatchisundaram and Michael, 2010; Meenatchisundaram et al., 2011;
Sentila et al., 2011). In continuation of the above mentioned work, in this proposal, we
intend to use the IgY based antivenom for development of electrochemical biosensor using
nanobiotechnology.
Although studies have been carried on antivenom interaction with native venom
using radioactive labeling (Thwin et al., 1996) and ELISA, (Dong et al., 2003), till now to
our knowledge there are very few studies on development of electrochemical biosensors to
differentiate snake venom based on antigen specific antibodies. The present study aims in
utilizing the difference in the composition of these haemostatic active proteinase
compounds from one snake venom to the other for monoclonal antisnake venom IgY
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production. The interaction of noble metal nanoparticles with these IgY antibodies for
biosensor development has not been carried out so far and the process remains novel.
16.2 (a) Rationale of the study supported by cited literature (b) Hypothesis (c) Key
questions.
(a) Rationale of the study
Snake bite remains a public health problem in many countries. It is estimated that
the incidence of snake envenomation could exceed 5 million per year (Chippaux, 1998). In
Asia, it has been estimated that a million snakebites occur each year, of which
approximately 50% are envenomed, resulting in 1,00,000 annual deaths. In India,
approximately 15000 people die every year due to snake bite (Meenatchisundaram and
Michael, 2009) and many cases remain unreported. At present the physicians have to wait
for symptoms caused by envenoming due to lack of specific detection kit and forced to give
polyvalent polyclonal antivenom. The use of polyvalent polyclonal antivenom is not
recommended by WHO, hence in an International conference on snakes, venoms and
snakebite 2008 it was concluded, diagnosis of snakebite is essential in treating envenomed
patients. Since in most snake bite incidence, the patient retains no information on the type
of snake which has bitten, a development of biosensor to detect the possible toxin present
in the bitten site could help to treat patients with specific anti-snake venom antibody
instead of giving him a polyclonal antibody. Such treatment could enhance the survival rate
of patients and easy recovery and avoid cross reactivity and hypersensitivity caused by the
polyvalent antibodies.
(b) Hypothesis
With the development of nanotechnology, nanoparticles have attracted substantial
interest because of their unique optical, electrical, thermal and catalytic properties. These
properties originating from quantum-size dimensions could change with their sizes and
shapes. Now nanoparticles have been extensively used in construction of electrochemical
sensor. It was reported that Au nanoparticles displayed high catalytic activity in
catalyzing carbon monoxide(CO) and methanol oxidation (Biswas et al., 1995), which
was related to the band gap of a metallic-insulator transition for particles in the range of a
few nanometers (Yang et al., 2001). Based on its excellent catalytic activity and good
biocompatibility GNPs have been used as electrode modifier to develop DNA and
protein/enzyme-sensing systems or other electrochemistry sensors by all kinds of
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immobilization methods (Park et al., 2002, Lioubashevski et a., 2004). Because of these
properties, they find potential applications in biomedical field (Li et al., 2011). When
these gold nanoparticles are coated onto glassy carbon electrode having smooth surface,
large electrochemical window, higher hydrogen over potential and excellent stability, the
sensitivity of these electrode increases (Li et al., 2011).
In this proposal, we intent to exploit the biotin-avidin complexation for the tagging of
antibodies to nanoparticles coated onto glassy carbon electrode and sandwhich ELISA
model for the venom detection. In brief, streptavidin-conjugated gold nanoparticles will
be deposited onto glassy carbon electrode over which biotinylated IgY antivenom
antibodies will be allowed to bind. This lead to the formation of array of antibodies
tagged to the glassy carbon electrode. The unbound areas will be masked using 1% BSA.
The binding of antibodies to the electrode will lead to the change in
conductivity/impedance. When venom is added to the IgY-AuNP-classy carbon
electrode, antigen antibody interaction takes place and strong binding of venom to the
antibody occurs. Followed by a repeated washing to remove unbound components, the
secondary antibody tagged with gold nanoparticles will be added. The excess of unbound
secondary antibodies will be washed and removed. After the immunological reactions,
when the electrode is immersed in 0.1 mol/L HCl solution for the electrochemical
detection, in which AuNPs can be electro-oxidized to produce AuCl4- (Ambrosi et
al., 2007). Finally, this strategy will be applied in the detection of venom in the sample.
Key questions:
1. What will be an alternative if the proposed mechanism doent work?
The investigators will try to tag quantum dots to the antibodies which can be used to
develop immuno-fluorescence sensor.
2. How will you process the blood/venom sample for biosensor?
Initially commercially available venom will be tested for its efficiency. If the sensor
works properly, the blood samples/bite site swab will be collected and resuspended in
appropriate buffer and used. The blood samples will be centrifuged and blood
components will be removes before subjecting it to the immunosensor.
3. How do you test your specificity of venom and IgY based antibody?
Before developing a sensor, Ouchterlony double diffusion method/ ELISA will be
used to detect the selectivity and specificity of antigen towards antibodies.
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16.5 Current status of research and development in the subject (both international
and national status)
International status
Snake bite and IgY antibodies
Out of 3500 known species of snakes in the world only one tenth are venomous. The
venoms of most species have been found to be mixtures of several toxic proteins and
enzymes with diversified and complicated pharmacological effects. Swaroop and Grab
(1954) have attempted to assess the global incidence of snake bites and rate of mortality
due to snake bites. They estimated around 30,000 and 40,000 people die of snake bite in
the world. Lallo et al., [1995] calculated around 50,000-1,00,000 people die of snake bite
in the world. These authors relied on data provided by government hospitals. A number
of more recent studies have suggested that these figures greatly underestimated the
problem because, in many parts of the rural tropics especially in India , snake -bite
victims do not go to hospitals but die at home under the care of traditional healers. The
only specific treatment of snake venom poisoning is the administration of antivenom.
Until now, anti snake venom for neutralizing snake bite venom has been made by
injecting horses with small quantities of venom to produce an immune reaction.
Antibodies are then harvested from the animal's blood. Thally and Carroll (1990)
described a new, avian source of antivenoms that precludes the complications of equine
antivenom, and an efficient and gentle means for preparing antivenoms composed solely
of venom specific antibodies (IgY). Immunoglobulin IgY is the major antibody produced
by chickens (Gallus domesticus). Theakston et al.,(1992) (WHO Lab) compared the
purity and efficacy of affinity purified avian antivenoms with commercial equine crotalid
antivenoms and concluded that the purity, efficacy and ease of manufacture of avian
antivenoms and their inability to fix mammalian complement make them an attractive
alternative to equine and other mammalian antivenoms. Almedia et al., (1998) reported
that adult leghorn chickens hyperimmunized with low doses of Bothrops and Crotalus
venoms produce specific antibodies which recognise components of these venoms and
neutralize their toxic and lethal effects. Secondly that the serum levels of antibodies
mainly as IgY immunoglobulin isotypes, remain high for long periods and are
immediately transferred to the yolk and thirdly that high yields of yolk IgY rich
preparations with strong neutralizing properties could be obtained. Chicken IgY does not
react with anti mammalian antibodies in human serum such as rheumatoid factors and
human anti-mammalian anti-IgG. In immunological assays the interference caused by
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these antibodies can be problematic particularly as the sensitivity of the assay increases.
Thus, if chicken IgY is used, interference by anti-mammalian IgG antibodies is
eliminated (Lindmark et al., 1983).
