Chemical Sensors and Microinstrumentation - American Chemical

Chapter 9

A Fiber-Optic Electrode for Optoelectrochemical Biosensors Masuo Aizawa, Masaru Tanaka, and Yoshihito Ikariyama

Downloaded by AUBURN UNIV on November 17, 2016 | http://pubs.acs.org Publication Date: August 24, 1989 | doi: 10.1021/bk-1989-0403.ch009

Department of Bioengineering, Tokyo Institute of Technology, O-okayama 2-12-1, Meguro-ku, Tokyo 152, Japan Afiber-opticelectrode was fabricated for the simultaneous generation and transmission of electrochemical luminescence by preparing a transparent electrode on the optical and surface of afiber-optic.The opto-electrochemical properties of the micro-optical device were characterized in solutions containing the compounds required for luminol luminescence. The validity of sensitive measurement of electrochemiluminescence to be employed in a homogeneous immunoassay was evaluated by using potential step excitation of luminol in the presence and in the absence of hydrogen peroxide. As the highfluxof information density is one of the advantages offiber-opticcables over other types of information carriers,fiber-opticcommunications have been providing a powerful means of data transmission with their extremely high bit rates. In the case of chemical sensors, several advantages of thefiberscan additionally be exploited, namely such capabilities as remote sensing, miniaturization, electromagnetic immunity, and in situ measurement can be employed (12). Optical biosensors are the devices that can transduce the information concerned with a detenninant into optical signals such as the changes in absorbance, fluorescence, and luminescence (3-5). Generally these biosensors are the union of optoelectronic devices and biomaterials such as enzymes and antibodies in their solid matrix-bound forms (6). Fiber-optic chemical sensors for substances such as protons, oxygen, and carbon dioxide have been developed. These sensors respond to specific substances in accordance with the fluorescent changes of fluorescent probes. An opticalfiber-opticfor pH can be coupled to enzyme sensors when the enzymes cause a change in pH, which results in a change in fluorescent property of the probe compounds. These optical sensors are named Optrodes", and were extensively reviewed by Seitz (1). An optoelectronic enzyme sensor, a combination of a photodiode and an enzyme membrane, has been constructed by using a luminescence-generating enzyme (7). The enzyme photodiode is applied to the determination of enzyme substrates such as hydrogen peroxide and glucose with the use of the luminescent reaction between luminol and hydrogen peroxide. On the other hand, enzyme-immobilized membranes are coupled withfiber-optics,when sensitive determinations of biological substances are required (8). We have also made a series of works on the luminescent immunoassay by using the luminescent reaction of luminol (9-13). Labeling agents such as hemin (10-12) and peroxidase (9.13) are employed for the competitive and sandwich assays of antigenic substances such as insulin, /^-microglobulin and human albumin. Recently the authors have proposed a new homogeneous immunoassay based on electrochemical luminescence, by employing aromatic hydrocarbons such as pyrene as an electrochemically active label (14). The principle of the proposed immunoassay is schematically 009J-61567^/0403™01^$06.00/0 © 1989 American Chemical Society

Murray et al.; Chemical Sensors and Microinstrumentation ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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shown in Figure 1. The pyrene-labeled antigen emits luminescence by electrodic reaction, while the labeled antigen completed with its antibody generates less luminescence depending on the antibody concentration, because antibody molecules prevent the labeled antigen from contacting the electrode surface. Although the generated photons are transmited to a photon counter through a fiber-optic, we have not succeeded in sensitizing the electrochemihiminescence-based homogeneous immunoassay due to the low quantum efficiency of the luminescent reaction of pyrene. In order to improve the sensitivity of the homogeneous immunoassay we have developed a fiber-optic electrode for the efficient generation and transmission of photons, and we have exploited luminol which has a much higher quantum efficiency than pyrene as a candidate for an electrochemiluminescent probe. Spectroelectrochemistry, a combination of optical spectroscopy and electrochemistry has provided a powerful means for elucidating complex redox processes near solution-electrode interfaces (15.16). A union of transparent electrodes with fiber-optics provides a new strategy for designing new biosensing devices, provided they can be assembled into one instrument (fiber-optic electrode) that will generate and transmit a luminescence signal in response to the substance to be determined. We report here the performance characteristics of the fiber-optic electrode by applying the device to the electrochemiluminescence of luminol to clarify the feasibility of luminol as an electrochemiluminescent label for a sensitive homogeneous immunoassay.

