[雙語翻譯]--醫(yī)學(xué)外文翻譯--fc-cho標(biāo)記免疫球蛋白igg構(gòu)建的芯片流動(dòng)免疫分析的電化學(xué)探針（原文）

1、Electrochemical Probe for On-Chip Type Flow Immunoassay: Immunoglobulin G Labeled With FerrocenecarboaldehydeMina Okochi, Hiroko Ohta, Tsuyoshi Tanaka, Tadashi MatsunagaDepartment of Biotechnology and Life Sciences, Toky

2、o University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan; telephone: 81-42-388-7020; fax: 81-42-385-7713; e-mail: tmatsuna @cc.tuat.ac.jpReceived 2 June 2004; accepted 13 August 2004Pu

3、blished online 25 February 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20313Abstract: Labeling of ferrocenecarboaldehyde (Fc-CHO) to immunoglobulin G (IgG) via formation of Schiff-base and i

4、ts reduction was investigated for construction of an electro- chemical probe for miniaturized amperometric flow immu- noassay. Approximately eight molecules of Fc-CHO were labeled to IgG and the reversible redox property

5、 of ferrocene was observed. Labeling efficiency improved by over three times as compared to the conventional method using ferro- cenemonocarboxylic acid (Fc-COOH). Also, binding affinity of IgG labeled with Fc-CHO to its

6、 antigen, IgE, was in- vestigated by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance assay. IgG labeled with Fc-CHO that retained eight ferrocene moiety showed sufficient binding affinity to its a

7、ntigen and the current re- sponse obtained in the flow electrochemical detection sys- tem increased by 14-fold as compared with IgG labeled with Fc-COOH when applying the potential of 390 mV vs. Ag/AgCl. The minimum dete

8、ctable concentration of IgG la- beled with Fc-CHO was 0.06 AM. IgG labeled with Fc-CHO demonstrate biochemical and electrochemical properties that are useful for electrochemical immunosensors. B 2005 Wiley Periodicals, I

9、nc. Keywords: electrochemical probes; on-chip; immunoas- say; ferroceneINTRODUCTIONImmunoassay is one of the most important methods used in clinical diagnoses, environmental analyses, and biochem- ical studies. Immunosen

10、sors can be categorized as optical, electrochemical, and microgravimetric assay, based on the detection principle applied (Luppa et al., 2001). Chemilumi- nescent or fluorescent detection with enzyme-linked immu- nosorbe

11、nt assay (ELISA) has been the most practically used; however, it requires precise detection devices for miniatur- ized systems with a small sample volume. The electrochem- ical detection method is suitable for sensor min

12、iaturization and automated detection, since it is highly sensitive, lowcost, low power requirements, and has high compatibility with advanced micromachining technologies. Developments in miniaturization of chemical and b

13、io- technological processes have a significant impact on all aspects of diagnostic testing. Miniaturized immunosensors, which combine the analytical power of microfluidic devices with the high specificity of antibody-ant

14、igen interactions, have been intensively developed (Bernard et al., 2001; Kojima et al., 2003; Lim et al., 2002, 2003; Saleh and Sohn, 2003; Sato et al., 2002; Wang et al., 1998, 2002; Wang and Jin, 2003). Ferrocene deri

15、vatives have often been used as electrochemical signaling probes for immunoassay (Lim et al., 2002, 2003; Padeste et al., 2000; Wang et al., 2002) as well as the DNA hybridization assay (Fan et al., 2003; Kim et al., 200

16、3; Long et al., 2003; Takenaka et al., 1994, 2000, 2003; Wang et al., 2003). Labeling of ferrocene derivatives to enzymes such as glucose oxidase has been intensively studied and used as mediators in biosensors (Degani a

17、nd Heller, 1987, 1988, 1989; Gleria et al., 1986; Suzawa et al., 1994). Also, electroactive label of IgG with ferrocene- monocarboxylic acid (Fc-COOH) by chemical crosslinkers, sulfo-N-hydroxysulfosuccinimide (NHS) and 1

18、-ethyl-3-[3- dimethylaminopropyl]-carbodiimide hydrochloride (EDC), has been commonly used (Lim et al., 2002, 2003). Only two to three ferrocene moiety has been stably introduced to IgG and its binding affinity was not w

19、ell characterized. Therefore, a new labeling method is required for intro- ducing a higher number of ferrocene moiety to IgG for sensitive detection. In the present study, IgG was labeled with ferrocene- carboaldehyde (F

20、c-CHO) for sensitive detection. The elec- trochemical property of IgG labeled with Fc-CHO was investigated and the number of bound ferrocene moiety on IgG was estimated by atomic absorption spectroscopy. Also, binding af

21、finity of IgG labeled with Fc-CHO to its antigen was characterized. Using Fc-CHO, it was possible to obtain a higher electrochemical signal due to a higher number of labeled ferrocene moiety on IgG.B 2005 Wiley Periodica