IgY, like mammalian IgG, is a reasonably stable protein. Diluted in saline-
containing substances that preserve the protein structure, IgY antibody activity can be
stored at 2–4 8C. When lyophilized, the IgY antibody activity is not diminished even
after several months of storage at temperatures of _20 8C or less, or even for 1 month at
37 8C. IgY, however, is not very stable at temperatures of higher than 70 8C, and at pH
below 4.0 (Larsson et al., 1999). IgY can be stored for over 10 years in 0.15M NaCl
containing 0.02% NaN3 at 4 8C (Shimitzu et al., 1994). The IgY antibodies, such as
mammalian IgG, can be labeled by routinely described methods with biotin or
horseradish (Olovsson and Larsson, 1993; Larsson et al., 1999).
Advantages of IgY antibodies
The advantages of chicken antibodies over mammalian antibodies include: (a)
reduction in animal use, since chickens produce larger amounts of antibodies than
laboratory animals; (b) the elimination of painful blood collections in animals; (c) the
utility of IgY in many immunological assays without loss of specificity and sensitivity;
(d) the considerably lower cost of feeding and handling of chickens than mammalians; (e)
crude egg may be used as an antibody source. Items (a) and (b) meet the
recommendations of the European Centre for the Validation of Alternative Methods
(ECVAM), which specify that yolk antibodies should be used instead of mammalian
antibodies for animal welfare reasons (Schade et al.,2005).
IgY and immunodiagnostics
The IgY concentration in the serum of adult hens is approximately 5–7 mg/ml.
One hen of a high egg-laying strain can produce around 20 eggs per month. Such
amounts correspond to 2 g of IgY per month equivalent, therefore, to the IgY content of
300 ml of serum or 600 ml of total blood (Shimitzu et al., 1994). Such amounts of blood
only can be obtained from large mammals. Chicken antibodies, therefore, constitute a
much less expensive vehicle for use in diagnostic proposals. It is a well-known concept
that the immune response is more potent when the distance between the antigen source
and the immune system increases. Therefore, to obtain immuno reagents containing
antibody titers against mammalian antigens, chickens are better and cheaper than
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mammals (Larsson et al., 1999). Furthermore, chicken antibodies recognize more
epitopes when mammalian proteins are used as antigens, than the corresponding
mammalian antibodies (Svendsen and Hau, 1996). The absence of immunological cross-
reactivity between chicken IgY and mammalian IgG (Hadge and Anbrosuius, 1984),
determined by the evolutionary distance, reinforces the advantages of using IgY over IgG
as the first antibody in some types of immunological reactions. For instance, in
immunohistochemical analysis the usually common cross-reactions observed between
tissue IgG and epitopes, shared by the primary antibody and recognized by the secondary
mammalian antibody, are not observed when IgY is used as the secondary antibody
(Larsson and Lindahl, 1993). Chicken antibodies exhibit high avidity (109 L/mol) even
after the first immunization. In order to reach similar avidity values (1010 L/mol), sheep
must receive four boosters (Wooley and Landon, 1995). Chickens can be immunized
through different routes, as desired by the immunization protocols (Wooley and Landon,
1995). The injection of the antigen by the intramuscular route results in higher antibody
levels by day 28 after immunization, and the resulting antibodies also exhibit higher
specificity, being over 10 times more specific when compared with chickens immunized
with the same antigen but by the sub-cutaneous via (Wooley and Landon, 1995).
Chickens, immunized by the intramuscular via, continue producing specific antibodies
during more than 200 days (Horton et al., 1984). Chickens can also tolerate the use of
common immunological adjuvants, such as Freund’s adjuvant, Specol, Hunters, TiterMax
and lipopeptide Pam3-Cys-(lys)4 (Losch et al., 1986). The percentage of antigen specific
antibodies in one egg yolk is close to 10% (Thalley and Carrol, 1990; Akita et al., 1998).
Immunosensor for diagnosis
Identification of biting species is critical for the administration of potent monovalent for
the management of snakebites. Several immunoassays have been reported for the
detection of snake venom/toxin in animals and human victims and enzyme linked-
immunosorbant assay (ELISA) has been suggested to be more practical than other system
(Ratanabanangkoon et al., 1987). Bioassays, immunodiffusion, immunoelectrophoresis,
immuno¯uoresence, haemagglutination, radioimmunoassay (RIA), enzyme-linked
immunosorbent assay (ELISA) etc. have been developed for venom detection, and
ELISA is used for venom antibody detection (Theakston, 1983). In spite of numerous
efforts, few immunodiagnostic kits have been developed and tested. Though ELISA is
widely used for the detection of snakebite victims, there remain some practical
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difficulties in making the tests executable in the field (Selvanayagam and
Gopalakrishnakone, 1999). Recent advancements in immunosensor technology offer a
potential alternative for the diagnosis of snake envenomation. The advantages of immune
sensor include simplicity, miniaturization and automation, as well as the capacity for high
throughput screening (Scouten et al., 1995).
Ion-sensitive field effect transistor (ISFET) sensor was developed by Bergveld
(1970). The application of enzymes as the selecting agent in ISFET-based sensing
systems leads to the development of highly selective sensors (Caras and Janata, 1980).
Such enzyme-modified ISFETs (EnFETSs) can in principle be constructed with any
enzyme that produces a change in pH upon conversion of the substrate (Kimura and
Kuriyama, 1990). By combining ISFET with a membrane that contains antibody, the
sensor can thus detect antigen_/antibody interaction. ISFET biosensor is already used in
medical diagnostics, fermentation process control and environmental monitoring
(Kerkhof et al., 1995; Tsuruta et al., 1995b). An ELISA system, which uses a pipette tip
as the solid phase and a pH-FET as the detector has been developed for the detection of
PCR products (Tsuruta et al., 1995a). Recently a magnetoimmunosensor system
incorporating pH sensitive ISFET has also been reported (Sole et al., 1998). Beta -
Bungarotoxin (b-BuTx), a presynaptic neurotoxin isolated from Bungarus multicinctus
venom was used as a model analyte for the development of ISFET immunosensor
(Selvanayagam et al., 2002). The venom detection kit (VDK) issued by Commonwealth
Serum Laboratories (CSL), Australia is the only commercial diagnostic kit for the
detection of snake venoms. The efficacy of this kit for the detection of venoms of five
major venomous snakes of Australia, viz., brown snake (Pseudonaja textilis), death adder
(Acanthophis antarcticus), king brown snake (Pseudechis australis), taipan (Oxyuranus
scutellatus) and tiger snake (Notechis scutatus) has been evaluated by numerous clinical
studies (Mead and Jelinek, 1996; Southern et al., 1996).
National status
In India polyvalent anti- snake venom (ASV) is available which contain
antibody against cobra, Russell’s viper, common krait and saw-scaled viper. Five lakh
ASV vials are manufactured by the four institutes (Haffkine Institute, Mumbai , Serum
institute, Pune, King Institute, Chennai, Central Research Institute, Kasauli). Exact total
amount of venom injected by snake at the time of bite is unknown but fatal dose is known
viz. cobra 120 mg, Russell’s viper 150 mg, krait 60 mg and Echis carinatus 80 mg (4.6
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mg injected at the time of bite i.e much less as compare to fatal dose). But the amount of
venom neutralized by one ml of polyvalant ASV is known viz. cobra 0.6mg, Russell’s
viper 0.6mg, krait 0.45 and Echis carinatus 0.45 mg. Empirically total ASV required is
200, 250, 134 and 10.22 ml. respectively. However, the clinical features and outcomes
are not as simple as predicted, because every bite does not result in complete
envenomation. In India commercially produced ASV is relatively cheap as compared to
Western countries (Bawaskar, 2004). One vial of ASV costs Rs.400. Majority of snake
bite victims are reported to government hospitals because ASV is given free of cost, still
many victims delays hospitalization and vital time is killed by attending to Mantrik.