Experimental

Section

F a b r i c a t i o n of fiber-optic e l e c t r o d e . Platinum was sputtered on the flat end surface of a plastic fiber-optic (diameter : 2 mm, length : 150 cm) courteously supplied by Mitsubishi Rayon Co. Sputtering was performed by a Hitachi minisputter for 5 min at 15 mA. The platinum counter electrode was prepared as illustrated in Figure 2. The thin platinum layer for the working electrode was connected to a lead wire with silver paste, while that for the counter electrode was connected with solder. The contact points were then used in place and insulated by sealing with epoxy resin. A photon counter of Hamamatsu Photonics consisted of a photon counter (Type C 767) and a power source (Type 752-01). Electrode potential was controlled by a function generator and a potentiostat (Hokuto Denko Co.) against a Ag/AgCl reference electrode. C h a r a c t e r i z a t i o n o f f i b e r - o p t i c e l e c t r o d e . Luminol was prepared in a phosphate-buffered solution of p H 7.0 (0.1M). Luminol was dissolved in the buffer under sonication. Both the photon counter output and the current output from the fiber-optic electrode were digitized (8 bit) and monitored by a Hitachi digital memory scope (Type VC-6O20). The digitized data were stored on a floppy disk by a N E C microcomputer (Type PC-9801 VX2) through a GP-BB interface. The data were processed in the microcomputer to reduce background luminescence. The experimental setup of measuring apparatus for electrochemiluminescence detection and the following data processing is illustrated in Figure 3.

Results and Discussion Electrolytic luminescence of luminol was investigated with the fiber-optic electrode in a solution containing 1 m M luminol. Figure 4 shows a cyclic voltammogram of the solution used to study the electrochemiluminescence of luminol. The luminescence was observed from +0.4 V and reached a maximum at +0.65 V in the neutral solution when the potential was scanned from negative to positive potential However, no luminescence was generated without the preceding negative potential application, the reason for which is described below. After repeated potential scanning, the fiber-optic electrode generated reproducible current and luminescence. From the transparency (20 %) of the electrode, the efficiency of the photon collection was estimated to be ca. 2 %. The estimated efficiency was calculated by considering only the photons emitted toward the fiber.


9. AIZAWA ET AL.

Fiber-Optic Electrodefor Optodectrochemiad Biosensors


•5 / "

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° / s

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F i g u r e 1.

Principle of homogeneous immunoassay based on electrochemical immunoassay. In the presence of antibody the labeled antigen emits less photons due to the steric hindrance of antibody in electrodic reaction.


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Sup 1· Solitioa Figure

2. Schematic illustration of the configuration of afiber-opticelectrode for the generation and simultaneous transmission of electrochemiluminescence.


9. AIZAWA ET AL,

Fiber-Optic Electrode for Optoelectrochemical Biosensors 133

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Computer

Digital Storage QsciUoscope


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Printer Optical Data Rmetion

Current Data

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Fbtentiostat PMT RE

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Figure 3. Experimental apparatus for the characterization offiber-opticelectrode. The cell volume was less than 2 mL, therefore the potential of thefiber-opticelectrode was referred to the reference electrode (Ag/AgCl) through a salt bridge.



134


-15 -1.0 -05 0.0 05 1.0 Potential / V vs. Ag/AgCl F i g u r e 4. Cyclic voltammogram of a solution containing luminoL Electrochemical luminescence of luminol was also shown with respect to the current-voltage relationship. Every solution was prepared in 0.1 M phosphate buffer (pH 7.0), and the scan rate was 100 mV/sec

Putentiot / V v s A g A g Q F i g u r e 5. Differential pulse polarogram of luminol solution. The fiber-optic electrode was employed for the oxidation of luminol. A phosphate-buffered solution (0.1 M , p H 7.0) was used as an electrolyte, and the scan rate was 5 mV/sec.



9. AIZAWACTAL.

Fiber-Opik Electrode for Optoelectrochemical Biosensors

As negative potential application is a prerequisite electrolytic process for the luminol electrochemiluminescence, it seems to us that a greater luminescence should be generated by a potential step mode from a negative potential to a positive one. Electrolytic oxidation of luminol was studied by differential puke voltammetry, and luminol was found to be oxidized at +0.65 V on the surface of the fiber-optic electrode at the neutrally buffered solution as shown in Figure 5. Furthermore, a greater luminescence was obverved at more negative potentials when the positive potential was fixed at +0.65 V. However, the platinum-sputteredfiber-opticelectrode denatured rapidly at a potential less than -1.5 V. Fixing the negative potential at -1.5 V , and scanning positively gave greater luminescence at + 0.65 V, at which potential luminol was oxidized. Figure 6 illustrates the relation between the luminescence intensity and a negative potential scan. The interval of a step-wise potential application for the excitation of luminol was also investigated. Figure 7 shows that a duration of the negative potential application of 5 s and that of the positive potential application of 15 s was the best condition for the greatest luminescence generation. Under the optimum condition, electrochemiluminescence of luminol was detected with the fiber-optic electrode in the concentration range from 10" to 1 0 M as shown in Figure 8. We have also studied the role of dissolved oxygen, because the dissolved oxygen may be easily reduced to hydrogen peroxide at a potential of -1.5 V. Monitoring of the dissolved oxygen was carried out by a dark-type oxygen electrode. The relation between dissolved oxygen and luminescence is shown in Figure 9. After the removal of dissolved oxygen by bubbling nitrogen gas, little luminescence was observed. This finding led us to perform another potential-step excitation from 0 V to +0.65 V in the presence of hydrogen peroxide. The electrochemical luminescence generated by the application of a positive potential of +0.65 V was proportional to the concentration of luminol in the concentration range from 10** to xlO" M in the presence of 2 mM hydrogen peroxide, as shown in Figure 10. Catalase (330 u/mL) addition annihilated the luminol electrochemiluminescence, while superoxide dismutase gave no effect on the electrochemical luminescent reaction. The wide dynamic range of the calibration curve was obtained due to the efficient generation and transmission of the electrochemiluminescence of luminol by afiber-optic,electrode combination. Kuwana et a l reported the electrochemical luminescence of luminol mainly in alkaline solutions by using a conventional three-electrode apparatus and a photon counter (17-19). The fiber-optic electrode described here, however, substantially improves the sensitivity in detecting the electrochemical luminescence of luminol in very low concentration at neutral p H . The micro-optical device coupled with the electrocheiniluminescence seems promising as a signal-transducing and transmitting element for a homogeneous immunosensing system with high sensitivity (Fig. 1) because of the high quantum efficiency and the simple labeling of luminol. 8