22、ls, Inc.Correspondence to: Tadashi Matsunaga Contract grant sponsor: NEDO Contract grant number: 30027jected to quench the unreacted sites. HBS-EP buffer (10 mM HEPES, 150 mM NaCl, 3.4 M EDTA, and 0.005% (v/v) Tween20) w

23、as used as constant running buffer. Following binding and regeneration the sequence was repeated to see the interaction of goat antihuman IgE IgG labeled with Fc-CHO and its antigen, human IgE. IgG la- beled with Fc-CHO

24、and nonlabeled IgG at a protein con- centration in the range of 0.04–0.59 AM was injected for 60 AL at a flow rate of 25 Al/min. Injection of analyte was performed for 180 sec for monitoring association curves and dissoc

25、iation curves were monitored for another 180 sec by injection of HBS-EP buffer. Regeneration of the sensor after injection of goat antihuman IgE IgG was conducted with a 48-sec pulse of 10 mM glycine-HCl buffer (pH 2.6).

26、 Real-time reference curve subtraction over a noncoated sur- face was employed.Electrochemical Detection of IgG Labeled With Fc-CHO and Fc-COOHCyclic voltammetry of goat antihuman IgE IgG labeled with ferrocene was perfo

27、rmed in 100 Al of sample using an electrochemical analyzer (model 832A, Bioanalytical Sys- tems (BAS), W. Lafayette, IN) at a scan rate of 100 mV/s. A glassy carbon electrode with a diameter of 1.0 mm (BAS), a platinum w

28、ire, and silver/silver chloride (Ag/ AgCl/KCl) were used as a working, a counter, and a refer- ence electrode, respectively. Flow amperometric detection was performed in a radial flow cell (model 11-3456, BAS). PBS was f

29、lowed at a rate of 170 Al/min and 50Al of goat antihuman IgE IgG labeled with Fc-CHO and Fc-COOH was flowed into the flow cell and the current response was measured using amperometric detector (model LC-4C, BAS).RESULTSL

30、abeling of IgG With Fc-CHO and Fc-COOHFor construction of an electrochemical immunoassay probe, labeling of goat antihuman IgE IgG with Fc-CHO and Fc-COOH was investigated. When Fc-CHO was added to an IgG solution at a m

31、ild alkaline condition (pH 9.3), it reacted with free amino groups to form an unstable Schiff- base type compound. After this reaction step, this Schiff- base compound was reduced with sodium borohydride. The number of f

32、errocene moiety bound to goat antihuman IgE IgG increased with an increased amount of Fc-CHO in the reaction mixture (Fig. 2). A maximum labeling number of ferrocene moiety was obtained when Fc-CHO was reacted at a molar

33、 ratio of 1:400 (IgG:Fc-CHO) in the reaction mixture. The maximum mean number of ferrocene moiety bound to goat antihuman IgE IgG was eight. The number of ferrocene moiety bound to goat antihuman IgE IgG was reproducible

34、 with three experiments and a mean number of seven to eight ferrocene moiety were bound to individualIgG. At a higher concentration of Fc-CHO in the reaction mixture (when IgG and Fc-CHO reacted at a molar ratio of 1:500

35、), aggregation of IgG was observed and the bound number of ferrocene moiety in the filtrate decreased. When labeling goat antihuman IgE IgG with Fc-COOH via reac- tion of sulfo-NHS and EDC, the maximum number of bound fe

36、rrocene moiety was 2 to 3 per individual IgG. It was shown that Fc-CHO could efficiently bind to IgG by choosing an optimum pH and reducing Schiff base.Electrochemical Properties of IgG Labeled With Fc-CHO and Fc-COOHCyc

37、lic voltammograms of goat antihuman IgE IgG labeled with Fc-CHO was carried out in PBS at a scan rate of 100 mV/s using a glassy carbon electrode at a protein con- centration of 3.4 mg/mL. Oxidation and reduction peaks a

38、ppeared at 390 and 320 mV vs. Ag/AgCl, respectively (Fig. 3). For goat antihuman IgE IgG labeled with Fc- COOH (protein concentration, 2.5 mg/mL), only a slight oxidation and reduction peak appeared at 350 and 280 mV (da

39、ta not shown). A higher electrochemical signal could be obtained using goat antihuman IgE IgG labeled with Fc- CHO, which retains a higher number of ferrocene moiety. Oxidation and reduction peaks were reproducible with

40、an error of 15% for three individually prepared IgG labeled with Fc-CHO. Also, IgG labeled with Fc-CHO was stable for at least a few days and a change in redox potential or redox peak current was not observed.Binding Aff

41、inity of Goat Antihuman IgE IgG Labeled With Fc-CHO to AntigenFigure 4 shows the dependence of the mean number of bound ferrocene moiety per IgG and its binding affinityFigure 2. Relationship between the mean number of b