Paul et al (2003) reported an interesting finding of morbidity and mortality which is high
in group received high dose of ASV (120 ml) as against the group who received low dose
(60 ml) (Paul et al., 2004). Similar experience reported from Vellore and Rajastan
(Thomas and Jacob, 1985; Kothari et al., 2001). Kulkarni and Anees (1994) from
Karnataka studied 630 snake bite cases in pediatric and majority of his cases recovered
with 80 ml of ASV. Further reduction of requirement of ASV can be achieved by
encouraging ASV producers in India to prepare ASV from venoms obtained from snakes
caught from relevant areas of the country (Bhawaskar and Bhawaskar, 2002).
Manjula J. Kusum et al., (2006) generated antivenom specific antibodies in white
leghorn chicken and their egg yolks. They recently reported that the purity, efficacy and
ease of manufacture of avian antivenoms and their inability to fix mammalian
complement make them an attractive alternative to equine antivenoms. They also
specified that over 10-15 mg of venom specific antibodies can be obtained from an
immunized chicken's egg yolk. To our knowledge and search, we have not found any
publication for snake venom detection using electrochemical approach in India.
16.6 The relevance and expected outcome of the proposed study
The outcome of the proposed project will lead to the development of a technology
to differentiate venom proteins based on IgY antibody-nanoparticle interaction and will
ultimate lead to the development of a gold nanoparticle based electrochemical
immunosensor.
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16.7 Preliminary work done so far
Preliminary work carried out by PI (Dr.R.Selvakumar)
Dr.R.Selvakumar has been working in the field of Nanobiotechnology at PSG
Institute of Advanced Studies since 2009. He has successfully synthesized many types of
nanoparticles using microorganism and have 4 publications on the same. At present,
citrate capped gold nanoparticles have been synthesized using chloroauric acid standard
reduction method and have been analyzed using HRTEM (Fig.1).
Fig. 1: Citrate capped gold nanoparticles synthesized at our laboratory
The impedance analysis of the gold nanoparticles were carried out using PARSTAT
impedance analyzer and its I-V characteristics were analyzed (fig 2 and 3).
Fig.2: impedance analysis of gold nanoparticles synthesized in our lab
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Fig.3: I-V (i) and CV (ii) characteristics of gold nanoparticles synthesized in our laboratory Preliminary work carried out by Co- PI (Dr.A.Michael)
The Co-principal investigator has got extensive research experience in the field of
microbiology and immunology. He has been working on chicken antibodies from 1994
onwards. His Ph.D work itself was on immunodiagnosis of beta- haemolytic streptococci
using Chicken egg yolk antibodies as an alternative source to rabbit antibodies.
Through student project scheme he carried out research work on the generation of
antivenom in immunized chicken and their egg yolk. As a follow up study now 2 M.Phil
and 1 Ph.D., students of his are working on different aspects of antivenom generation in
Chicken. He is well versed in basic applied microbiological techniques, and specializes
in the field of medical microbiology, Immunology, Serology, Animal tissue culture
techniques, production of monoclonal and polyclonal antibodies and their
characterization immuno assays, purification and labelling of antibodies- Chicken egg
yolk antibodies production and their purification methods. During his research work he
underwent training/ participated in workshops in various prestigious institutions such as
National Institute of Immunology ( Total period 3 months ) WHO Collaborating Centre
for Streptococcal Diseases and Lady Hardinge Medical College, New Delhi, Christian
Medical College, Vellore, Indian Institute of Science and National Institute of Mental
Health and Neurosciences, Bangalore, Tuberculosis Research Centre, King Institute,
Cancer Institute, Center for Biotechnology - Anna University and Biotechnology Unit-
Veterinary University, Chennai to learn some techniques and hands on experience. He
was instrumental in bringing some immunological techniques to this centre. He has also
been one of the internal faculties involved in conducting of short term training programs
i) ii)
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on basic microbiological techniques for college teachers and work shop on
immunotechnology conducted by our department.. He has so far guided 120 M.Sc
Applied Microbiology students project work and is at present guiding 40 M.Phil and 7
Ph.D students.
Preliminary work carried out by Co- I (Dr.P.Rani)
The co-investigator (Dr.P.Rani) has got extensive research experience in the field
of Biosensors and is working on development of diagnostic marker kit for the early
detection of Alzheimer’s Disease sponsored by DBT. She has successfully completed a
project on development of biosensor for water and air quality measurement funded by
DRDO.
References: 1. Chippaux JP (1998) Bull WHO. 76, 515- 524. 2. S. Meenatchisundaram & A. Michael, Ind.J. Sci Technol . 2(10) 69-73. 3. Maung-Maung-Thwin, P. Gopalakrishnakone, R. Yuen, C. H. Tan (1996) Toxicon, 34(2) 183-199. 4. L.Dong , L. K. Quyen , K.H.Eng , P. Gopalakrishnakone, (2003) J. Immuno. Methods. 282;13–3. 5. Paul V, Pratibha, Prahalad KS , Eraly J, Francis S, Lewis F. JAPI 2004;52:14-7. 6. Kothari D, Bomb BS, Bolya YK, Srivastva S, et al. JAPI 2001;49:57 7. Kulkarni ML, Anees Ind Pedtr 1994;31:1239-43. 8. Bawaskar HS, Bawaskar PH. Lancet 2002;360:1703. 9. Manjula J. Kusum.P, Sairam A.K, Murthy P.B. & Subbarao .P.V. Journal of Cell and Tissue Research Vol.6(2): 733-738.2006. 10. Thomas PP and Jacob J. BMJ1985;291:177-78. 11. Paul V, Pratibha, Prahalad KS , Eraly J, Francis S, Lewis F.JAPI. 2004;52:14-7. 12. Bawaskar HS, JAPI•VOL. 52•JANUARY 2004.11-13. 13. Paul V, Prahld KA , Earali J, Francis S, Lewis F. JAPI 2003;51:163-66. 14. LaIloo DG et al. Royal Society Tropical Medicine and Hygiene, 1995, 89: 178- 182. 15 Theakston et al., Joumal of tropical medicine and 84:hygiene, 199-202. 1981. 16. Schade,R., Calzado,E.G., Sarmiento,R., Chacana,P.A., Porankiewicz- Asplund,J., Terzolo,H.R.,2005. Altern.Lab.Anim.33,129–154. 17. Shimitzu,M.,Fitzsimmons,R.C.,Nakai,S.,1998. J.FoodSci.53,1360–1366. 18. Svendsen,B.,Hau,J.,1996. Scand.J.Lab.Ann.Sci.23,85–91. 19. Hadge,D.,Anbrosuius,H.,1984..Mol.Immunol.21,699–707. 20. Larsson,A.,Lindahl,T.,1993. Avian Immunology in Progress. INRA, Paris, pp.97– 102. 21. Wooley, J.A.,Landon,J.,1995. J. Immunol. Methods178, 253–265. 17. Specific objectives
To develop egg yolk antibodies (IgY) against purified venom in chicken Purify IgY antibodies based on affinity chromatography and to study its
immunological interactions with native venom of different snakes.
