Detection of Hepatitis B Surface Antigen Using Immunovoltammetry with Nano2Magnetic Microsphere

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1 ACTA CHIMICA SINICA Vol No a b Ξ a a Ξ ( a ) ( b ) ng ml ng ml - 1 Detection of Hepatitis B Surface Antigen Using Immunovoltammetry with Nano2Magnetic Microsphere CHENG Qiong a b PENG Tu2Zhi Ξ a LIU Ai2Li a ( a Department of Chemistry Xixi Campus Zhejiang University Hangzhou ) ( b College of Biological and Chemical Engineering Jiaxing College Jiaxing ) Abstract A novel method for detecting hepatitis B surface antigen ( HBsAg) using nano2magnetic polymer carboxyl microsphere was reported The surface of the nano2magnetic polymer carboxyl microsphere covalently bound with hepatitis B surface antibody ( HBsAb) and then captured HBsAg and hepatitis B secondary antibody labeled with horseradish peroxide enzyme The microsphere with sandwich complex was separated magnetically from sample solution and the enzyme labeled at the complex catalyzed the reaction of 22aminohydroxybenzene and hydrogen peroxide in the buffer solution The electroactive product 22hydroxy232aminophenoxazine was detected using differential pulse voltammetry The peak current was linear with the concentration of HBsAg in the range of and ng ml - 1 respectively The detection limit was found to be 0106 ng ml - 1 The method has been used to detect the concentration of hepatitis B virus in human serum and the sensitivity was much higher than that of enzyme2linked immunosorbent assay ( ELISA) used in diagnostic analysis Keywords nano2magnetic functional microsphere hepatitis B surface antigen immunoassay electrochemical detection ( OH COOH CHO NH 2 ) [1] [2 5] [6 9] Ξ E2mail : edu cn ; Tel : ; Fax : Received May ; revised and accepted August (Nos ) (No 2003C21024)

2 2448 Vol L ( ) 2 h 011 mol L - 1 Tris2HCl % Tween 20 1 % (BSA) 24 h [10] ( ELISA) ( RIA) RIA BSA 50 ELISA L 50 L ( ) min 011 mol L - 1 Tris2HCl % Tween ml mol L ml mol L ml 011 mol L - 1 ph = min Telfon ELISA (DPV) 50 mv s PPMS29 ( Quantum Design ) ; J EM22010 ( HR) ( NEC ) ; S ( ) ;CHI2440 ( CHI ) ; E2 200 ( ) ; G2560E ; 1126 emu g % ; Teflon ; [15] HBsAb ( 512 mg ml - 1 ) ; HBsAb HRP ( 417 mg ml - 1 ) ; HBsAg ( 5 mg ml - 1 ) ; ; nm HBsAg ( 015 ng ml ; 1 ng ml ; 2 ng ml ; 5 ng ml ) (32 ) mg ml - 1 ( EDC) N2 (NHS) Sigma h [ 11] Fe 3 O nm 3 ml 7 g 60 ml 15 ml 2 h ELISA 24 h 10 min 20 ml 2 ml 018 : V H 2 SO 4 30 % H 2 O 2 (3 1) [14] mol L - 1 EDC NHS 1 h 1 h ml 3 g h [16] 2 h [12] L 20 g L mol L - 1 EDC [13] NHS 500 L 15 min ph 712

3 No 24 : 2449 ph ph ph 9 10 ph 7 ph [17] h 30 min ( 1) 30 min 32 ph L V V min ELISA 30 min L 50 L ( 3) ng ml - 1 ( ) 1 :a 0 ; b 80 ng ml - 1 Figure 1 Cyclic voltammograms of electroactive catalyzed substrate Concentration of antigen : a 0 ; b 80 ng ml : 2 32 ng ml - 1 Figure 2 Formation and oxidation of 22hydroxy232aminophenoxazine Figure 3 Differential pulse voltammograms of different antigen concentrations ph ph ph 3 : a 0 ; b 0 2 ; c 0 5 ; d 1 0 ; e 10 ; f 30 ; g 80 Concentration of the antigen : a 0 ; b 0 2 ; c 0 5 ; d 1 0 ; e 10 ; f 30 ; g 80 ng ml - 1

