Anal. Chem. 1989,61, 633-636
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TECHNICAL NOTES Electrochemical Manipulation of Fluorescence and Chemiluminescence Signals at Fiber-optic Probes David A. VanDyke’ and Hung-Yuan Cheng* Physical and Structural Chemistry9 Smith Kline and French Laboratories, 709 Swedeland Road, King of Prussia, Pennsylvania 19406 Immobilization of a chemical or biochemical reagent on the end of a fiber-optic probe has proven to be a fruitful strategy in the development of chemical sensors (1,2). With a judicious selection of the reagent phase and proper manipulation of the reactions, one can obtain advantages in selectivity o r l a n d sensitivity for a specific mode of measurement. We recently reported a fiber-optic-based spectroelectrochemical probe for back-scattered reflectance measurement of ascorbate in a tissuelike environment and demonstrated the technique of electrochemical manipulation of spectral signals for improving the selectivity of fiber-optic-assisted spectroscopy (3). Conceptually, the incorporation of electrically conductive material in the fabrication of a fiber-optic probe may be viewed as the immobilization of a tunable and in situ regenerated redox “reagent” on the end of the probe. In this technical note we will demonstrate that the spectroelectrochemical approach can be extended t o fiber-optic fluorescence and chemiluminescence measurements using model systems which we believe have important implications in bioanalytical sensor development. Two new probe designs will be described.
EXPERIMENTAL SECTION Chemicals and Solutions. All solutions containing DNA (deoxyribonucleic acid) were prepared in pH 7.4 PBS (phosphate-buffered saline) containing 10 mM phosphate, 1mM EDTA (ethylenediaminetetraacetic acid), 1mM Ca2+,and 0.2 mM Mg2+. Calf thymus DNA was purchased from Pharmacia as the sodium salt. Ethidium bromide was purchased from Sigma and used as received. Luminol(3-aminophthalhydrazide)was purchased from Aldrich. Hydrogen peroxide was obtained as the 30% solution from J. T. Baker. All solutions used in the chemiluminescence work were prepared in high-purity water from a Millipore/Continental water purification system. All other regents were of analytical reagent grade. Instrumentation. The Oriel 78300 system spectrophotometer equipped for fiber-optic spectroscopy has been described previously (3). For fluorescence measurements, 1/8-m monochromators were used both in the excitation beam and in the emission beam, and an R928 photomultiplier tube (PMT) was used as the detector. The raw fluorescence intensity data was acquired with an Apple I1 computer/interface and transferred to a VAX computer for background subtraction and plotting using RS/1 software. Filters were used to reduce stray light: For the work with ethidium bromide, long-pass filters with 50% transmittance a t 495 and 530 nm were used in the excitation and emission beams, respectively. The excitation wavelength was 525 nm. For the luminol chemiluminescencework, no monochromation was required and the flow-cell or fiber-optic probe was mounted directly in front of the window of the cooled PMT housing (from Products for Research) containing a UV-extended PMT. The Oriel Model 7070 photometer output was recorded on a strip chart recorder. The flow injection analysis system for the determination Current address: Department of Chemistry, University of Pennsylania, Philadelphia, PA. 0003-2700/89/0361-0633$01 SO/O
of H202,similar to that described elsewhere ( 4 , 5 ) ,was made of components available in our laboratory. Fiber-optic Probes/Electrodes. Two different designs of fiber optic/spectroelectrochemical probes were used in this work. A first design (Figure 1) for stationary fluorescence measurements consisted of 12 200 pm diameter core fused silica fibers (Polymicro Technologies) sealed into the tip of a disposable pipet with black sealing wax. The tip was then polished with successively finer grades of polishing paper, followed by final polishing with alumina. Six fibers from around the circumference of the probe were selected and gathered to serve as the emission collection arm of the probe, while the remaining six were gathered to serve as the excitation arm. The working electrode was a cylinder 5 mm in diameter and 2 cm high formed out of 52 mesh platinum gauze. A platinum wire woven down through one side of the cylinder served as the electrical contact. For measurements, the working electrode was first wedged into position in an electrolytic cell, and then the fiber-optic probe, which was attached to a micropositioner, was lowered to a selected depth into the center of the electrode cylinder. Care was taken to ensure that the original position of the probe within the cylinder was reproduced after each solution change, and that no air was trapped beneath the probe tip. A second design (Figure 2), which incorporated optical fibers and a “cage” platinum working electrode, was intended for flow injection analysis (as well as for stationary solution measurement) and represents a modification of an absorbance-based probe designed by Coleman et al. (6). The cage electrode assembly was constructed from a 2.5-cm length of 1.57 mm o.d., 1.32 mm i.d. platinum tubing (Johnson Matthey, Seabrook, NH). Several 0.5 mm diameter holes were drilled around the circumference near the bottom of the tubing to allow the passage of solution. A small section of 1mm diameter platinum wire was polished