16
Studies on cross reactivity of anti-IgY antibodies with other components of the venom.
Liganding of IgY antibodies to noble metal nanoparticles and studies on change in electrochemical and optical properties using impedance spectroscopy, ELISA reader and other suitable techniques.
Quantitative and qualitative analysis of the IgY tagged noble metal nanoparticles for its specificity in interaction with various haemostatic proteinase compounds
Studying the possible application of the mechanism of antigen bounded antibody interaction with noble metal nanoparticles and glassy carbon electrode for electrochemical based biosensor development.
18. Work Plan: should not exceed 3-4 pages (the section can be divided according to the specific aims and under each specific aim, the following should be stated clearly as sub headings) 18.1 Work plan (methodology/experimental design to accomplish the stated
aim)
Over all experimental design:
Affinity based purification of IgY
Purified IgY antibodies
venom
Antigen injection
Healthy chickens
Anti venom IgY
Venom-antivenom IgY interaction studies
IgY liganding
Nobel metal nanoparticles
Nanoparticle coated Silicon/quartz substrate
Silicon/quartz substrate Electrode
Venom
Change in Impedance/ optical property
Application in biosensor
17
Detailed experimental design for biosensor development:
Glassy carbon electrode
Connectivity of the participating institutions and investigators (in case of
multi-institutional projects only)
Three major institutions (PSG Institute of Advanced Studies (PSG IAS),
PSG College of Arts and Science(PSG CAS) and PSG College of Technology
(PSG CT) will participate in the proposed research. PSG institutions, one of the
pioneers in the field of education for more than seven decades are committed to
Arrangement of Streptavidin conjugated gold nanoparticles onto electrodes
Biotinylated Purified IgY snake venom antibodies
Venom from bitten site/blood
Gold conjugate having IgY snake venom antibodies
Electrochemical impedance analysis
Addition of 0.1 mol/L HCl to electro-oxidized AuNPs to produce AuCl4-
18
provide world class engineering and science education through UG, PG and
research programs. They are well recognized by industry and R&D institutions.
PSG institutions have the reputation of being among the top institutions imparting
high quality education and have excellent industry-academia interaction.
The PIs will develop gold nanoparticles and tag with antibodies using the
facilities at PSG IAS. The Physical and biological characterization of such tagges
nanoparticles will be characterized at PSGIAS by the PI. The Co- PI
(Dr.A.Michael) from PSGCAS will take care on the isolation and purification of
IgY antibodies from egg yolk and provide for the experiment. The Co-I (Dr. P.
Rani) will take part in enhancing the sensitivity of the experiment by finding
appropriate system that will increase the signal yield for impedance analysis.
This work will be carried out at PSGCT. The facilities available in all the three
institutes will be used by the investigators.
18.3 Alternate strategies (if the proposed experimental design or method does
not work what is the alternate strategy)
The immuno fluorescence approach will be considered (as per the
following experimental design) as an alternative if the proposed technique doesn’t
work.
Streptavidin treated glass matrix
Biotinylated Purified IgY snake venom antibodies
Venom from bitten site/blood
19
19. Timelines: (Please provide quantifiable outputs)
Period of
study
Achievable targets
6 Months Literature review, purchasing chemicals and
Appointment of JRF
12 Months Preparation and characterization of nanoparticles,
immunization of chicken and IgY production
18 Months Purification of IgY, Immunological characterization,
Optimization of conditions for IgY- gold nanoparticle
conjugation and deposition on electrode
24 Months Preparation of immunosensor and analysis using
electrochemical impedance spectroscopy.
30 Months Testing of biosensors for sensitivity and selectivity
36 Months Documentation, publication and review
CdSe quantum dot conjugate having IgY snake venom antibodies
Immuno fluorescence analysis using microscope
20
20. Name and address of 5 experts in the field
S.No Name Designation Address
1 Dr. S.K.Gupta Staff Scientist -VI & Head
Department of Gamete antigen laboratory, National Institute of Immunology, Aruna Asaf Ali marg, New Delhi -110 067. Ph: 6162281, 6188306,6183004 Fax :6162125/6177626.
2 Dr. G. Dhinakar Raj Professor
Department of Biotechnology, TamilNadu Veterinary and Animal Sciences University, Madras Veterinary College, Vepery, Chennai-600 007.Ph:5381506, 8274055(Res). Fax: 5381886,
3 Dr. K. Ramasamy Vice Chancellor
Karpagam University, Eachanari Post, Coimbatore-641021 Email: [email protected], Phone : +91-422-6471113, 6471114, 6471115. Fax : +91-422-2611043, 2611646.
4. Dr.D.Mangalaraj Professor and Head
Department of Nanoscience and Technology,
Bharathiar University, Coimbatore-641046, Email: [email protected] phone: 09894762141
5 Dr. (Mrs) P. Renuka Devi
Assistant Professor and
Head (IC)
Biotechnology centre, Anna University of Technology, Coimbatore-641047 Email: [email protected]: Phone: 09791044994
PART IV: BUDGET PARTICULARS
Over all Budget (In Rupees)
A. Non-Recurring (e.g. equipments, accessories, etc.)
Sub-Total (A): Rs.7,00,000.00
S.
No
Item Year 1 Year 2 Year 3 Total
1 96 well plate ELISA
reader and -80 deep
freezer
7,00,000.0 - - 7,00,000.00
Total 7,00,000.0 - - 7,00,000.00
21
B. Recurring
B.1 Manpower (See guidelines at Annexure-III)
S.
No
Position
No.
Consolidated
Emolument
Year 1 Year 2 Year 3 Total
1 2 JRF
(M.Sc.,
Microbiolog
y/Biotechno
logy/Nanosc
ience)
Rs. 16,000.00 (1st
and 2nd year)
Rs.18,000.00 (3rd
year) + 15% HRA
4,41,600.00 4,41,600.00 4,96,800.00 13,80,000.0
0
Sub-Total (B.1) =13,80,000.00
B.2 Consumables
S. No Item
Quantity (approxim
ate) Year 1 Year 2 Year 3 Price
1 Glassware and Plasticware
Depends upon the requirement of Experiments
50,000 - - 50,000
2 Venoms 50,000 25,000 25,000 1,00,000
3
Experimental animals (Chicken, Mice), Immunochemicals reagents and Purification columns
1,00,000 25,000 - 1,25,000
4 electrodes Chemicals 50,000 50,000 50,000 1,50,000
Sub Total
( B2) 2,50,000 1,00,000
75,000 4,25,000
Sub-Total (B.2) = Rs 4,25, 000.00
22
Other items Consolidated Emolument
Year 1 Year 2 Year 3 Total
B.3 Travel 30,000.00 20,000.00 20,000.00 70,000.00 B.4 Contingency
20,000.00 10,000.00 20,000.00 50,000.00
B.5 Overhead (10%)
1,92,500
Sub-total of B (B.1+B.2+B.3+B.4+B.5)
13,80,000.00+ 4,25,000.00+ 70,000.00+ 50,000.00+ 1,92,500
21,17,500.00
Grand Total (A + B)
7,00,000.00+ 21,17,500.00
28,17,500.00
Total: Rs. 28, 17, 500.00 (Rupees twenty eight lakhs and seventeen thousand
and five hundred only)
INSTITUTION WISE BUDGET: A. Non-Recurring (PSG INSTITUTE OF ADVANCED STUDIES)
Sub-Total (A): Rs.7,00,000.00
B.1 Manpower (PSG INSTITUTE OF ADVANCED STUDIES)
S.
