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Biosensors and Bioelectronics 25 (2009) 592–598

Contents lists available at ScienceDirect

Biosensors and Bioelectronics

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olecularly imprinted nanopatterns for the recognitionf biological warfare agent ricin

antwana Pradhan, M. Boopathi ∗, Om Kumar, Anuradha Baghel, Pratibha Pandey,.H. Mahato, Beer Singh, R. Vijayaraghavanefence Research and Development Establishment, Jhansi Road, Gwalior 474002, India

r t i c l e i n f o

rticle history:eceived 3 September 2008eceived in revised form 9 March 2009ccepted 25 March 2009vailable online 5 April 2009

eywords:IP

WA

a b s t r a c t

Molecularly imprinted polymer (MIP) for biological warfare agent (BWA) ricin was synthesized usingsilanes in order to avoid harsh environments during the synthesis of MIP. The synthesized MIP was utilizedfor the recognition of ricin. The complete removal of ricin from polymer was confirmed by fluorescencespectrometer and SEM–EDAX. SEM and EDAX studies confirmed the attachment of silane polymer on thesurface of silica gel matrix. SEM image of Ricin-MIP exhibited nanopatterns and it was found to be entirelydifferent from the SEM image of non-imprinted polymer (NIP). BET surface area analysis revealed moresurface area (227 m2/g) for Ricin-MIP than that of NIP (143 m2/g). In addition, surface area study alsoshowed more pore volume (0.5010 cm3/g) for Ricin-MIP than that of NIP (0.2828 cm3/g) at 12 nm porediameter confirming the presence of imprinted sites for ricin as the reported diameter of ricin is 12 nm.

icin

anopatternsanoporesrotein

The recognition and rebinding ability of the Ricin-MIP was tested in aqueous solution. Ricin-MIP reboundmore ricin when compared to the NIP. Chromatogram obtained with Ricin-MIP exhibited two peaks dueto imprinting, however, chromatogram of NIP exhibited only one peak for free ricin. SDS-PAGE resultconfirmed the second peak observed in chromatogram of Ricin-MIP as ricin peak. Ricin-MIP exhibited

f 1.76

an imprinting efficiency oabrin.

. Introduction

Molecular imprinting allows the creation of artificial recogni-ion sites in synthetic polymers (Wulff and Sarhan, 1972; Arshadynd Mosbach, 1981). These sites are tailor-made in situ by co-olymerization of functional monomers and cross-linkers aroundhe template molecules. The print molecules are subsequentlyemoved from the polymer, leaving accessible complementaryinding sites in the polymeric network. Molecularly imprintedolymers (MIPs) have considerable potential for applications in thereas of clinical analysis, medical diagnostics, environmental mon-toring and drug delivery (Allender, 2005). Molecular imprintingechnology allows the creation of synthetic receptors with bind-ng constants comparable to natural receptors. In addition, MIPsre capable of withstanding much harsher conditions such as highemperature, pressure, extreme pH, and organic solvents when

ompared to proteins and nucleic acids. These materials are also lessxpensive to synthesize and can be manufactured in large quanti-ies with good reproducibility (Haupt and Mosbach, 2000).

∗ Corresponding author. Tel.: +91 751 2341848x162; fax: +91 751 2341148.E-mail address: mannanboopathi@yahoo.com (M. Boopathi).

956-5663/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.bios.2009.03.041

and it also showed 10% interference from the structurally similar protein

© 2009 Elsevier B.V. All rights reserved.

Recent reviews summarizes the utility of MIPs in manyfields including protein imprinting (Hansen, 2007; Bossi et al.,2007), biomedical devices (Chaterji et al., 2007), chemical sens-ing (Holthoff and Bright, 2007), solid phase extraction (Baggiani etal., 2007), affinity-based chromatography (Haginaka, 2008), sam-ple treatment (Pichon, 2007), therapeutic monitoring (Hillberget al., 2005) and others. Moreover, MIPs are also used for manypurposes such as adsorption of 17�-estradiol (Wei and Mizaikoff,2007), reflectometric interference spectroscopic sensing of atrazine(Belmont et al., 2007), separation of ephedrine enantiomers(Guerreiro et al., 2008), hanging mercury drop MIP modified elec-trode for differential pulse cathodic stripping voltammetric analysisof creatine (Lakshmi et al., 2007), fructosyl valine recognition(Rajkumar et al., 2007), solid phase extraction of photosynthesis-inhibiting herbicides (Breton et al., 2007), biorecognition solventextraction agents (Castell et al., 2006), recognition of atrazine(Lavignac et al., 2006), fabrication of bilirubin-specific sensor(Huang et al., 2007), adsorption study of dansylated amino acidsby surface plasmon resonance (Li and Husson, 2006), choles-

terol recognition (Boonpangrak et al., 2006), recognition of copper(Baghel et al., 2007) recognition of chemical warfare agent sulfurmustard (Boopathi et al., 2006), recognition of ochratoxin A (Turneret al., 2004) and also in catalysing a trans esterification process(Meng et al., 2004).

