2004 62 24 2447 2450 ACTA CHIMICA SINICA Vol 62 2004 No 24 2447 2450 a b Ξ a a Ξ ( a 310028) ( b 314001) 32 012 110 110 500 ng ml - 1 01060 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 310028) ( b College of Biological and Chemical Engineering Jiaxing College Jiaxing 314001) 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 012 110 and 110 500 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 : tzp @zju edu cn ; Tel : 0571288273689 ; Fax : 0571288273690 Received May 25 2004 ; revised and accepted August 24 2004 (Nos 29975024 20275034) (No 2003C21024)
2448 Vol 62 2004 50 L ( 1 1000) 2 h 011 mol L - 1 Tris2HCl2 0105 % Tween 20 1 % (BSA) 24 h [10] ( ELISA) ( RIA) RIA 1 2 3 BSA 50 ELISA L 50 L ( 1 2000) 37 10 min 011 mol L - 1 Tris2HCl2 0105 % Tween 20 1 2 4 011 ml 110 10-2 mol L - 1 015 ml 410 10-3 mol L - 1 015 ml 011 mol L - 1 ph = 712 37 30 min Telfon ELISA (DPV) 50 mv s - 1 1 1 1 PPMS29 ( Quantum Design ) ; J EM22010 ( HR) ( NEC ) ; S2570 2 1 ( ) ;CHI2440 ( CHI ) ; E2 200 ( ) ; G2560E ; 1126 emu g - 1 011 % ; Teflon ; [15] HBsAb ( 512 mg ml - 1 ) ; HBsAb HRP ( 417 mg ml - 1 ) ; 90 100 HBsAg ( 5 mg ml - 1 ) ; ; nm HBsAg ( 015 ng ml - 1 0208 ; 1 ng ml - 1 0207 ; 2 ng ml - 1 0207 ; 5 ng ml - 1 0301) 212 232(32 ) 510 10-3 mg ml - 1 ( EDC) N2 (NHS) Sigma 1 2 1 2 1 2 h [ 11] Fe 3 O 4 8 10 nm 3 ml 7 g 60 ml 15 ml 2 h ELISA 24 h 10 min 20 ml 2 ml 018 : - 0150 0 V H 2 SO 4 30 % H 2 O 2 (3 1) [14] 2 110 10-3 mol L - 1 EDC NHS 1 h 1 h ml 3 g 75 10 h [16] 2 h [12] 2 2 1 2 2 2 2 1 20 L 20 g L - 1 110 10-3 mol L - 1 EDC [13] NHS 500 L 15 min ph 712
No 24 : 2449 ph 4 5 - ph 6 7 8 ph 9 10 ph 7 ph 712 32 [17] 32 2 2 3 2 h 30 min ( 1) 30 min 32 ph 712 50 L 32-01270 V - 01320 V 2 2 4 10 min ELISA 30 min 2 2 5 50 L 50 L ( 3) 012 110 110 500 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 - 1 32 2 : 2 32 ng ml - 1 Figure 2 Formation and oxidation of 22hydroxy232aminophenoxazine Figure 3 Differential pulse voltammograms of different antigen concentrations 2 2 2 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
2450 Vol 62 2004 y ( A) = 21474 x ( ng ml - 1 ) + 011313 r = 019908 y ( A) = 0106218 x (ng ml - 1 ) 2 Wang J ; Liu G ; Merkoci A Anal Chim Acta 2003 + 31323 r = 019961 3 482 149 0106 ng ml - 1 10 ng ml - 1 6 3 Partington K M ; Jenkinson E J ; Anderson G J 4138 4145 4104 Immunol Methods 1999 223 195 4137 4110 4132 A RSD = 319 % 4 Porter J ; Pickup R W J Microbiol Methods 1998 33 221 5 Yu L S L ; Uknalis J ; Tu S2I J Immunol Methods 2 2 6 HBsAg 2001 256 11 ( HBsAg) 6 Gehring A G ; Brewster J D ; Irwin P L ; Tu S2I ; e ( HBeAg) ( HBcAg) Houten L J V J Electroanal Chem 1999 469 27 HBsAg 7 Yazdankhah S P ; Hellemann A2L ; Ronningen K ; Olsen E Vet Microbiol 1998 62 17 HBsAg 8 Nibbeling H A M ; Van Etten L ; Fillie Y E ; Deelder HBsAg A M Acta Trop 1997 66 85 HBsAg HBsAg 9 Yu H ; Raymonda J W ; McMahon T M ; Campagnari HBsAg A A Biosens Bioelectron 2000 14 829 HBsAg 012 015 110 ng ml - 1 10 Chen Z2Z ; Wang K2M ; Yang X2H ; Huang S2S ; 690 1587 2521 na Huang H2M ; Li D ; Wang Q Acta Chim Sinica 2003 HBsAg 669 61 137 (in Chinese) 1547 2493 na ( HBsAg 2 2 7 11 Qiu G2M ; Sun Z2H Chin J Chem Reagent 1993 15 (4) 234 (in Chinese) 1 100 1 500 ( 1993 15 (4) 234 ) HBsAg 019 ng ml - 1 0121 ng ml - 1 12 Hong X2P ; Peng T2Z Chin J Anal Chem 2003 31 (7) 789 (in Chinese) ELISA 1 100 HBsAg 018 ng ml - 1 ( 2003 31 (7) 789 ) 1 500 13 Prioult G ; Turcotte C ; Labarre L ; Lacroix C ; Fliss I Int Dairy J 2000 10 627 ELISA 14 Yang Q2J ; Xu D2K ; Jiang X2P ; He W ; Liu Z2 3 2002 30 (1) 56 ) HBsAg 15 He X2X ; Wang K2M ; Tan W2H ; Liu B ; Li D ; ELISA Huang S2S Chem J Chin Univ 2003 24 (1) 40 (in Chinese) Richardson D J Trends Anal Chem 2000 19 (9) 530 17 Zhang S2S ; Chen H2Y ; Jiao K Sci China Ser B 1999 29 (1) 83 (in Chinese) ( (B ) 1999 29 (1) References 1 Sieben S ; Bergemann C ; Lubbe A ; Brockmann B ; Rescheleit D J Magn Magn Mater 2001 225 175 2003 61 137 ) H ; Wang Y ; Ma L2R Chin J Anal Chem 2002 30 (1) 56 (in Chinese) ( ( 2003 24 (1) 40 ) 16 Ferretti S ; Paynter S ; Russell D A ; Sapaford K E ; 83 ) (A0405257 CHENG B ; DONG H Z )
Graphical Abstract Vol 62 2004 Preparation and Characterization of MnOOH Nanorods in Reverse Micelles DONG Xi2Yan ; ZHANG Xing2Tang ; CHENG Gang ; LI Yun2Cai ; DU Zu2Liang Acta Chimica Sinica 2004 62 (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 2004 62 (24) 2444 Detection of Hepatitis B Surface Antigen Using Immunovoltammetry with Nano2Magnetic Microsphere CHENG Qiong ; PENG Tu2Zhi ; LIU Ai2Li Acta Chimica Sinica 2004 62 (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