4 2450 Vol y ( A) = x ( ng ml - 1 ) r = y ( A) = x (ng ml - 1 ) 2 Wang J ; Liu G ; Merkoci A Anal Chim Acta r = ng ml ng ml Partington K M ; Jenkinson E J ; Anderson G J Immunol Methods A RSD = 319 % 4 Porter J ; Pickup R W J Microbiol Methods Yu L S L ; Uknalis J ; Tu S2I J Immunol Methods HBsAg ( HBsAg) 6 Gehring A G ; Brewster J D ; Irwin P L ; Tu S2I ; e ( HBeAg) ( HBcAg) Houten L J V J Electroanal Chem HBsAg 7 Yazdankhah S P ; Hellemann A2L ; Ronningen K ; Olsen E Vet Microbiol HBsAg 8 Nibbeling H A M ; Van Etten L ; Fillie Y E ; Deelder HBsAg A M Acta Trop HBsAg HBsAg 9 Yu H ; Raymonda J W ; McMahon T M ; Campagnari HBsAg A A Biosens Bioelectron HBsAg ng ml Chen Z2Z ; Wang K2M ; Yang X2H ; Huang S2S ; na Huang H2M ; Li D ; Wang Q Acta Chim Sinica 2003 HBsAg (in Chinese) na ( HBsAg Qiu G2M ; Sun Z2H Chin J Chem Reagent (4) 234 (in Chinese) ( (4) 234 ) HBsAg 019 ng ml ng ml Hong X2P ; Peng T2Z Chin J Anal Chem (7) 789 (in Chinese) ELISA HBsAg 018 ng ml - 1 ( (7) 789 ) Prioult G ; Turcotte C ; Labarre L ; Lacroix C ; Fliss I Int Dairy J ELISA 14 Yang Q2J ; Xu D2K ; Jiang X2P ; He W ; Liu Z (1) 56 ) HBsAg 15 He X2X ; Wang K2M ; Tan W2H ; Liu B ; Li D ; ELISA Huang S2S Chem J Chin Univ (1) 40 (in Chinese) Richardson D J Trends Anal Chem (9) Zhang S2S ; Chen H2Y ; Jiao K Sci China Ser B (1) 83 (in Chinese) ( (B ) (1) References 1 Sieben S ; Bergemann C ; Lubbe A ; Brockmann B ; Rescheleit D J Magn Magn Mater ) H ; Wang Y ; Ma L2R Chin J Anal Chem (1) 56 (in Chinese) ( ( (1) 40 ) 16 Ferretti S ; Paynter S ; Russell D A ; Sapaford K E ; 83 ) (A CHENG B ; DONG H Z )

5 Graphical Abstract Vol Preparation and Characterization of MnOOH Nanorods in Reverse Micelles DONG Xi2Yan ; ZHANG Xing2Tang ; CHENG Gang ; LI Yun2Cai ; DU Zu2Liang Acta Chimica Sinica (24) 2441 MnOOH nanorods were synthesized in quaternary water/ oil ( W/ O) microemulsion solution containing CTAB n2butanol heptane and water which were about 10 nm in diameter and more than 200 nm in length Figure ( a ) is transmission electron microscopy ( TEM) micrographs Figure ( b) is high resolution transmission electron microscopy ( HRTEM) image and electron diffraction analysis ( ED) patterns Results showed that the obtained products were monoclinic manganese oxyhydroxide (MnOOH) A QM2MM Study on DNA Cleavage by Seryl2 Histidine Dipeptide ZHONG Ru2Gang ; ZHAO Li2Jiao ; ZHAO Yu2 Fen Acta Chimica Sinica (24) 2444 Detection of Hepatitis B Surface Antigen Using Immunovoltammetry with Nano2Magnetic Microsphere CHENG Qiong ; PENG Tu2Zhi ; LIU Ai2Li Acta Chimica Sinica (24) 2447 The results show that seryl2histidine dipeptide can combine with the trinucleotide by the interaction of three hydrogen2bonds but histidyl2serine combines with the trinucleotide by single hydrogen bonds only hydrolysis mechanism Differential pulse voltammograms of the electroactive substrate with different HBsAg concentrations : a 0 ; b 0 2 ; c 0 5 ; d 1 0 ; e 10 ; f 30 ; g 80 ng ml - 1 This binding mode suggests a possible phosphodiester bond A novel method for detecting hepatitis B surface antigen ( HBsAg ) using nano2 magnetic polymer carboxyl microsphere was reported The surface of the nano2magnetic polymer carboxyl microsphere covalently bound with hepatitis B surface antibody (HBsAb) and then captured HBsAg and hepatitis B secondary antibody labeled with horseradish peroxide ( HRP ) The microsphere with sandwich complex was separated magnetically from sample solution and the HRP labeled at the complex catalyzed the reaction of 22 aminohydroxybenzene and hydrogen peroxide in the buffer solution The electroactive product 22hydroxy232 aminophenoxazine was detected using differential pulse voltammetry Executive editor PAN Bing2Feng ; computer typesetter DONG Hua2Zhen