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S. Pradhan et al. / Biosensors an

The present work is centered on the imprinting of protein andhe following are some of the earlier reported literature for themprinting of protein such as myoglobin (Lin et al., 2007), albuminBonini et al., 2007), lysozyme (Matsunaga et al., 2007), lysozyme,ibonuclease A and myoglobin (Lin et al., 2006), bovine serum albu-

in (Guo et al., 2006), hemoglobin and bovine serum albuminGuo et al., 2005), microperoxidase, Cytochrome C, lactoperoxi-ase and horseradish peroxidase (Piletsky et al., 2001). The presenttudy utilizes silanes as functional monomer and cross linker forhe imprinting of ricin. Silane based monomers are utilized in thistudy based on their major advantage including the stability ofhe imprints when compared to the imprinted soft-gels which areubjected to swelling. The following are some of the earlier stud-es reported based on silanes for the imprinting of proteins. Therst attempt for protein imprinting reported in literature using sil-

ca materials as monomer and cross linker was revealed by theosbach group (Glad et al., 1985). Subsequently, the imprinting

f proteins onto silicates and polysiloxanes was explored (Ventonnd Gudipati, 1995a,b). The experimental outcomes revealed sil-ca materials as a very promising one for protein imprinting. Innother study, silica-based monomers were subjected to createmprinted polymer for ricin (Lulka et al., 2000). Moreover, template-mprinted nanostructured surfaces for protein recognition (Shi etl., 1999), lyzozome (Pembroke, 2000), protein-imprinted poly-er surfaces for template recognition (Shi and Ratner, 2000), and

rotein-imprinted polysiloxane scaffolds for lyzozome or ribonu-lease A (Lee et al., 2007) were also reported. Another approachdopted for protein imprinting was conjugating silica materialased on template immobilization, where the protein templateas not free in the polymerization mixture but it was covalently

nchored to the supporting silica beads (Shiomi et al., 2005).Ricin is a potent toxin isolated from the seeds of castor bean

lant Ricinus communis and it is also classified as biological warfaregent. The ricin molecule is comprised of two glycoprotein chains

and B of equal size (MW: ca. 62 kDa) that are joined by disul-de bond (Olsnes and Phil, 1973). The B chain binds to galactoseesidues present on various cell surface glycoproteins and glycol-pids in addition to triggering endocytosis of toxin. The A chaineaches cytosol through Golgi complex after the reduction of theisulfide bond. Ricin A chain exhibits an RNA N-glycosidase activityhich hydrolyses a specific adenine residue from a highly con-

erved loop region of 28S rRNA (Endo and Tsurugi, 1987). This RNA-glycosidase activity results in loss of protein elongation and pre-

umably subsequent death of the exposed cell. Ricin endocytosisay also occur through another recognition process which involves

he interaction of mannose containing carbohydrate side-chains ofhe toxin with mannose receptors. Ricin is known to have diverseffects on cells of different organs like liver, kidney, pancreas,ntestines and parathyroid (Sadani et al., 1997). The mechanismsf ricin toxicity may include ribosome inactivation, disturbance

n calcium–magnesium balance, release of cytokines, acute phaseeactions, oxidative stress in the liver and apoptosis (Olsnes, 2004).icin binds to cells, internalises through surface clathrin-coated pits

nto endosomes, and eventually translocates through intracellularembranes into the cytosol, where it irreversibly inactivates ribo-

omes inhibiting protein synthesis (Barbieri et al., 1993). Based onhe above effects, the recognition of ricin is very vital one. Our estab-ishment is working for the defenses against chemical and biological

arfare agents, hence, the present study was undertaken by utiliz-ng the well-known molecular imprinting technology using silaness monomer and cross linker. Silane based monomers advantages