No
Position
No.
Consolidated
Emolument
Year 1 Year 2 Year 3 Total
1 1JRF
(M.Sc.,
Microbiolog
y/Biotechno
logy/Nanosc
ience)
Rs. 16,000.00 (1st
and 2nd year)
Rs.18,000.00 (3rd
year) + 15% HRA
2,20,800.00 2,20,800.00 2,48,400 6,90,000
S.
No
Item Year 1 Year 2 Year 3 Total
1 96 well plate ELISA
reader and -80 deep
freezer
7,00,000.0 - - 7,00,000.00
Total 7,00,000.0 - - 7,00,000.00
23
B.1 Manpower (PSG College of Arts and Science)
S.
No
Position
No.
Consolidated
Emolument
Year 1 Year 2 Year 3 Total
1 1JRF
(M.Sc.,
Microbiolog
y/Biotechno
logy/Nanosc
ience)
Rs. 16,000.00 (1st
and 2nd year)
Rs.18,000.00 (3rd
year) + 15% HRA
2,20,800.00 2,20,800.00 2,48,400 6,90,000
B.2 Consumables (PSG INSTITUTE OF ADVANCED STUDIES)
S. No Item Quantity
(approximate) Year 1 Year 2 Year 3 Price
1 Glassware and Plasticware
Depends upon the requirement of Experiments
50,000 - - 50,000
2 Electrodes, Chemicals 50,000 50,000 50,000 1,50,000
Sub Total (
B2) 1,00,000 50,000
50,000 2,00,000
Sub-Total (B.2) = Rs 2,00, 000.00
B.2 Consumables (PSG College of Arts and Science)
S. No Item
Quantity (approximat
e) Year 1 Year 2 Year 3 Price
1 Venoms 50,000 25,000 25,000 1,00,000
2
Experimental animals (Chicken, Mice), Immunochemicals reagents and Purification columns
1,00,000 25,000 - 1,25,000
Sub Total
( B2) 1,50,000 50,000
25,000 2,25,000
Sub-Total (B.2) = Rs 2,25, 000.00
24
(PSG INSTITUTE OF ADVANCED STUDIES)
(Rupees seventeen lakhs and forty five thousand only) (PSG COLLEGE OF ARTS AND SCIENCE)
(Rupees Ten lakhs seventy two thousand and five hundred only)
Other items Consolidated Emolument
Year 1 Year 2 Year 3 Total
B.3 Travel 15,000.00 10,000.00 10,000.00 35,000.00 B.4 Contingency
10,000.00 5,000.00 10,000.00 25,000.00
B.5 Overhead (10%)
95000
Sub-total of B (B.1+B.2+B.3+B.4+B.5)
6, 90,000.00+ 2,00,000.00+ 35,000.00+ 25,000.00+ 95,000
10,45,000
Grand Total (A + B)
7,00,000.00+ 10,45,000.00
17,45,000.00
Other items Consolidated Emolument
Year 1 Year 2 Year 3 Total
B.3 Travel 15,000.00 10,000.00 10,000.00 35,000.00 B.4 Contingency
10,000.00 5,000.00 10,000.00 25,000.00
B.5 Overhead (10%)
92,500
Sub-total of B (B.1+B.2+B.3+B.4+B.5)
6, 90,000.00+ 2,25,000.00+ 35,000.00+ 25,000.00+ 97,500
10,72,500
Grand Total (A + B)
Nil+ 10,72,500.00
10,72,500
25
YEAR WISE ALLOTTED TO THE TWO INSTITUTES
(Rupees twenty eight lakhs and seventeen thousand and five hundred only)
Justification for budget:
The above mentioned budget is requested for the proposed work since the
experiments require two JRFs who need to work parallel with IgY production and
purification and for development of immunosensor.
Justification for manpower requirements
In our proposed research work, 1 JRF’s are required to perform the
experiments from nanoparticles preparation, characterization, antibody
conjugation, sensor development etc studies. The other research fellows is
required for immunization, polyclonal antibody generation in chicken against
snake venoms, purification and characterization of egg yolk antibodies, assay,
evaluation and efficacy analysis in animals.
Justification for travel
As a part of this project, PIs would like to visit laboratories involved in
similar research activities for discussion as well as to attend seminars and
conferences to present research papers based on the work proposed.
Justification for expensive consumables
The venom and the antibody purification columns are very costly and needs to
have high purity for the experiment. Glass ware and plastic ware are required for the
laboratory work. Venoms are required for antivenom production and also for clinical in
vitro and in vivo studies. Immunochemicals and reagents are required for the separation
and assay of antibodies. Purification columns are required for purification. Experimental
animals are required for the project work.
Institute Year 1 Year 2 Year 3 Total PSG Institute of Advanced Studies
11,40,800 2,85,000 3,18,400 17,45,000
PSG college of Arts and Science
4,93,300 2,85,800 2,93,400 10,72,500
Total 28,17,500.00
26
Justification for other cost (contingency)
Cost involved in installation of equipments,Lb maintance, transport of
materials and any other expenditure not anticipated at this time will be covered
under the head of other costs.
V: EXISTING FACILITIES
Resources and additional information
1. Laboratory:
a. Manpower:
The PIs and Co-Is are available for the project work. In PSG IAS, various
research projects are being undertaken as part of the student projects (B.Tech, and
M.Tech) as well as for M.Phil and Ph.D. degrees. Students from different
disciplines are available for doing research project in the area of
Nanobiotechnology. Moreover, two Junior Research Fellows will be hired (for 3
years) specifically for this project.
b. Equipments:
Important equipments available at PSG IAS and other PSG institutions related
to the project are listed below.
Equipment facilities at the PIs institute
Equipment available Model
Equipments at PSG Institute of Advanced Studies (In PI’s Lab)
1. Multimode scanning probe microscopy with nanoindendation facility
NT-MDT, Russia
2. Bench top AFM/STM Nanosurf, Switzerland
3. HR-TEM with EDX and CCD camera JEOL, Japan
4. EDS for HR-TEM Oxford Instruments, UK
5. Clean air type II A2 biosafety cabinet Clean Air, India
6. High speed refrigerated centrifuge (upto 30,000 rpm) Sigma, Germany
7. Rotary evaporator Buchi, Switzerland
8. Millipore water purification system Millipore, India
9. Temperature controlled incubated shaker Scigenics, India
27
10. High temperature hot air oven NSW-145, India
11. Phase contrast microscope Carl Zeiss, Germany
12. High speed emulsifier Remi, India
13. UV-VIS spectrophotometer T90+ UK
Equipments at PSG College of Technology
At the Department of Chemistry
1. FT-IR spectrometer Shimadzu-Japan
2. Differential scanning calorimeter Perkin-Elmer-USA
3. Electrometer (Model 6517) Keithley-USA
4. Atomic absorption spectrometer (AAS) Shimadzu-Japan
5. Optical polarizing microscope Euromax-The Netherlands
At the Department of Metallurgical Engineering
X-Ray diffraction unit Philips
SEM with EDAX JEOL-Japan
At Department of Microbiology, PSGCAS
Laminar flow hood chambers Indian
Incubators and BOD Incubators Indian
Sterilizers - Autoclave, Hot air oven Indian
Water bath Indian
Spectronic 20 Bausch and Lomb
High speed centrifuge Remi R24
Eppendorf centrifuge Plastocraft
Balance Anamed
Refrigerators Indian
Transilluminator Imported
Microscope - Phase contrast and Dark field Carl-zeiss
Microscope- Bright Field Olympus
Millipore water system - RO and Milli Q Imported
Minigel Electrophoresis system Broviga
Gas liquid chromatography Chemito
28
2. Other resources such as clinical material, animal house facility, glass house,
experimental garden, pilot plant facility etc.
