Potentiometric Diphtheria Immunosensor Based on a Glassy Carbon Electrode Modified with Colloidal Gold and Anti2Diph

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1 , ACTA CHIMICA SINICA Vol 62, 2004 No 20, ΞΞ ( ) (AET) ( GCE), (NG), (Cys) ( GA) (anti2diph), (Diph), ng ml - 1, 8 9 mv/ decade, , 5 2 ng ml - 1,,,,, Potentiometric Diphtheria Immunosensor Based on a Glassy Carbon Electrode Modified with Colloidal Gold and Anti2Diph TANG, Dian2Ping YUAN, Ruo Ξ CHAI, Ya2Qin LIU, Yan ZHONG, Xia DAI, Jian2Yuan ( College of Chemistry and Chemical Engineering, Southwest China Normal University, Chongqing ) Abstract A novel potentiometric immunosensor for the detection of diphtheria antigen (Diph) has been developed by means of self2assembly to covalently immobilized diphtheria antibody (anti2diph) on a glassy carbon electrode At first, the modification of glassy carbon electrode ( GCE) with nano2gold (NG) by chemical adsorption of 22aminoethane thoil (AET) was achieved, and then cysteine (Cys) molecules were adsorbed on the nano2gold surface Finally, diphtheria antibodies were covalently immobilized on the electrode by glutaraldehyde ( GA) The modification procedure was electrochemically monitored by characterization of the electrode surface The performance and influencing factors of the resulting immunosensor were studied in detail The resulting immunosensor exhibited fast potentiometric response to Diph and the detection limit of the immunosensor was from 24 ng ml - 1 to 600 ng ml - 1 with correlation coefficient of and a detection limit of 5 2 ng ml - 1 More than 91 % of the results of the bioproduced samples obtained by this method were in agreement with those by enzyme2linked immunosorbent assay ( ELISA) Keywords diphtheria, glassy carbon electrode, colloidal gold, potentiometric immunosensor (Diphtheria),,, ( ELISA) [1 ( RIA),2],,, ; [] [4], -,,, [5 7], Ξ E2mail : edu cn ; Tel : ; Fax : Received December 2, 200 ; revised May 10, 2004 ; accepted June 24, 2004 (No ) (No )

2 No 20 : 206, Sigma, USA), (AET, Sigma, USA), (Cys, 1 BRANSONIC 200 ( BRANSON ULTRASCHALL ) ), ( GA,25 %), (PVB),, (PBS, ph 7 0) :NaCl 8 0 g, Na HPO g, KH 2 PO g, KCl 0 2 g, 1000 ml MP 20 ( Metter Toledo ),phs24c ( ),IM 6e ( 1 2 ZAHNER Elektrik ),CHI 660A ( NG/ AET/ GCE CHI ), AB2042S ( Metter Toledo ), Scheme 1 ( 16 nm) [8 ] GCE( 2 mm) 10 % HNO 2 5 % K 2 CrO V 1 min, GC ( anti2diph) ( 800 g ( OH) ( COOH),, 1 % ml - 1, Diph) ( ), AET, h,, (HBsAg, ), (BSA, 16 nm 6 h, NG/ AET/ GCE 1 Scheme 1 Peparation process of the immunoelectrode on the surface of GCE electrode

3 2064 Vol 62, NG/ AET/ GCE 10 mmol L - 1 Cys, h, Cys, Cys ; 5 % GA h, 2, PBS ph 7 0 anti2diph,4,, ; 0 25 % BSA 7 1 h,,,, 4 ( Scheme 1) 1 1 1,,, ph 7 0 PBS V mol L - 1 K [ Fe (CN) 6 ]/ K 4 [ Fe2 1 2 (CN) 6 ] mol L - 1 KCl + PBS (ph 7 0), PBS (ph 7 0), Figure 1 Cyclic voltammograms of substrate at different electrodes ( E 0 ),,, ( E 1 ), E = E 1 - E 0 with 0 ng, 600 ng ml - 1 Diph in mol 2 (a) bare GCE; (b) AET/ GCE; (c) Cys/ NG/ AET/ GCE; (d) anti2diph/ GA/ Cys/ NG/ AET/ GCE; ( e ) anti2diph/ GA/ Cys/ NG/ AET/ GCE treated ml - 1 Diph ; (f) the same electrode as (d) but treated with the 0 1 mol L - 1 KCl + PBS (ph 7 0) solution L - 1 K [ Fe (CN) 6 ]/ K 4 [ Fe (CN) 6 ] +, Fe ( CN) , mol L K [ Fe (CN) 6 ]/ K 4 [ Fe ( CN) 6 ] mol L - 1 KCl ng ml - 1 Diph, mol L - 1 PBS (ph 7 0), V min, min, CV 1, 1 a, GCE Fe2, E, 4 - / (CN) - 6, E p,a = 0 27 V, E p,c = Diph 0 14 V, i p,a / i p,c = 1 GC AET, Cys/ NG, anti2diph/ GA, ( 1 b d), 1 Fe (CN) - 6 GC, Table 1 Potentiometric response time of the immunosensor in negative GC, GC and positive serum PBS solution (ph 7 0), i d < i c < i b < i a 0 Response time/ min ng ml min, Negative serum/ mv 1 e, anti2diph Diph Positive serum/ mv ,, Fe ( CN) 6, 2, Fe ( CN) 6,, Diph, Diph 17, 1 7 mv, ( :600 ng ml - 1 ),, ( 5 1 mv), Fe (CN) - 6, (, 1 f), mv s - 1 Diph ph 7 0, K [ Fe (CN) 6 ],