nclude not requiring harsh environment during imprinting process

hich are normally required during imprinting with conventionalrganic monomers; moreover, formation of free radicals of aminocids of protein is avoided by not using any harsh free radical initia-ors here. Hence, the removal of protein template is very easy and

lectronics 25 (2009) 592–598 593

also the three-dimensional conformation of protein is retained inthe MIPs (Ou et al., 2004). To our knowledge, this is the first workin which a Ricin-MIP was synthesized for ricin recognition using 3-aminopropyltriethoxysilane as monomer and tetraethoxysilane ascross linker. In addition, very extensive characterization of Ricin-MIP and NIP by SEM–EDAX was conducted to know the differencebetween the image pattern and elemental composition, respec-tively. BET nitrogen adsorption study was carried out in order toknow the surface area, pore volume and pore width of Ricin-MIPand NIP. Column liquid chromatography studies to know the effectof imprinting on the adsorption of ricin and sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) studies to confirmthe adsorption of ricin on Ricin-MIP.

2. Materials and methods

2.1. Chemicals and reagents

R. communis seeds used for ricin purification were obtained fromthe local market. The following chemicals and reagents are used inthis study after appropriate purifications wherever necessary. 3-Aminopropyltriethoxysilane (99%), tetraethoxysilane (99.9%), acrylamide (99%), bisacryl amide (99%), glycine (99%), lactose (99%) andsodium dodecyl sulfate (99%) are supplied by Sigma–Aldrich, India.Tris free base was obtained from Himedia, India. Acetic acid (100%),methanol (99.9%) and hydrochloric acid (37%) are product of Merck,India. Silica gel (99%) (CDH, India), Coomassie Brilliant Blue (BDH,England), bromophenol blue (99%) and glycerol (99%) (Qualigens,India) and other reagents used in this work were of analytical grade.Millipore water (18 M� cm at 25 ◦C) was used for the preparationof solutions after appropriate pretreatment.

2.2. Instruments

Spectrofluorimeter (Shimadzu RF 5000, Japan), chromatograph(Bio-Rad laboratories, USA, model Biologic HR), gel electrophoresis(Bio-Rad Laboratories, USA, Mini-Protean 3), surface area ana-lyzer (Autosorb 1C-AS1C-RGA2, Quanta Chrome, USA), ESEM–EDX(Quanta400-ESEM with EDAX–FEI, The Netherlands) and Eutechinstruments pH meter (pH-510, Singapore) were utilized in thisstudy.

2.3. Ricin purification

Ricin was purified in the laboratory from R. communis seeds asdescribed earlier after taking appropriate precautionary measures(Kumar et al., 2003). Briefly, defatted castor seed meal was treatedwith 5% acetic acid and crude ricin was extracted. Further, affinityand gel filtration purification of ricin was carried out according to(Griffiths et al., 1995). The crude ricin was loaded on affinity col-umn on acid treated sepharose-4B matrix (0.1 M HCl, 3 h at 50 ◦C)in NaCl solution (0.5 M). The column was washed with PBS for sev-eral hours (till the optical density reached below 0.05) to removethe unbound proteins. The matrix-bound lectins were eluted with(0.5 M) NaCl solution containing [3-d-galactose (0.1 M)]. Fractionswere collected manually, and absorbance was recorded at 280 nm.Protein containing fractions were pooled and concentrated. Sub-sequently, the concentrate was separated on the basis of the sizedifference using bio-gel (A—0.5 m gel) chromatography.

2.4. Synthesis of Ricin-MIP and NIP

MIP for ricin was synthesized by adopting two-step proce-dure based soft silane polymerization technique. The MIP forricin was constructed on the surface of silica particles (diame-ter 60–120 �m) by using a mixture of two organic silanes by

5 d Bioelectronics 25 (2009) 592–598

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dopting an earlier reported method (Glad et al., 1985). Briefly,.4 g of silica (silica gel 60–120 �m) was mixed with 750 �l of 3-minopropyltriethoxysilane and 250 �l tetraethoxysilane at 4 ◦C.emplate protein 250 �l of ricin, 1 mg/ml was added and mixedith the silica/silane slurry and kept at 4 ◦C for 8 h to allow the

mprints to form on the surface of the silica gel. The resultant mix-ure was transferred to a glass filter and thoroughly washed firstith distilled water; second with 10 mM Tris buffer (pH 7.4) con-

aining 0.5 M NaCl and finally again with distilled water in ordero get MIP. The sequential removal of ricin from the polymer was

onitored by using a spectrofluorimeter. NIP was synthesized withut ricin by adopting the same protocols those are used for thereparation of Ricin-MIP.