CPCSEA approved central animal house facilities are also available. Rodents,
non rodents and sheep are available as experimental animals. One operation
theatre with all necessary facilities to carry out surgery in smaller animals is
available in the animal house.
29
PART VI: DECLARATION/CERTIFICATION It is certified that
a) the research work proposed in the scheme/project does not in any way duplicate the work already done or being carried out elsewhere on the subject.
b) the same project proposal has not been submitted to any other agency for financial support.
c) the emoluments for the manpower proposed are those admissible to persons of corresponding status employed in the institute/university or as per the Ministry of Science & Technology guidelines (Annexure-III)
d) necessary provision for the scheme/project will be made in the Institute/University/State budget in anticipation of the sanction of the scheme/project.
e) if the project involves the utilisation of genetically engineered organisms, we agree to submit an application through our Institutional Biosafety Committee. We also declare that while conducting experiments, the Biosafety Guidelines of the Department of Biotechnology would be followed in toto.
f) if the project involves field trials/experiments/exchange of specimens, etc. we will ensure that ethical clearances would be taken from concerned ethical Committees/Competent authorities and the same would be conveyed to the Department of Biotechnology before implementing the project.
g) it is agreed that any research outcome or intellectual property right(s) on the invention(s) arising out of the project shall be taken in accordance with the instructions issued with the approval of the Ministry of Finance, Department of Expenditure, as contained in Annexure-V.
h) we agree to accept the terms and conditions as enclosed in Annexure-IV. The same is signed and enclosed.
i) the institute/university agrees that the equipment, other basic facilities and such other administrative facilities as per terms and conditions of the grant will be extended to investigator(s) throughout the duration of the project.
j) the Institute assumes to undertake the financial and other management responsibilities of the project. Signature of Project Coordinator Signature of Executive Authority (Dr.R.Selvakumar) of Institute/University with seal Date : Date : Signature of Principal Investigator: Signature of Co-PI: (Dr.R.Selvakumar) (Dr.A.Michael) Date: Date: Signature of Co-I (Dr.P.Rani) Date:
30
PART VII: PROFORMA FOR BIOGRAPHICAL SKETCH OF INVESTIGATORS
Provide the following information for the key personnel in the order listed on PART II.
Follow this format for each person. DO NOT EXCEED THREE PAGES Name: Dr. R.Selvakumar Designation: Assistant Professor in Nanobiotechnology Department/Institute/University: PSG Institute of Advanced Studies, Peelamedu, Coimbatore - 4 Date of Birth: 24/05/1981 Sex (M/F): M SC/ST: Not applicable Education: (Post-Graduation onwards & Professional Career)
S. No.
Institution Place
Degree Awarded
Year Field of Study
1 Bharathiar University
M.Sc. 2003 Applied Microbiology
2 Bharathiar University
Ph.D. 2009 Biotechnology
A. Position and Honors Position and Employment (Starting with the most recent employment)
Sl No.
Institution Place
Position From (Date)
To (date)
1 PSG Institute of Advanced studies, Coimbatore, India
Assistant Professor
June 2009
Till date
Honors/Awards:
Received DST-International Travel Grant for attending Asia Nano2008 conference at Singapore
Received Certificate of Recognition from Vice Chancellor and Syndicate of Bharathiar University for presenting paper at Asia Nano2008 conference at Singapore
CSIR-SRF (Senior Research Fellow) awarded by Council for Scientific and Industrial Research, Government of India.
Senior Research Fellow awarded by Defence Research and Developmental Organization, Government of India.
Junior Research Fellow awarded by Defence Research and Developmental Organization, Government of India.
Received Academic Award for Distinction in B.Sc., Microbiology.
31
Professional Experience and Training relevant to the Project:
I have 2.5 years of research experience in the synthesis of biological and chemical nanoparticle and in Nanobiotechnology. From the date of joining I have been working on development of biosensor for cancer marker detection for which I have been granted a minor project worth Rs 25,000 by Villgrow-PSG. Three B.tech students are doing research project under me for the development of above mentioned sensor. Myself and Dr. Rani (Co-Investigator) are working for the development of visual detection kit for snake venom using immunosensor method. B. Publications: 12 (Peer reviewed, International: 11 and National: 1)
Books: 0(Book chapter); Patents: 0; Reviews: 0 Conference presentation: 13 (International: 4 and National: 9)
Selected peer-reviewed publications (Ten best publications in chronological order):
1. R.Selvakumar, K.Karthikeyan and P.Radhakrishnan. 2011. Analysis on surface nanostructures present in hindwing of dragon fly (Sympetrum vulgatum) using atomic force microscopy. Micron (Accepted). (Selected for special issue on “Atomic Force Microscopy in Biology & Biomedicine” in Micron) (SCI indexed; impact factor: 1.649)
2. R.Selvakumar, S.Aravindh, C.P.Kaushik, V.G.Katarani, Vidya.S.Thorat, Prema Gireesan, V. Jayavignesh, K. Swaminathan and Kanwar Raj, 2011. Screening of silver nanoparticles containing carbonized yeast cells for adsorption of few long- lived active radionuclides. Journal of Radioanalytical and Nuclear Chemistry. 288:629–633. (SCI indexed; impact factor: 0.777)
3. R.Selvakumar, N. Arul Jothi, V. Jayavignesh, K. Karthikaiselvi, Geny Immanual Antony, P.R. Sharmila, S.Kavitha and K. Swaminathan. 2011. As(V) removal using carbonized yeast cells containing silver nanoparticles, Water Research, 45: 583-592 (SCI indexed; impact factor: 4.546)
4. R.Selvakumar, S.Kavitha, M.Sathishkumar, V.Jayavignesh and K.Swaminathan. 2010. Liquid phase separation of As(V) from aqueous solution using pretreated
Paecilomyces variotii biomass. Separation Science and Technology, 45, 776-785. (SCI indexed; impact factor: 1.015)
5. S.Kavitha, R.Selvakumar, M.Sathishkumar, K.Swaminathan, P.Lakshmanaperumalsamy, A.Singh and S.K.Jain. 2009. Nitrate removal using ‘ Brevundimonas diminuta MTCC 8486 from ground waters. Water Science and Technology, 60(2): 517–524. (SCI indexed; impact factor: 1.056)
6. M. Sathishkumar, A.R. Binupriya, D. Kavitha, R. Selvakumar, R. Jayabalan, S.E.Yun. 2009. Adsorption potential of maize cob carbon for 2, 4-dichlorophenol removal from aqueous solutions: Equilibrium, kinetics and thermodynamics
