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Gold Nanoparticles for Colorimetric detection of
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N R Tiwari et al Advances in Bioscience and Biotechnology 1 2010 322 329 323. of enzyme Penicillin G acylase Acylases are a group of was obtained from Thomas Baker India and Molychem. enzymes that catalyze the cleavage of carbon nitrogen India respectively Penicillin G pen G was obtained. bonds in amides Penicillin acylases are members of the from Hindustan Antibiotics Limited Pune India En. N terminal nucleophile Ntn hydrolase superfamily zyme penicillin G acylase PGA was purified from the. which share a common fold around the active site and a gram positive bacteria Arthrobacter viscosus ATCC. catalytic residue in the N terminal position 24 25 15294 The enzyme was produced extracellularly in the. These enzymes are used mainly in the pharmaceutical culture broth The cells were separated by centrifugation. industry for the production of 6 Aminopenicillinic acid and the clear broth was used for further purification by. 6 APA a key intermediate in the production of semi hydrophoric chromatography octyl sepharose followed. synthetic penicillins and cephalosporins which are now by anion exchange chromatography Q sepharose Pu. far the most widely used antibiotics In addition these rity of the enzyme was checked by native as well as. enzymes are very useful as biocatalysts in some impor SDS PAGE not shown for the brevity The concentra. tant reactions like peptide synthesis 26 27 and also in tion of enzyme used was 0 7 mg ml so in 10 l of solu. the resolution of clinically active compounds 28 29 tion 7 g of enzyme is present. Penicillin acylases specifically catalyse hydrolysis of. 2 2 CTAB Capped Gold Nanoparticles, Penicillins For each type of penicillin there exists an. enzyme which performs this hydrolysis reaction Peni CTAB capped gold nanoparticles were synthesized by. cillin G acylase PGA is a specific enzyme for an im reducing aqueous solution of 5 10 4 M HAuCl4 10 ml. portant drug Penicillin G Pen G The hydrolysis reac containing 0 2 M CTAB with 1 10 2 M ice cold NaBH4. tion of penicillins by penicillin acylases leads to break 0 6 ml The solution was stirred for 3 hours and then. ing of its molecules into different byproducts thereby kept at room temperature for 3 days 10 l of enzyme. leading to consumption of penicillins All the enzymatic solution was added to 2 ml of gold nanoparticles solu. assays quantify consumption of the respective penicillins tion followed by addition of 100 l of substrate pen G. or detect the formation of byproducts over time Enzy 100 mg ml The solution was kept at 40 deg for 10 min. matic activity which is the measure of amount of active Absorption spectra were recorded after each step. enzymes in the solution is then determined using such. different kinds of assays The earliest procedures to as 2 3 PGA Enzyme Assay. say penicillin acylase activity are tedious and time con The enzyme activity of PGA was determined by meas. suming 7 30 33 Some of these include paper chroma uring the amount of aminopenicillic acid 6 APA pro. tography 6 gas chromatography 34 assays using duced in a reaction mixture containing 10 mg ml 1 peni. 6 nitro 3 phenylacetamido benzoic acid NIPAB 35 cillin G in 0 1 M phosphate buffer pH 7 0 when incu. titrimetric determination using pH stat 8 etc Methods bated at 40 C 36 The 6 APA produced was estimated. stated above either require expert operators or expensive spectrophotometrically at 415 nm after reaction with. instruments or reagents e g NIPAB p dimethylaminobenzaldehyde PDAB according to 7. Our method consists of detecting 6 Aminopenicillin modified by 37 One unit of PGA activity is defined as. acid which is a byproduct in hydrolysis of pen G and in the amount of enzyme required to produce 1 mole of. turn assaying PGA without any chromogenic reagent 6 APA per minute under the assay conditions 40 C and. There is a distinct color change observable in the gold pH 7 0. nanoparticle solution with naked eyes from initial red to. final blue when the pen G hydrolysis reaction is per 2 4 Characterization. formed by PGA in the presence of gold nanoparticles Absorption spectra of the samples were recorded in so. Although various enzymes have also been detected by lution form on Perkin Elmer lambda 950 instruments in. using nanoparticles as probes there are no reports of the range 200 to 800 nm TEM images are acquired on. detection of Penicillin acylases using nanoparticles We Philips CM200 instrument with an accelerating voltage. indeed use various sophisticated techniques like TEM of 120 KV For TEM analysis samples were drop coated. UV VIS spectroscopy in order to investigate the bio. on copper grids and were allowed to dry for 45 minutes. sensing mechanism using gold nanoparticles However. once this test is acceptable in practice only colorimetric 3 RESULTS AND DISCUSSION. changes can be used to detect the presence of PGA, CTAB capped nanoparticles have rarely been investi. 2 EXPERIMENT gated for biosensing purposes due to the fact that CTAB. bilayers are not easily displaced by biomolecules and. 2 1 Reagents hence are difficult to handle as far as bioconjugation is. Chloroauric acid trihydrate HAuCl4 3H2O and CTAB concerned On the other hand CTAB capped nanoparti. Copyright 2010 SciRes ABB, 324 N R Tiwari et al Advances in Bioscience and Biotechnology 1 2010 322 329. cles are chemically more stable and would have a longer completely The experiment was also performed with. shelf life In the present report we could observe the re different concentration of enzymes Figure 1 b The. action between enzyme penicillin G acylase PGA and red shift was found to gradually increase on increasing. pen G in the solution of CTAB capped gold nanoparti the concentration of enzyme from 0 007 mg ml to 0 7. cles mg ml The concentration of pen G in this case was. Plasmon resonance band of CTAB capped gold same as in Figure 1 a i e 100 mg ml The spectral shifts. nanoparticles was observed at 527 nm Gold nanoparti are thus sensitive for enzyme concentration as low as. cles synthesized here are covered with a bilayer of 0 007 mg ml Figure 1 c shows gradual shifts in the. CTAB 38 Inner layer is bound to the gold surface via plasmon band as the amount of pen G is increased from. the headgroup and is connected to the outer layer 10 to 100 l 1 to 10 mg of pen G The extinction spec. through the hydrophobic interactions while the trum started changing after an addition of 10 l of pen G. headgroups of the outer layer are in the aqueous medium and on addition of 100 l pen G it red shifted by 18 nm. Headgroup of the CTAB chain consists of an amine The total red shift obtained after adding 100 l of pen G. group Hence protonated amine group is present on the varied in the range of 17 to 23 nm on repeating the ex. outermost surface of CTAB capped gold nanoparticles periment several times Also the spectrum was recorded. No change in the absorption peak position of the gold four hours after the addition of pen G Figure 1 d The. was observed on addition of enzyme PGA 10 l 0 7 plasmon band was found to be gradually shifted towards. mg ml However on addition of 100 l of pen G 100 the longer wavelength 572 nm and decreased in inten. mg ml the plasmon band shifted from 527 nm to 545 sity The gradual shift in the absorption band is attributed. nm Figure 1 a After the addition the solution was to slow removal of remaining CTAB layers from gold. kept at 40 C for 10 min to allow the reaction to occur nanoparticles After the complete displacement of CTAB. Figure 1 a changes in spectra on addition of different amounts of Pen G when the enzyme conc is 0 69. mg ml b Changes in spectra after addition of different conc of enzyme when the Pen G concentration is 100. mg ml c Shift in extinction maximum of Au nanoparticles Vs amount of Pen G 100 mg ml and d Spectra. acquired after 4 hour of addition of Pen G Inset shows change in the color of the Au nanoparticle solution 1. before and 2 after the enzyme pen G addition, Copyright 2010 SciRes ABB. N R Tiwari et al Advances in Bioscience and Biotechnology 1 2010 322 329 325. the particles precipitate at the bottom of the bottle leav. ing clear supernatant Peak at 322 nm is attributed to the. absorption due to the penicillin G Broadening of plas. mon band along with decrease in the extinction intensity. was also observed, The spectral shifts in the surface plasmon band are.