.5. Characterization of Ricin-MIP and NIP

The mere silica gel and the synthesized Ricin-MIP and NIP wereharacterized very extensively. Spectrofluorimeter was used for theetermination of ricin during its removal from polymer and alsouring the recognition of ricin. Ricin fluorescence measurementsere made at 295 nm (excitation) and 340 nm (emission). Surface

rea of the synthesized Ricin-MIP and NIP was deduced using sur-ace area analyzer in BET nitrogen adsorption mode after degassingnd drying the sample appropriately. The surface morphologicalmage and elemental composition of mere silica gel and Ricin-MIPnd NIP were obtained from SEM and EDAX, respectively.

.6. Ricin recognition studies with Ricin-MIP and NIP

Recognition of ricin by Ricin-MIP was performed by the adsorp-ion of ricin from aqueous solution in batch mode experiments (pH.4 Tris buffer). Ricin-MIP of 50 mg was added into the vial contain-ng ricin 100 �g/mL. Contents of the vial were allowed to equilibrateor 60 min at 25 ◦C. In all experiments, the amount of Ricin-MIP wasept constant as 50 mg/4 mL ricin solution. After the rebinding, con-entration of free ricin in the aqueous phase was measured by usingpectrofluorimeter with appropriate prior sample pretreatments.he response of the Spectrofluorimeter equipment was periodi-ally checked with known standard ricin solution in order to knowts correctness. The adsorption studies were performed five timesnd statistical treatment was also carried out for the obtained data.dsorption values were calculated as the difference between theicin concentration in the solution before and after adsorption. In aimilar way, NIP was also subjected to adsorption studies in ordero know its ricin adsorbing ability and also to find out imprintingfficiency of the Ricin-MIP. The imprinting efficiency was calculatedy adopting an earlier reported method (Boopathi et al., 2006).

.7. Interference study with Ricin-MIP

To know the effect of interference on the Ricin-MIP from othertructurally related protein abrin, adsorption study was carried outy preparing a solution containing 100 �g/mL of abrin which washen treated with 50 mg of the Ricin-MIP in batch mode at 25 ◦C.fter a specified time of adsorption (60 min), concentration of the

emaining abrin in the solution was obtained by utilizing Spec-rofluorimeter.

.8. Chromatographic adsorption of the target protein

1 mg/ml of ricin was added to a chromatographic column com-

acted with Ricin-MIP or NIP. The column was incubated along withhe ricin for 1hr at room temperature and then the column wasashed with phosphate buffer saline. The chromatographic peaksere monitored with UV detection at 280 nm. After getting the firsteak of unbound protein, the column was washed with PBS and

Fig. 1. Stepwise removal of ricin form the polymer using Tris buffer of pH 7.4 asobtained form Spectrofluorimeter.

after obtaining the baseline (lower than 0.05 absorbance unit) thebound ricin was eluted with 0.4 M lactose. The fractions collectedfor each peak were mixed dialysed against PBS and were subjectedto SDS-PAGE analysis, protein was visualized using Coomassie Bril-liant Blue.

3. Results and discussion

3.1. Synthesis of MIP for ricin

MIP was prepared as discussed in the experimental part on sil-ica gel by using 3-aminopropyltriethoxysilane as monomer andtetraethoxysilane as cross linker. MIP for ricin is developed in thestudy based on silane chemistry due to the unique advantages asso-ciated with the silica imprinted polymers such as not requiringharsh environments including high temperature, non requirementof free radical initiating catalysts and also not having the swellingnature upon exposure to water. However, the utilization of conven-tional organic monomers requires harsh radical initiators duringthe synthesis of MIPs. The radical initiator also produces radicalsof amino acids present in the protein and thereby it forms cova-lent bonding with polymer. This leads to a difficulty during proteintemplate removal from the polymer and also it affects the three-dimensional structure of protein (Ou et al., 2004). Hence, silanebased Ricin-MIP was prepared in this study. After the preparationof polymer, ricin was removed from the polymer as discussed in theexperimental part in order to get Ricin-MIP, the removal was mon-itored by Spectrofluorimeter and the result is presented as Fig. 1.In a similar fashion, control non-imprinted polymer (NIP) was alsosynthesized without ricin.