modeling. Journal of Chemical Engineering, 147: 265-271 (SCI indexed; impact factor: 3.074)
32
7. R.Selvakumar, S.Kavitha, M.Sathishkumar and K.Swaminathan. 2008. “Arsenic adsorption by polyvinyl pyrrolidone K25 coated cassava peel carbon from aqueous solution” Journal of Hazardous Materials, 153(1-2): 67-74. (SCI indexed; impact factor: 3.723)
8. S. Kavitha, R. Selvakumar and K. Swaminathan. 2008. As(V) adsorption onto Polyvinyl pyrrolidone K25 doped pretreated Aspergillus clavatus biomass from
aqueous solution. Separation Science and Technology. 43 (15): 3902-3915. (SCI indexed; impact factor: 1.015)
9. M.Sathishkumar, A.R.Binupriya, D.Kavitha, R.Selvakumar, K.K. Sheema
S.E.Yun and K.Swaminathan. 2008. Organic micropollutant removal in liquid phase using carbonized silk cotton hull. Journal of Environmental Sciences, 20(9):
1046-1054. (SCI indexed; impact factor: 1.513)
10. R.Selvakumar, S.Kavitha and K.Swaminathan. 2007. Adsorption of As(v) from aqueous solution by chemically doped coir pith carbon, Indian Journal of Chemical Technology. 14: 276-282. (SCI indexed; impact factor: 0.373) List maximum of five recent publications relevant to the proposed area of work:
1. R.Selvakumar, S.Aravindh, C.P.Kaushik, V.G.Katarani, Vidya.S.Thorat, Prema Gireesan, V. Jayavignesh, K. Swaminathan and Kanwar Raj, 2011. Screening of silver nanoparticles containing carbonized yeast cells for adsorption of few long- lived active radionuclides. Journal of Radioanalytical and Nuclear Chemistry. 288:629–633. (SCI indexed; impact factor: 0.777)
2. R.Selvakumar, N. Arul Jothi, V. Jayavignesh, K. Karthikaiselvi, Geny Immanual Antony, P.R. Sharmila, S.Kavitha and K. Swaminathan. 2011. As(V) removal using carbonized yeast cells containing silver nanoparticles, Water Research, 45: 583-592 (SCI indexed; impact factor: 4.546) Place: Coimbatore Signature of Investigator Date:
33
PART VII: PROFORMA FOR BIOGRAPHICAL SKETCH OF INVESTIGATORS
Provide the following information for the key personnel in the order listed on
PART II. Follow this format for each person. DO NOT EXCEED THREE PAGES
Name: Dr. A. MICHAEL Designation: Associate Professor and Head, Dept. of Microbiology
Department/Institute/University: PSG College of Arts and Science Date of Birth: 21/ 5/ 1962 Sex (M/F): M SC/ST: Not applicable Education: (Post-Graduation onwards & Professional Career) Sl No. Institution Place Degree
Awarded Year Field of Study
1 PSG College of Arts & Science, Coimbatore
M. Sc 1986
Applied Microbiology
2 Voluntary Health Services, Chennai
Diploma in Medical technology
1987 Medical technology
3
Bharathiar University, Coimbatore
Ph. D. 1999
Microbiology
4 Bharathiar University, Coimbatore
D.Sc. 2011( registered) Microbiology
A. Position and Honors: Position and Employment (Starting with the most recent employment)
S. No.
Institution Place
Position From (Date)
To (date)
1 PSG College of Arts and
Science, Coimbatore
Associate Professor and Head, Dept. of Microbiology
1.1.2007 Till date
2 PSG College of Arts and Science, Coimbatore
Reader and Head, Dept. of
Microbiology 24.7.2006 31.12.2006
3 PSG College of Arts and Science, Coimbatore
Reader in Microbiology 25.10.1999 24.7.2005
4 PSG College of Arts and
Science, Coimbatore
Selection Grade Lecturer in
Microbiology 21.9.1998 25.10.1999
5 PSG College of Arts and Science, Coimbatore
Senior Lecturer in Microbiology 20.9.1993 21.9.1998
34
Honors/Awards:
1. Awarded ‘PSG Management Institutional Gold Medal’ for the excellent performance on my Ph.D. Research work on “Immunodiagnostics of Group A Streptococci by latex agglutination and coagglutination assay with polyvalent chicken and rabbit antibodies” for the academic year 2000-2001.
2. Awarded ‘PSG Management Institutional Gold Medal’ for excellence in
Research for the years 2000, 2002 and 2003.
3. Awarded ‘Outstanding Teacher -2010’ for my contribution to the teaching and promotion of research in Applied Microbiology at 8th Annual Conference of IAAM held at Bharathidasan University, Tiruchirappalli, September 2010.
4. Recently awarded ‘Life Time Achievement Award’ for my 25 years of teaching
and contribution to Microbiology at 9th Annual Conference of IAAM held at Periyar University, Salem on 14th and 15th October 2011.
5. I got the best paper award along with Dr.N.Kannan and Dr.B. Appalaraju for my
work on Chicken antibodies as an alternative source for serodiagnosis of streptococcal isolates during the XXIV National Congress of Indian Association of Medical Microbiologists, held from 17.11.2000 to 19.11.2000 at Belgaum, Karnataka.
Professional Experience and Training relevant to the Project:
I got my Doctorate degree on “Immonodiagnosis of Group – A streptococci by latex agglutination and coagglutination assay with polyvalent chicken and rabbit antibodies”. I was always interested in alternative methods of diagnosis and treatment of infectious diseases. I have been working on Chicken egg yolk antibodies since 1998. We have generated antibodies in chicken against various bacterial, viral, fungal infectious agents and against Indian Poisonous Snake Venoms. Producing antibodies in laying hens is commercially feasible, because the eggs from immunized chicken provide a continuous daily source of polyclonal antibodies and this convenient approach offers greater compatibility with animal protection regulation. With my experience in the field of antibody engineering, I would like to develop a Center of Excellence in Immunotechnology, which should be a national facility. It would be my contribution towards the development of our country.
B. Publications: 22 (Peer reviewed, International: 16 and National: 6)
Books: 6(Book chapter); Patents: 0; Reviews: 0 Conference presentation: 16 (International: 2 and National: 14)
Others: 5
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Selected peer-reviewed publications (Ten best publications in chronological order)
1. 1. S.Meenatchisundaram, G.Parameswari, Michael A and S.Ramalingam.“Studies on pharmacological effects of Russell’s viper and Saw-scaled viper venom and its Neutralization by chicken egg yolk antibodies” - International immunopharmacology journal. 2008; 8:1067–1073.
2. S.Meenatchisundaram, G.Parameswari, Michael A and S.Ramalingam.
“Neutralization of pharmacological effects of cobra and krait venom by chicken egg yolk antibodies’ Toxicon. 2008; 52:221–227.
3. B.Muralikrishnan, S.Shajith Anoop, K.Karthikeyan, K.Nanthakumar and
Michael A. Comparative studies on generation of anti-venom in chicken using bentonite and adjuvant coated venoms of common venomous snakes in India. African Journal of Microbiology Research. 2009. Vol 3(7).