accompanied by changes in the color of gold nanoparti. cle solution from red to blue Figure 1 d inset Color. of the solution started changing from red to dark pink. after an addition of 10 l of the pen G However on the. addition of 25 l Pen G the color was purple The purple. color intensified on further addition of pen G The figure. shows the change in color after adding 100 l of pen G. The prominent shift in the plasmon band of gold, nanoparticles can be attributed to the aggregation of. nanoparticles Figure 2 shows TEM images of gold, nanoparticles before and after the addition of the enzyme. and the pen G Size of gold nanoparticles was found to. be 15 nm Particles are well dispersed before the addi. tion of enzyme and pen G On addition of the enzyme. and pen G to gold nanoparticle solution aggregation of. particles is observed TEM image red shift in the plas. mon band and the color change in the gold nanoparticle. solution together indicate aggregation of gold nanoparti b. Figure 2 TEM images of a gold, Reaction occurring between the enzyme and the pen G nanoparticles and b gold nanoparticles. is schematically shown in Scheme 1 PGA specifically enzyme substrate. catalyses the hydrolysis of the amide bonds in pen G. Cleavage of amide bond in pen G by PGA is accompa immediately however the plasmon band shifted from. nied by the formation of 6 aminopenicillanic acid 527 to 565 nm after keeping the solution for two hours. 6 APA and phenylacetic acid PAA Addition of mixture of 6 APA and PAA same concen. In order to confirm the mechanism of aggregation of trations as mentioned above to gold nanoparticle solu. gold nanoparticles due to enzyme pen G reaction we tion caused the plasmon band to shift from 527 to 545. added just the products of this reaction to the solutions nm immediately and to 600 nm finally after three hours. 6 APA PAA and mixture of 6 APA and PAA were added This was also accompanied by broadening of the plas. separately to the nanoparticle solutions in the absence of mon band This trend is very much similar to that ob. enzyme and pen G and spectral changes were recorded tained in the presence of enzyme and substrate pen G. Figure 3 Interestingly the color changes were ob see Figure 1 The spectral and colorimetric changes. tained in the presence of 6 APA only PAA 100 l were obtained immediately when both acids were to. 2 5mM alone was not able to induce the color changes gether added to the gold nanoparticle solution which is. Figure 3 b On addition of 6 APA 100 l 2 5 mM also happening when the reaction is done in the presence. no change in the spectral peak position was obtained of enzyme and the pen G This indicates that the gold. nanoparticles are sensing hydrolysis of the pen G Hence. the combination of acids produced during the hydrolysis. and the amount of 6 APA produced should be the key. factor in determining the spectral and colorimetric. changes produced in the nanoparticle solution due to the. enzyme substrate chemistry This is further supported by. the fact that the extent of red shift increases when we. increase the concentration of enzyme PGA Figure 1 b. This is due to the increase in the amount of 6 APA pro. Scheme 1 Penicillin G Acylase catalysed hydrolysis of Peni duced. cillins The mechanism of sensing hydrolysis reaction can be. Copyright 2010 SciRes ABB, 326 N R Tiwari et al Advances in Bioscience and Biotechnology 1 2010 322 329. red shift in the plasmon band when compared to the iso. lated gold nanoparticles This phenomenon is attributed. to the coupling between the dipole modes of plasmons of. different particles As the interparticle distance is de. creased more red shift in the plasmon band is observed. due to an increase in the extent of coupling A decrease. in the interparticle distance after the completion of en. zyme pen G reaction is clearly observed in the TEM. images Figure 2 Along with the coupling of plasmon. modes contribution to the red shifting of plasmon band. also comes from increased scattering of nanoparticle. aggregates in the longer wavelength region 25, a Some of the previously reported work 1 have used.
thiol containing Cys functionalized gold nanoparticles. and an assembly directing actuator to control the assem. bly of nanoparticles through pi stacking interactions. Upon hydrolysis by enzymes disassembly was observed. due to increased repulsion between NH3 groups and, removal of the hydrophobic interactions between the. actuator This disassembly caused blue shift in the ex. tinction spectrum of gold nanoparticles It is noteworthy. that we have not used any assembly directing ligand to. control the assembly of gold nanoparticles Also in our. case the cleavage of pen G is actually leading to a red. shift in the plasmon resonance band This indicates that. the products formed by the enzyme pen G reaction de. crease the electrostatic repulsion between particles. causing their aggregation, In order to check the effect in the absence of enzyme. Figure 3 a UV Vis spectra of gold nanoparticles PGA UV Vis spectrum of gold nanoparticles was ac. Gold Nanoparticles for Colorimetric detection of hydrolysis of antibiotics by penicillin G acylase Neha R Tiwari1 sis of an antibiotic penicillin G pen G and subse quent enzyme detection using gold nanoparticles is presented Gold nanoparticles capped with Cetyl trimethyl ammonium bromide CTAB are synthe sized using chemical route The particles could be used for detection of

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