proforma – i proforma for submission of...

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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 antisnake 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


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


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


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)




10,000.00 5,000.00 10,000.00 25,000.00

B.5 Overhead (10%)

95000


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




10,000.00 5,000.00 10,000.00 25,000.00

B.5 Overhead (10%)

92,500


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


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


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


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


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)


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


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


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

38

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)


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:

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