4. Meenatchisundaram. S, Priyagrace. S, Vijayaraghavan. R, Velmurugan. A,
Parameswari. G and Michael A. Antitoxin activity of Mimosa pudica root extracts against Naja naja and Bangarus caerulus venom, A Journal of the Bangladesh Pharmacological Society (BDPS), 2009; 4: 105-109
5. Meenatchisundaram. S and Michael A. Preliminary Studies on Antivenom Activity of Mimosa Pudica Root Extracts against Russell’s viper and Saw Scaled Viper Venom By In Vivo and In Vitro Methods. Pharmacologyonline. 2009; 2: 372-378.
6. S.Meenatchisundaram, R.Selvakumaran, G.Parameswari and Michael A. Comparison of Antivenom Potential of Chicken Egg Yolk Antibodies Generated against Betonite and Adjuvent Coated Venoms of Common Poisonous Snake in India, Bangl. J. Vet. Med. 2009; 7(1): 259-267.
7. S. Meenatchisundaram and Michael A. Comparison of four different purification
methods for isolation of anti Echis carinatus antivenom antibodies from immunized chicken egg yolk, Iranian Journal of Biotechnology, 2010; Vol.8, No. 1.
8. S.Meenatchisundaram and Michael A., Antitoxin activity of Mucuna pruriens aqueous extracts against Cobra and Krait venom by in vivo and in vitro methods, International Journal of PharmTech Research, 2010; Vol.2, No.1: pp 870-874.
9. Meenatchisundaram.S Michael A. T.Subbraj, T.Diraviam, and V.Shanmugam, Isolation, Purification and Neutralizing potential of chicken egg yolk immunoglobulin (IgY) against mastitis causing Escherichia coli in dairy cows in Coimbatore District, International Journal of Drug Development & Research. 2011; Vol. 3(2):147-153.
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10. Sentila, R., Karthika, S., and Michael A. 2011. Generation of egg yolk antibodies in chicken (IgY) against Streptococcus mutans and its in-vitro neutralization efficacy. Archives of applied science, 3(5):404-412.
List maximum of five recent publications relevant to the proposed area of work:
1. S.Meenatchisundaram, R.Selvakumaran, G.Parameswari and Michael A. Comparison of Antivenom Potential of Chicken Egg Yolk Antibodies Generated against Betonite and Adjuvent Coated Venoms of Common Poisonous Snake in India, Bangl. J. Vet. Med. 2009; 7(1): 259-267.
2. S. Meenatchisundaram and Michael A. Comparison of four different purification
methods for isolation of anti Echis carinatus antivenom antibodies from immunized chicken egg yolk, Iranian Journal of Biotechnology, 2010; Vol.8, No. 1.
3. S.Meenatchisundaram and Michael A., Antitoxin activity of Mucuna pruriens aqueous extracts against Cobra and Krait venom by in vivo and in vitro methods, International Journal of PharmTech Research, 2010; Vol.2, No.1: pp 870-874.
4. Meenatchisundaram.S Michael A. T.Subbraj, T.Diraviam, and V.Shanmugam, Isolation, Purification and Neutralizing potential of chicken egg yolk immunoglobulin (IgY) against mastitis causing Escherichia coli in dairy cows in Coimbatore District, International Journal of Drug Development & Research. 2011; Vol. 3(2):147-153.
5. Sentila, R., Karthika, S., and Michael A. 2011. Generation of egg yolk antibodies in chicken (IgY) against Streptococcus mutans and its in-vitro neutralization efficacy. Archives of applied science, 3(5):404-412.
Place: Coimbatore Signature of Investigator Date:
37
PART VII: PROFORMA FOR BIOGRAPHICAL SKETCH OF INVESTIGATORS
Provide the following information for the key personnel in the order listed on PART II.
Follow this format for each person. DO NOT EXCEED THREE PAGES Name: Dr. P. Rani
Designation: Associate Professor Department/Institute/University: Department of Biotechnology, PSG College of Technology Date of Birth: 10.04.1965 Sex (M/F): M SC/ST: Not applicable
Education: (Post-Graduation onwards & Professional Career) Sl No. Institution
Place Degree Awarded
Year Field of Study
1 Bharathiar University, Coimbatore
M. Sc. 1988 Biochemistry
2 Avinashilingam Deemed University, Coimbatore
M.Phil 1989 Biochemistry
3 Indian Institute of Technology, Chennai
Ph.D 1996 Selenium Biochemistry
B. Position and Honors: Position and Employment (Starting with the most recent employment)
Sl No.
Institution Place
Position From (Date)
To (date)
1 Department of Biotechnology, PSG College of Technology
Assistant Professor
2004 Till date
2 Department of Biotechnology, PSG College of Technology
Lecturer, 2001 2003
3 Dept of Biotechnology, Kongunadu College of Arts and Science
Lecturer, 1996 2001
Honors/Awards:
Recipient of National Merit Scholarship from 1981-86 First prize for proficiency in M.Sc Recipient of old Medal in M.Sc Best poster presentation at the Society of Biological Chemists (I) held in
Calcutta, 1991 JRF and SRF from Indian Institute of Technology, Madras (1990- 1995) CSIR Research Associate Fellowship (1995-1996) Best paper presentation- International Conference on New Horizons in
Biotechnology, Nov 2007 BRSI-NIIST, Trivandrum, India
Professional Experience and Training relevant to the Project: I have been granted with two project for the development of biosensors for water and air quality measurement (funded by DRDO) and an ongoing project on “Development of
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diagnostic marker kit for the early detection of Alzheimer’s Disease” (Funded by DBT). Other than the above I have experience in the following areas
Development of ELIZA based immunosensors Mammalian selenoproteins characterization Characterization of plant antioxidants Oxidative damage and neurological disorders Biotechnology based extension service to rural community development
B. Publications: 12 (Peer reviewed, International: 5 and National:7)
Books: 0(Book chapter); Patents: 0; Reviews: 0 Conference presentation: 8 (International: 2 and National: 6)
Selected peer-reviewed publications (Ten best publications in chronological order): 1. Evaluation of lignocellulosic wastes for production of edible mushrooms P. Rani, N. Kalyani and K. Prathiba Applied Biochemistry and Biotechnology, 151, 151-159, 2008
2. Evaluation of antioxidant properties of Berries P.Rani, K.Meena Unni and J.Karthikeyan Indian Journal of Clinical Biochemistry, 19(2), 117 - 125, 2004
3. Enzymatic and non-enzymatic antioxidants in selected Piper species J.Karthikeyan and P.Rani Indian Journal of Experimental Biology 41(2) , 135-140, 2003
4. Structural and thermodynamic consequences of introducing -aminoisobutric acid in the S- peptide of ribonuclease S Girish. S. Ratnaparkhi, Satish Kumar Awasthi, P.Rani, P.Balaram and R.Varadarajan Protein Engineering, 13(10) , 697 -702, 2000
5. Evidence for Altered Structure and Impaired Mitochondrial Electron Transport Function in Selenium Deficiency P.Rani, and K.Lalitha Biological Trace Element Research, 51 (3), 225-234, 1996.
6. Mitochondrial Selenium – 75 uptake and Regulation Revealed by Kinetic Analysis K.Lalitha, and P.Rani Biological Trace Element Research, 49(1), 21-42,1995.
7. Metabolic Relevance of Selenium in the Insect Corcyra cephalonica – Uptake of 75 Se and Subcellular Distribution K.Lalitha, P.Rani, and V.Narayanaswami Biological Trace Element Research, 41(3),217-233,1994. List maximum of five recent publications relevant to the proposed area of work: Nil Place: Coimbatore Signature of Investigator Date: