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Biochemical Engineering Journal 47 (2009) 132–135

Contents lists available at ScienceDirect

Biochemical Engineering Journal

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novel proteinaceous photolinker for simultaneous binding to an inert surfacend a biomolecule

aroj Kumar, Dileep Kumar Kannoujia, Azmi Naqvi, Pradip Nahar ∗

nstitute of Genomics and Integrative Biology (CSIR), Mall Road, Delhi 110007, India

r t i c l e i n f o

rticle history:eceived 18 March 2009eceived in revised form 9 June 2009ccepted 10 July 2009

a b s t r a c t

A simple and versatile method is developed for covalently binding a protein ligand onto a matrixirrespective of functional groups either on the ligand or the matrix. Prerequisite of the method isa novel proteinaceous photolinker having multiple light-activable functional groups. We have madephotoreactive-BSA – a proteinaceous photolinker by the reaction of bovine serum albumin (BSA) withexcess of 1-fluoro-2-nitro-4-azidobenzene (FNAB). When an enzyme is placed on an inert polystyrene

eywords:hotoimmobilizationnzyme-Fluoro-2-nitro-4-azidobenzenehotolinkerovalent immobilization

matrix in presence of photoreactive-BSA and exposed to light the later forms highly reactive nitrenes someof which bind to the matrix and the rest to the ligand resulting simultaneous formation of covalent bondswith the matrix and the enzyme. The method is further exemplified by performing ELISA by covalentbinding of antigen or antibody on a polystyrene microtiter plate in just 30 min using photoreactive-BSA.ELISA carried out in less than 3 h using photoreactive-BSA showed comparable results with that of con-ventional ELISA carried out in 18 h. Thus the method is potentially useful for rapid ELISA or covalent

onto

nzyme-linked immunosorbent assay immobilization of ligands

. Introduction

In recent years, covalent immobilization of a biomolecule onsolid matrix has become the subject of intense research as it is

apid, stable and efficient. Covalent method of immobilization isow gaining attention even in diagnostics involving ELISA [1–5].

t leads to analytical advances in that the signal to noise ratioan be increased by thorough washing of the microplates afterinding. Among covalent methods, photolinker-mediated immobi-

ization of biomolecule is fast becoming popular due to its simple,on-invasive and mild procedure [2–9]. Photolinker can be hetero-ifunctional having a photoreactive and a thermoreactive groups.hotoreactive group yields highly activated species (nitrene, car-ene or oxygen radical) on exposure to light and binds to anyolecule irrespective of its functional group. Thermoreactive group

f the photolinker on the other hand binds to a molecule hav-ng an active functional group. However, in all these cases prior

o covalent immobilization a matrix or a ligand has to be acti-ated to get active functional group to facilitate covalent linking.rotein-based photolinker is a new class of photolinker having sev-ral photoactivable groups, some of which binds to the biomolecule

Abbreviations: FNAB, 1-Fluoro-2-nitro-4-azidobenzene; HRP, Horseradish per-xidase; BSA, Bovine serum albumin; HELISA, Heat-mediated enzyme-linkedmmunosorbent assay.∗ Corresponding author. Tel.: +91 11 27667439; fax: +91 11 27667471.

E-mail address: pnahar@igib.res.in (P. Nahar).

369-703X/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.bej.2009.07.009

an inert surface without prior activation.© 2009 Elsevier B.V. All rights reserved.

and some to the matrix when exposed to light. Advantage of usinga protein molecule as a base includes a large number of functionalgroups available in a single molecule for formation of covalent link-ages to incorporate several molecules of a photolinker. Sigrist andco-workers has immobilized biomolecules on inert surfaces in pres-ence of light using such type of linker prepared by the reaction ofBSA and 3-(trifluoro methyl)-3-(m-isothiocyanophenyl) diazirine[10–12]. However, this method is not popularized due to tediousand time consuming multistep procedure for preparation of suchlinking agent. Hence, there is a need to prepare photolinker polymerby a cheap and simple method.

Here, we report a novel proteinaceous photolinker preparedby a simple reaction of BSA and FNAB both of which are easilyavailable. Photoreactive-BSA is used for simultaneous binding ofbiomolecule and the matrix irrespective of functional groups eitheron the matrix or the biomolecules.

2. Materials and methods

2.1. Materials

Horseradish peroxidase (HRP), goat anti-human IgG, human

IgG, anti-human IgG-peroxidase, bovine serum albumin and o-phenylenediamine dihydrochloride were purchased from Sigma,USA. 96-Well polystyrene (PS) microtiter plates were purchasedfrom Greiner, Germany. All other chemicals were of analytical gradeand were purchased from SRL, India. Photoirradiation was carried

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ut at a wavelength of 365 nm in an UV Stratalinker, model-2400Stratagene, USA). 1-Fluoro-2-nitro-4-azidobenzene was preparedy published procedure [7]. Phosphate buffered saline (PBS) wasrepared by adding 0.85% NaCl to phosphate buffer (0.01 M, pH.2). Washing buffer was prepared by adding 0.1% Tween-20 toBS (0.01 M, pH 7.2). Absorbance values were expressed as theeans ± S.D. for three determinations.

.2. Optimization of FNAB concentration and reaction time forreparation of photoreactive-BSA

Six conical flasks, each containing a solution of 128 mg of BSAn 600 �l of H2O were heated at 55 ◦C after adding 4, 8, 16, 32, 64nd 128 mg of FNAB respectively. After 1 h photoreactive-BSA wasrecipitated by 20 ml of ethanol. Supernatant was removed afterentrifugation and the precipitate was washed thrice by 10 ml of2H5OH. Washings were removed after centrifugation and decanta-ion. Photoreactive-BSA was dried and kept in a dark bottle at 4 ◦C.ontrol experiment was carried out without FNAB. Reaction timeas optimized by doing the reaction of BSA (128 mg/600 �l of H2O)

nd FNAB (64 mg) at 55 ◦C for 35, 45, 60, 90, and 120 min, respec-ively. Efficacy of photoreactive-BSA was checked by immobilizingRP into wells of a PS microtiter plate by adding photoreactive-BSAnd exposing to UV light for 40 min. Immobilized HRP was assayedy 100 �l of substrate-dye buffer (5 mg of o-phenylenediamineihydrochloride and 5 �l of 30% H2O2 in 12 ml of 0.2 M cit-ate buffer, pH 5). After 5 min color development was stoppedy addition of 20 �l of 5% H2SO4. Absorbance was recorded at90 nm.

.3. Optimization of UV irradiation time forhotoreactive-BSA-mediated immobilization of HRP onto an inerturface

HRP solution (1 �g/100 �l of PBS/well) was taken into wells ofS microtiter plates and photoreactive-BSA (5 mg/well) was addedo it. The plates were irradiated by UV light for 10, 20, 30, 40,nd 50 min, respectively, followed by washing with washing buffer.mmobilized HRP was assayed as described in section 2.2. For con-rol experiment reaction was carried out in dark.

.4. Photoreactive-BSA-mediated enzyme-linked immunosorbentssay (ELISA)

Photoreactive-BSA (50 mg) and anti-human IgG (250 �l of5 �g/ml) were added to 1 ml of PBS. From this solution 100 �las poured into a well of a microtiter plate and exposed to UV

ight for 30 min. The well was then thoroughly washed with wash-ng buffer. The well was blocked with 2% of bovine serum albumin200 �l/well) by incubating at 40 ◦C for 40 min. After washing,uman IgG (0.25 �g/100 �l of PBS) was loaded into the well and

ncubated for 45 min at 50 ◦C. Goat anti-human IgG-horse radisheroxidase (100 �l of 1:5000 dilution in PBS) was loaded intohe well and incubated for 45 min at 50 ◦C. Well was washed andssayed after adding substrate-dye buffer.

Conventional ELISA procedure was performed by immobilizingoat anti-human IgG for 11 h (over-night) at 4 ◦C, blocking for 1 h at7 ◦C, human IgG binding for 3 h at 37 ◦C and conjugate binding forh at 37 ◦C on PS surface. Experiment was carried out in triplicateells. Rabbit IgG was used as negative control sera.

.5. Inhibition study to determine the specificity of antibodies byhotoreactive-BSA-mediated ELISA

The solution of goat anti-human IgG (1 �g/well/100 �l of PBS)as added with 5 mg of photoreactive-BSA into wells of a PS

ing Journal 47 (2009) 132–135 133

microtiter plate and exposed to UV light for 30 min. After thestipulated time the wells were washed with washing buffer. Thegoat anti-human IgG immobilized solid phase was blocked by BSAat 40 ◦C in 40 min followed by washing with washing buffer. Adouble-dilution series of goat anti-human IgG (0.2–0 �g/100 �lof PBS) was taken in separate centrifuge tube to which an equalamount of human IgG (0.5 �g) was added and incubated for 1 hat 37 ◦C. From these solutions 100 �l was added into the solidphase and incubated for 45 min at 50 ◦C. After washing, remainingsteps of ELISA was carried out as described in Section 2.4. Simi-larly, conventional ELISA was also carried out but without usingphotoreactive-BSA.

3. Results and discussion

Photoreactive-BSA – a protein-based photolinker was preparedby introducing several FNAB molecules in a single BSA molecule.The reaction proceeded by the replacement of fluoro groups ofFNAB by the amino groups of BSA as schematically represented inFig. 1. When the photoreactive-BSA having several (at least two)photoreactive groups was exposed to UV light together with a lig-and and an inert surface, photoreactive azido groups of the reagentformed highly reactive nitrenes some of which got attached tothe surface and the rest to the ligand in an insertion reaction. UVlight of 365 nm was used which was non-invasive to biomolecules(Fig. 2).

The photoreactive-BSA was prepared by the reaction of BSA andFNAB in an aqueous solution at 55–60 ◦C for 60 min in an incubatorshaker. At this temperature insoluble FNAB melted and the reac-tion proceeded smoothly. Concentration of FNAB was important forpreparation of photoreactive-BSA. Attachment of more FNAB madethe BSA hydrophobic and became partly insoluble in water whichwas not desirable. However, when attachment of FNAB to BSA wasrestricted by taking BSA and FNAB at the ratio of 2:1 (w/w), watersoluble photoreactive-BSA was obtained. Such photoreactive-BSAgave maximum immobilization of HRP onto an inert PS surface(Fig. 3). The optimized time for preparation of photoreactive-BSA at55 ◦C was 90 min beyond which no further improvement of activityof photoreactive-BSA was found (data not shown).

UV exposure time was important for single step binding ofbiomolecule on an inert surface. Optimum time for generationof nitrene and its further reaction to biomolecule and the poly-mer surface was 30 min as seen from Fig. 4. We have found thatthe absorbance value of immobilization of HRP carried out usingphotoreactive-BSA in the presence of light was several folds greaterthan the immobilization carried out in the dark. This was expectedas there was no photo reaction in dark, hence no covalent bindingof protein onto the PS plate took place (Fig. 4).

Earlier we performed ELISA rapidly on an activated surface withbetter sensitivity [2]. We have also shown that ELISA could be car-ried out at elevated temperature using an activated surface. Suchheat-mediated ELISA (HELISA) is more sensitive and reduces theELISA timing to 3 h from conventional 15–20 h [3–5]. Minimumprerequisite of HELISA is covalent bonding of antigen or anti-body to solid surface. This report demonstrates the usefulness ofphotoreactive-BSA as an excellent means for covalent ELISA pro-cedure without requiring activation of either the surface or thebiomolecule. Thus, anti-human IgG was covalently bound to a wellof a PS microtiter plate when exposed to UV light for 30 min inpresence of photoreactive-BSA. Blocking, human IgG binding andanti-human IgG peroxidase binding were carried out at 40 ◦C for

40 min, at 50 ◦C for 45 min and at 50 ◦C for 45 min as described inthe HELISA procedure [3]. Further, the absorbance value obtained inthe present procedure carried out in less than 3 h was similar to con-ventional ELISA carried out in 18 h (Fig. 5). Similar results obtainedfrom both the procedures suggest that photoreactive-BSA mediated

134 S. Kumar et al. / Biochemical Engineering Journal 47 (2009) 132–135

Fig. 1. Schematic representation of the preparation of photoreactive-BSA.

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ig. 2. Schematic representation of immobilization of ligand onto solid support byxposed to UV light together with a ligand (i) and an inert surface (iii). Photoreactittached to the surface and the rest to the ligand in an insertion reaction and thus i

LISA did not alter the specificity of the assay. This was further con-rmed by doing an inhibition study with the goat anti-human IgG.s shown in Fig. 6, the reaction was completely inhibited in bothonventional and the present method of ELISA in the presence of

ig. 3. Optimization of FNAB concentration for the preparation of photoreactive-SA. Using photoreactive-BSA, HRP was immobilized into wells of a PS microtiterlate by UV light of 365 nm. Absorbance values were obtained after assaying thenzyme.

reactive-BSA. The photoreactive-BSA (ii) having several photoreactive groups, wasdo groups of the photoreactive-BSA forms highly reactive nitrenes, some of whichilizing ligand (i) to the matrix (iii) forming immobilized biomolecule (iv).

excess free goat anti-human IgG. However, with decreasing amount

of free goat anti-human IgG, the inhibition was reduced in boththe cases in a similar fashion. This clearly demonstrates that theimmobilization did not modify the antigenic determinants duringimmobilization.

Fig. 4. Optimization of reaction time for immobilization of HRP into PS wells usingphotoreactive-BSA. The reaction was carried out under UV light (�) and in dark (�).Immobilized HRP was assayed colorimetrically.

S. Kumar et al. / Biochemical Engineer

Fig. 5. Comparison between conventional ELISA (A) and photoreactive-BSA-mediated ELISA (B). Dark column represents the human IgG and hollow columnrepresents the rabbit IgG used as negative control sera.

Fig. 6. Inhibition study of goat anti-human IgG by conventional (�) andphotoreactive-BSA mediated (�) ELISA procedure.

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ing Journal 47 (2009) 132–135 135

4. Conclusion

Photoreactive-BSA – a protein-based photolinker could be usedfor single step immobilization of any biomolecule to any surfaceirrespective of their functional groups. The only prerequisite is thatthe matrix or the molecule must have carbon–hydrogen bonds.Hence, the method does not demand any prior activation of eithera matrix or a biomolecule.

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