Novel Concepts and Approach of Immunoassay and Cellular Analysis

19 12 2007 12 PROGRESS IN CHEMISTRY Vol. 19 No. 12 Dec., 2007 3 1 2 3 3 (1. 210009 ; 2., 210093), 3,, 2 : O657 ; Q503 : A : 10052281X(2007) 1221852209 Novel Concepts and Approach of Immunoassay and Cellular Analysis Yan Feng 1 Ju Huangxian 2 3 3 (1. Jiangsu Cancer Hospital, Nanjing 210009, China ; 2. Key Laboratory of Analytical Chemistry for Life Science, Ministry of Education, Nanjing University, Nanjing 210093, 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 1 1959 Yalow Berson 11 1798 Edward Jenner, 70 : 2007 8 3 (No. 20325518) (No. 20521503) (No. 20535010) (BS2006006, BS2006074) 3 3 e2mail :hxju @nju. edu. cn

12 1853 80 2,,,,,,,,, ( 2 500) gπml ngπml [3 ] 3 [4 ] [5, ],,, 3, 2 [1 ], 2,,, ( ) 311 10 7 [2 ], CEA [6 ] 2 2,

1854 19, 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 5194 0140 s - 1 [16 ], HRP CA 1929 HRP H 2 O 2, CEA hcg, CA 1929 015 120 ngπml 2168 UΠml CA 125 015 50 miuπml, 014 ngπml 013 miuπml CA 125 314 CA 125, CA 125 30 UΠml 1173 UΠml [8 ] SiO 2, (hcg),, hcg, HRP (AFP) 0126 114 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 125 210 75 UΠml 118 UΠml 313 1 (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 ]

12 1855 315 CA 1929 CA 125, HRP2CA 1929 HRP2CA 125, CA 1929 CA 125 [27 ], 012 014 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 ], 210 75 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,,

1856 19 [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, 510 100 UΠml 110 120 ngπml AFP, 35 min 510 100 ngπml 217 ngπml 4. 4 413 ( ),,,, 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 ]

12 1857,, MTT 2 2 2 2 = 4, 2 3 = 6, 2 4 = 8,3 3 = 9, UltraBind TM + 01830 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 199 2 HRP CEA K562Π ALP HRP 4 K562ΠADM P2 ( 5) [41 ] 37 min ( 4) [36 ] [39 ] K562 [40 ] 6 510 10 4 110 10 7 cellπml 110 5 10 4 cellπml, [42 ],, 2 110 10-12 510 10-11 molπl 218 10-13 molπl (0114 pgπml), 1175 ( 0114) 10 5,, 417 ( 0153) 10 10 LΠmol, 7 [37 ] AsPC21,, 2 R ct,, [38 ] [43 47 ] ph K562 N, N2 2 2 K562 52Flu 5 K562 C,K562 [43 ]

1858 19 [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 510 10 4 110 10 7 cellsπml [44 ] 2, 6 2 K562, 1134 10 4 1134 10 8 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, 510 10 3 510 10 7 cellsπml [47, ], 110 10 3 cellsπml CNF2CS K562, K562

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