8

-15

First

-10

-05

Step Fbtentiol /

-3

2

00

V vs. AgAgCt

F i g u r e 6. Relationship between the luminescent intensity and a potential scan in the negative direction.




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F i g u r e 8. Relationship between luminescent intensity and luminol concentration.



AIZAWÂ ET AL.

Fiber-Opik Strode for Optoelectrochemical Biosensors

10

20

30

AO

Dissolved Oxygen / PPM

Figure i . Effect of dissolved oxygen on luminol electrochemiluminescence.

-ao -12 -10 -8 -6 -4 -2 Log Figure

*r3 Luminol / mol-drn

J

10. Calibration curve for luminol in the presence of hydrogen peroxide.


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C H E M I C A L SENSORS AND MICROINSTRUMENTATION

Conclusion A variety of unique optical biosensing systems can be developed by paying attention to chemical and electrochemical luminescence. Microfabrication of an optical biosensing device can be carried out by taking optoelectric devices such as the photodiode and the fiber-optic A fiber-optic electrode, the combination of fiber-optic and electrode, is especially suitable for the microinstrumentation of sensitive immunosensing device because of the sophisticated union of fiber optic and electrode for the efficient generation and transmission of electrochemiuminescence. Intensive research and development of the opto-electrochemical biosensors is being done to open up a new vista of biosensor technology.


Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

17. 18. 19.

Seitz, W. R. Anal. Chem. 1984, 56, 16A. Petersen, J.I.;Vurek, G. G. Science 1984, 224, 123. Freeman, T. M.; Seitz, W. R. Anal. Chem. 1981, 53, 98. Lubbers, D. W.; Opitz, N. Sensors & Actuators 1983, 4, 641. Lippitoch, M.; Leiner, M. J. P.; Pusterhofer, J.; Wolfkeis, O. S. Anal. Chim. Acta 1988, 205, 1. Freeman, T. M.; Seitz, W. R. Anal. Chem. 1978, 50, 1242. Aizawa, M.; Ikariyama, Y.; Kuno, H. Anal. Lett. 1984, 17, 555. Ikariyama, Y.; Aizawa, M.; Suzuki, S. Appl. Biochem. Biotech. 1981, 6, 223. Aizawa, M.; Suzuki, S.; Kato, T.; Fujiwara, T.; Fujita, T. J. Appl. Biochem. 1980, 2, 190. Ikariyama, Y.; Suzuki, S.; Aizawa, M. Anal. Chem. 1982, 54, 1126. Ikariyama, Y.; Suzuki, S.; Aizawa, M. Enzyme Microb. Technol. 1983, 5, 215. Ikariyama, Y.; Suzuki, S.; Aizawa,M.Anal.Chim.Acta1984, 156, 245. Ikariyama, Y.; Aizawa, M. Proc. 2nd Sensor Symp. (IEEE of Jpn.) 1982, 97. Ikariyama, Y.; Kunoh, H.; Aizawa, M. Biochem. Biophys. Res. Commun. 1985, 128, 987. Heineman, W. R. Anal. Chem. 1978, 50, 390A. Kadish, Κ. M. Electrochemical and Spectrochemical Studies of Biological Redox Components : In Advances in Chemistry Series 20; American Chemical society : Washington, DC, 1982. Kuwana, T. J. Electroanal. Chem. 1963, 6, 164. Kuwana, T.; Epstein, B.; Seo, Ε. T. J. Phys. Chem. 1963, 67, 2243. Epstein, B.; Kuwana, T. Photochem.Photobiol.1965, 4, 1157.

RECEIVED March 9, 1989


Chemical Sensors and Microinstrumentation - American Chemical

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