3.2. Characterization of Ricin-MIP and NIP

The mere silical gel, MIP for ricin and NIP were characterizedusing Scanning Electron Microscopy (SEM) in order to know the sur-face morphological image. SEM image for silica gel was obtained inorder to know the polymerization on the silica gel matrix and alsoto compare it with the image of NIP. The SEM images are depictedas Fig. 2a (mere silica gel 40,000 magnification), Fig. 2b (NIP with40,000 magnification) and Fig. 2c (Ricin-MIP with 40,000 magni-

fication). It is inferred that Fig. 2a and b are different from eachother because Fig. 2a is only for silica gel and Fig. 2b is for silanepolymerized silica gel, hence, the morphological change in Fig. 2bis probably due to silane polymer on silica gel. This SEM image dif-ference between Fig. 2a and b is probably due to the formation

S. Pradhan et al. / Biosensors and Bioelectronics 25 (2009) 592–598 595

silica gel, (b) NIP and (c) Ricin-MIP.

otoMoNposiieRSt

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Fig. 2. SEM image for (a) mere

f silane polymer on the silica gel surface as only polymeriza-ion was performed on silica gel. In addition, in the SEM imagerdered nanopatterns (approximately 15 nm particle size) in Ricin-IP (Fig. 2c) are observed and this may be due to the imprinting

f ricin on the silica gel-polymer matrix. However, SEM image ofIP (Fig. 2b) is also exhibiting nanopatterns (approximately 35 nmarticle size) are not similar to which are observed in the imagef Ricin-MIP and this is probably due to the absence of imprintedites on the silica gel-polymer matrix. The SEM image of Ricin-MIPs entirely different from that of NIP; the differences between themages are probably due to the imprinting of ricin. Moreover, annhanced surface area was also observed in the SEM image of theicin-MIP than NIP. Thus, the ordered nanopatterns observed inEM image of Ricin-MIP are probably due to the ricin imprinting onhe polymer (Boopathi et al., 2006).

Nitrogen BET surface area characterization study was performedith the powder of Ricin-MIP and NIP to know the effect of imprint-

ng on their surface area, pore volume and pore width. Ricin-MIPxhibited more surface area (227 m2/g) when compared to NIP143 m2/g). BET surface area result indicates an enhancement inurface area for Ricin-MIP due to imprinting of ricin. Pore width

axima obtained for Ricin-MIP was 6.2 nm and NIP was 1.9 nm, this

esult also again confirms the nanopatterns those are observed inEM image of Ricin-MIP are due to the ricin imprinting. Pore volumef the Ricin-MIP and NIP was found to be 0.5010 and 0.2828 cm3/g,espectively at 12 nm pore diameter as shown in Fig. 3. It is inferred Fig. 3. Nitrogen adsorption pore size distribution curve for (a) NIP and (b) Ricin-MIP.

596 S. Pradhan et al. / Biosensors and Bioelectronics 25 (2009) 592–598

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matrix and this is in corroboration with fluorescence measurementstudy. A small amount of sodium is present in the EDAX spectrumof Ricin-MIP and NIP. This may be due to the adsorbed sodium ionson the polymer matrix which is a contribution from the buffer thatwas used for the ricin removal.

Table 1bElemental composition obtained for NIP from EDAX.

Element Weight % Atomic %

Carbon K 16.93 23.62Nitrogen K 05.92 07.08Oxygen K 51.28 53.71

Fig. 4. SEM-EDAX spectrum for (a)

rom pore volume value that more pore volume is exhibited byicin-MIP than that of NIP at 12 nm and it is well known that poreiameter of ricin is 12 nm from atomic force microscopic studiesSavvateev et al., 2003) and XRD studies (Montfort et al., 1987).his result confirms that the excess pore volume exhibited by Ricin-IP at 12 nm is due to the imprinting of Ricin on the silica gel

olymer matrix and also this is in corroboration with earlier studySavvateev et al., 2003).

Energy Dispersive X-Ray Analysis (EDAX) was performed tonow the elemental composition of Ricin-MIP, NIP and mere silicael. EDAX spectra are given in Fig. 4a for mere silica gel, Fig. 4b forIP and Fig. 4c for Ricin-MIP. The elemental compositions obtained

rom EDAX studies are presented as Table 1a (mere silica gel),able 1b (NIP) and Table 1c (Ricin-MIP). In Fig. 4a and Table 1a,nly carbon, silicon and oxygen (100%) are there for mere silica gel,he presence of carbon in silica gel is probably from the impurity in

ilica gel or from the adhesive tape used for holding the sample.owever, in Fig. 4b and Table 1b, carbon, nitrogen, oxygen, sili-

on and sodium (100%) are there. From Fig. 4b and Table 1b, onean understand the presence of nitrogen due to the availability ofmino silane polymer on the silica gel surface as aminosilane was

able 1alemental composition obtained for silica gel from EDAX.

lement Weight % Atomic %

arbon K 12.08 17.47xygen K 60.27 65.43ilicon K 27.64 17.09

otal 100.0% 100.0%

silica gel, (b) NIP and (c) Ricin-MIP.

used as monomer. In addition, one can infer the absence of sulfurin Fig. 4c and Table 1c are probably due to the complete removalof ricin from the polymer. The absence of sulfur in EDAX spectrum(Fig. 4c) confirms the complete removal of ricin from the polymer

Sodium K 01.26 00.92Silicon K 24.61 14.68

Total 100.0% 100.0%

Table 1cElemental composition obtained for Ricin-MIP from EDAX.

Element Weight % Atomic %

Carbon K 21.18 30.14Nitrogen K 06.98 08.52Oxygen K 37.84 40.43Sodium K 01.59 01.18Silicon K 32.40 19.72

Total 100.0% 100.0%

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ig. 5. Chromatogram obtained with (a) NIP and (b) Ricin-MIP for ricin rebindingtudies. Inset figure is SDS-PAGE for Ricin.

.3. Recognition of ricin by Ricin-MIP

Ricin recognition by Ricin-MIP was performed as indicatedn the experimental part. Ricin-MIP adsorbed 9.7 �g/mL (n = 5,.S.D. = 2.5%) of ricin from 100 �g/mL and NIP adsorbed 5.5 �g/mln = 5, R.S.D. = 2.9%) of ricin. Ricin-MIP adsorbed more ricin than NIPnd this observation is due to the imprinting of ricin in MIP. Themprinting efficiency (˛) of Ricin-MIP was calculated as reportedarlier and it was found to be 1.76. This ˛ value is comparable withn earlier study that was imprinted for lysozyme (Ou et al., 2004). Inrder to see the interference from structurally related other proteinith the synthesized Ricin-MIP, experiments were carried out as

iscussed in the experimental part and observed 10% interferencerom abrin.

To investigate the adsorption or recognition ability of Ricin-IP, the imprinted and non-imprinted polymer were individually

acked in the column. Ricin was loaded in the column and sub-equently performed chromatographic experiments as indicatedn the experimental part. As shown in Fig. 5a, only one peak wasbserved in the chromatogram for NIP, however, two peaks arebserved in the chromatogram in Fig. 5b when Ricin-MIP was useds column material. The difference in chromatograms in Fig. 5a andreveals that the only peak observed in Fig. 5a represents the non-

dsorbed ricin, however, the two peaks in Fig. 5b are due to the freeon-adsorbed ricin (first peak) and the specifically adsorbed ricin

second peak) that was eluted with elution buffer 0.4 M lactose. Inddition, with the NIP packed column no peak was observed withlution buffer 0.4 M lactose and this result confirms that there is noound ricin in NIP packed column. The fractions collected during

lectronics 25 (2009) 592–598 597

the chromatographic experiments at respective peak were concen-trated and subjected to SDS-PAGE study and confirmed as ricin.The images also depicted in the inset of Fig. 5a and b for NIP andRicin-MIP, respectively. This result again confirms the presence ofricin imprinted sites on the Ricin-MIP. Now work is progressing inour establishment by utilizing the synthesized Ricin-MIP as columnmaterial for the purification of ricin and also pre-concentration ofricin from dilute samples.

4. Conclusions

We have synthesized a Ricin-MIP on silica gel matrix for therecognition of ricin without using any free radical initiator. The MIPand NIP are characterized for their surface area, pore volume, porewidth and elemental composition. SEM image of the synthesizedRicin-MIP exhibited nanopatterns. In addition, Ricin-MIP showedmore surface area due to the imprinting when compared to non-imprinted control polymer. The pore width of the Ricin-MIP was innanometer size and the Ricin-MIP rebound more amount of ricinthan control polymer. This study brings a way for the recognition,pre-concentration and separation of ricin and other biological war-fare agents from various matrices.

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