4 No 20 : 2065,,, 4 S, ng ml - 1 mv, %,, E = ( 2) log[diph], r = ( 2) 2 5, 40 ng ml - 1,, HBsAg ( HCG, ),,, ( ) 2 1 Figure 2 Standard curve of potentiometric response to the Aizawa [9] log[diph] concentrations, ( Donnan ),,,, 5 1 mv 5 2 ng ( ), ml - 1, 5 2 ng ml - 1, Warburg ( ),, 5 1 mv, ;, ( ) 5 1 mv PBS,, ph 7 0, ; 2 4, ph 7 0,,,, RSD <,, 5 %,, 4, 5d, a 2 Diph Table 2 Potentiometric response results (mv) of different immunosensors in the sample containing Diph Immunosensor number E + SD RSD/ % First day Fifth day Ninetieth day a potentiometric response obtained in the presence of 28 6 ng ml - 1 Diph at ph 7 0 PBS Table Potentiometric response (mv) of the immunosensor in 40 ng ml - 1 diphtheria antigen containing different concentrations of interfering agent in ph 7 0 PBS solution Interfering agents C interfering agent / (ng ml - 1 ) E + SD RSD/ % HBsAg HCG

5 2066 Vol 62, Table 4 Potentiometric response of different methods on different Diph concentrations Sample number GCE2modified electrode ELISA ELISA 91 % [ ( ) / 80 ], Diph References HBsAg K [ Fe2 (CN) 6 ]/ K 4 [ Fe (CN) 6 ] Nyquist Figure AC impedance plots of immunosensor at different concentrations of diphtheria antigen (a) 0 ; ( b) 8 6 ng ml - 1 ; (c) 48 ng ml - 1 in ph 7 0 phosphate buffer solution containing 0 1 mol L - 1 K [ Fe (CN) 6 ]/ K 4 [ Fe (CN) 6 ] at frequency Hz at 20 ( Z vs Z at 220 mv vs SCE) 1 Sanchez, F G ; Diaz, A N ; Diaz, A F G Anal Chim Acta 1999, 78, Tao, Y2X ; Zhang, G2S ; Sun, Y ( Eds ) Determination of Clinical Immunology, Shanghai Science and Technology Press, Shanghai, 1986, pp 9 16 (in Chinese) (,,,,,, 1986, pp 9 16 ) Dong, S2Y ; Chen, G2Z ; Wang, H2R ; Gao, Y2Z ; Lu, M 2X ; Tian, B2Y Electrochemistry 2002, 8 ( 1 ), 66 ( in Chinese) (,,,,,,, 2002, 8 (1), 66 ), 4 Liang, R2P ; Cai, P2X ; Qiu, J 2D ; Zou, X2Y ; Xu, J 2, J ; Mo, J 2Y Chem J Chin Univ 200, 2, 79 (in Chinese) (,,,,,, 2, 200, 2, 79 ) ph 7 0 PBS 80 ( 5 Wang, J ; Tian, B ; Rogers, K R Anal Chem 1998, 70, ), ( 1682 E > 5 1 mv, E 5 1 mv ) 6 Purvis, D ; Leonardova, O ; Farmakovsky, D ; Cherkasov, V ( ELISA, ) Biosens Bioelectron 200, 11, 185, 4 7 Blackburn, G F ; Talley, D B ; Booth, P M ; Durfor, C ELISA 5, 48 N ; Martin, M T ; Napper, A D Anal Chem 1990, 62,, 5, 90 6 % (48/ 5) ; ELISA 27, 25, 2, Frens, G Nature 197, 241, % (25/ 27), 9 Aizawa, M Kagaku ( Kyoto) 1981, 6, 5 (A SHEN, H )