Novel Concepts and Approach of Immunoassay and Cellular Analysis

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1 PROGRESS IN CHEMISTRY Vol. 19 No. 12 Dec., ( ; 2., ), 3,, 2 : O657 ; Q503 : A : X(2007) Novel Concepts and Approach of Immunoassay and Cellular Analysis Yan Feng 1 Ju Huangxian (1. Jiangsu Cancer Hospital, Nanjing , China ; 2. Key Laboratory of Analytical Chemistry for Life Science, Ministry of Education, Nanjing University, Nanjing , China) Abstract The development of novel concepts and approach for clinic detection by combining analytical chemistry with clinic diagnosis has become one of research frontiers for human health. In this filed, immunoassay, especially the development of multianalyte immunoassay methods and cellular detection have attracted considerable attention. review focuses on the research progress in these areas in recent three years. The new concepts in immunoassay, including separation2free, quasi2reagentless and reagentless amperometric immunoassay, multichannel amperometric immunochips based on separated immunoreaction areas, flow2through multianalyte chemiluminescent immunosensing systems based on substrate zone resolution, channel resolution and channel2substrate zone two2dimensional resolution, new approach in cytosensing for electrochemically detecting cell concentration and amount on electrode surface, monitoring interfacial adhesion, proliferation and apoptosis of cells, studying exogenous effect and viability of living cells, and anti2tumor drug sensitivity test, are simply summarized. their applications in clinic diagnosis of cancer disease are also discussed. Key words immunoassay ; molecular diagnosis ; cytosensing ; cells ; tumor marker This Yalow Berson Edward Jenner, 70 : (No ) (No ) (No ) (BS , BS ) 3 3 e2mail edu. cn

2 ,,,,,,,,, ( 2 500) gπml ngπml [3 ] 3 [4 ] [5, ],,, 3, 2 [1 ], 2,,, ( ) [2 ], CEA [6 ] 2 2,

3 , HRP H 2 O 2, CA 125 [14 ] hcg [15 ], HRP hcg, HRP, 013 miuπml hcg [7 ] CA 1929, SiO 2 2, CA 1929 HRP CEA [16 ] hcg [17 ],HRP CA 1929 HRP s - 1 [16 ], HRP CA 1929 HRP H 2 O 2, CEA hcg, CA ngπml 2168 UΠml CA miuπml, 014 ngπml 013 miuπml CA CA 125, CA UΠml 1173 UΠml [8 ] SiO 2, (hcg),, hcg, HRP (AFP) miuπml [9,10 ] 312 AFP HRP2 HRP H 2 O 2 AFP HRP, H 2 O 2 HRP H 2 O 2 [7 10 ] 0174 ngπml [18 ], CEA,,, H 2 O 2, HRP CEA HRP2CEA 2H 2 O 2 CEA 0122 ngπml CEA,, 1 [19 ], CEA [11 ] [12 ] CA 125,, CA UΠml 118 UΠml (A), (B) HRP [19 ] H 2 O 2 Fig. 1 Schematic diagrams of ( A) three2electrode SPCE [13 ] TiO 2 system and (B) flow system. (a) nylon sheet, (b) silver ink, HRP 2 (c) graphite auxiliary electrode,(d) graphite working electrode, HRP (e) AgΠAgCl reference electrode, (f) insulating dielectric. P : peristaltic pump, V : eight2port rotary injection valve, EFC : electrochemical flow2through cell [19 ]

4 CA 1929 CA 125, HRP2CA 1929 HRP2CA 125, CA 1929 CA 125 [27 ], UΠml 4 [20 25 ] CEA,AFP,, CA 125 hcg 4, HRP, [20 22 ] 4 ( 2) [28 ], Kong 4 [24 ] 2 [25 ], [ Fe (CN) 6 ] 32Π42,,, ( ) TM 100 % ( ),,, TM [29 ],, [26 ] 411,, [30 32 ] [33 ] [34 ] UltraBind TM 2 (A), (B), [28 ] Fig.2 Schematic diagrams of (A) immunosensors array and, ( B ) automated multianalyte electrochemical immunoassay [30 ], ngπml system. (a) nylon sheet, (b) silver ink, (c) graphite auxiliary electrode, ( d) AgΠAgCl reference electrode, ( e ) graphite [32 ] working electrode, ( f ) insulating dielectric. P : peristaltic, pump, V : injection valve [28 ] 5 min 25 min, [35,36 ] 412 HRP,,

5 [35 30 min CA 1929 ], CA 125 CEA CA 1929 [33 ], 110 ngπml UltraBind TM, ; [34 ] AFP HRP2CA 125 (ALP)2CEA AFP, HRP ALP HRP,, ( 3) [35 ] CA 125 CEA, 2 2, UΠml ngπml AFP, 35 min ngπml 217 ngπml ( ),,,, 2 2, 415 2, [36 ] 3 [35 ] Fig. 3 Scheme of the flow2through multianalyte immunosensing system based on substrate zone2resolved technique. ( a) flow device, (b) flow2through analysis system and reactions occurred in the flow cell, ( c) substrate zones in the flow path. S: sample, RB : regeneration buffer, WB : wash buffer, S1 : HRP substrate, S2 : ALP substrate, V : multiposition valve, P : peristaltic pump, D : detector, W: waste [35 ] 4 2 Fig. 4 Scheme of the channel and substrate zone two2 dimensional resolution system for multiplex immunoassay of tumor markers panel. (a) flow cell, (b) flow2through system, (c) transect of flow cell for immunoassay, ( d) optical shutter and ( e ) near2simultaneous multianalyte immunoassay procedure. S1 : ALP substrate, S2 : HRP substrate, WB : wash buffer, RB : regeneration buffer, S: mixture of sample and tracer antibodies [36 ]

6 ,, MTT = 4, 2 3 = 6, 2 4 = 8,3 3 = 9, UltraBind TM V CA 125 CA 153 CA 199 CEA ALP CA 125 CA 199 HRP CA 153 CEA ALP CA 125 HRP CA 153 ALP CA HRP CEA K562Π ALP HRP 4 K562ΠADM P2 ( 5) [41 ] 37 min ( 4) [36 ] [39 ] K562 [40 ] cellπml cellπml, [42 ],, molπl molπl (0114 pgπml), 1175 ( 0114) 10 5,, 417 ( 0153) LΠmol, 7 [37 ] AsPC21,, 2 R ct,, [38 ] [43 47 ] ph K562 N, N2 2 2 K562 52Flu 5 K562 C,K562 [43 ]

7 [41 ] 5 P2 DPV AFM AP Fig. 5 Detection of P2glycoprotein on cell surface by electrochemical enzyme2linked immunoassay, DPV curves of 12NP at AP2p2gp2K562Π ADM2Au2chitosanΠGCE with different cell concentrations in ph 714 PBS, AFM image of K562ΠADM2Au2chitosan, and effect of concentration of AP conjugated antibody in incubation solution on DPV peak currents of K562ΠADM2 and K5622Au2chitosanΠGCE [41 ] MTT(32(4,52 )22,52 ) YDH2560, P2gp (p170) p170 K562A P2gp K562A, K562A cellsπml [44 ] 2, 6 2 K562, cellsπml [45 ] [46 ], Fig. 6 Formation of CNF2CS film and impedance sensor for CNF2CS, K562 cells, cyclic voltammograms of [ Fe (CN) 6 ] 3 - in 110 K562 molπl KCl at different electrodes and the Nyquist diagrams of, 6 [46 ] CNF Faradaic impedance spectra as well as calibration curve [46 ] K562, cellsπml [47, ], cellsπml CNF2CS K562, K562

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