and carefully epoxied into the bottom of the platinum tube so that the shiny surface of the wire was level with the bottom of the drilled holes. For H202work, gold-plating was carried out in lo4 g/mL Au3+/0.5 M HCl solution a t -0.15 V vs Ag/AgCl. A wire-wrap wire was wrapped tightly around the exterior of the tubing to allow electrical contact. A 1mm diameter hole was bored through the side of a 10-cm length of 3 mm 0.d. Pyrex tubing. This was then threaded down through the body of a 1mL plastic syringe, through holes in the rubber septum (plunger removed), until the hole in the glass tube was aligned within a needle guard “port”previously inserted into the syringe body. The glass tubing and the needle guard port were then sealed into position with epoxy. The platinum tube was then inserted into the bottom of the glass tube up to the point of the wire contact and fixed in place with epoxy. The desired number of optical fibers were bundled together by the use of heat-shrinkable Teflon tubing and were polished as described above. The bundle was then inserted down into the glass tube from the top until it reached a desired distance above the platinum wire “mirror”. The position of the fiber-optic bundle, which was either separated into two separate arms for fluorescence or retained as a single bundle for chemiluminescence, was fixed with sealing wax at the top of the glass tube. The flow of solution into the platinum tube could be achieved either by applying suction at the needle guard port or by pumping solution through the bottom holes in the platinum tubing. 1989 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 6, MARCH 15, 1989 light
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Figure 3. Emission spectra obtained with the probe in Figure 1: (a) 100 pM ethidium bromide in PBS; (b) addition of 40 pM calf thymus DNA: (c) 20 min after 0.85 V applied to solution b. The excitation wavelength was 525 nm. platinum rncs h
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Flgure 2. Simplified sketch of fiber-optic/electrochemical probe for electrcgenerated chemiluminescence measurements and flow injection analysis.
RESULTS AND DISCUSSION Fluorescence. Ethidium bromide exhibits a greatly enhanced quantum yield and a small shift in emission wavelength when intercalating with DNA. This property has been widely used for fluorometric assay of nucleic acids (7-10). However, background fluorescence from non-nucleic acid sources, especially for samples prepared from recombinant biological products, can pose problems (11). If the components in the ethidium/DNA system can be electrochemically manipulated, then the fiber-optic spectroelectrochemical probe
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Time (sec) Figure 4. Time course of DNAIethidium bromide fluorescence response. The conditions and probe used are the same as in Figure 3. The excitation and emission are at 525 and 610 nm, respectively. could provide a means for fluorescence background subtraction in situ. Cyclic voltammetry of ethidium bromide in PBS a t a graphite electrode showed an anodic wave near 0.67 V vs a Ag/AgCl reference. No corresponding reduction wave could be seen upon scan reversal even at a very high scan rate (>lo0 VIS). Addition of sodium bromide did not change the peak height, therefore eliminating the possibility that Br- contributed to the voltammetric peak. No electrochemical activity was observed in the -0.4 to + L O V region for a solution containing only calf thymus DNA. A cyclic voltammogram of a 1:l ratio (50 pM each) DNA base pakethidium solution was not different from that containing ethidium bromide alone. It was determined that an applied potential of 0.85 V should be sufficient to electrochemically perturb the DNAIethidium system. Figure 3a shows the emission spectrum (excitation a t 525 nm) of a 100 pM ethidium bromide PBS solution obtained with the platinum "mesh" fiber-optic probe shown in Figure 1. The solution is weakly fluorescent. With the addition of calf thymus DNA, the fluorescence quantum yield of ethidium increased significantly due to intercalation and hydrophobic interactions. As shown in Figure 3b, the increase in emission intensity is accompanied by a small shift in the peak position. This is consistent with results obtained with a conventional spectrofluorometer. Figure 3c shows the final spectrum after applying a potential of 0.85 V to the platinum mesh for 25 min. Almost all fluorescence signal was eliminated by the electrooxidation. This process cannot be reversed; Le., a reducing potential (-0.1 v) did not restore the fluorescence intensity.
ANALYTICAL CHEMISTRY, VOL. 61, NO. 6, MARCH 15, 1989
The effect of electrochemical manipulation on the fiberoptic probe can be more clearly seen in the response-time plot of Figure 4. The emission at 610 nm was plotted vs time. The signal increased immediately (with mixing) after the ethidium bromide solution was spiked with calf thymus DNA, and reached a steady level. With an applied potential of 0.85 V, the signal dropped quickly (without mixing) due to the depletion of ethidium/DNA complex in the light path of the probe. The result did not reveal explicitly whether the ethidium/DNA complex was being electrolyzed directly or only the ethidium bromide was being oxidized at the electrode. For the latter case, the ethidium/DNA complex would be depleted via a rapid equilibrium between the bound and unbound ethidium molecules. The rate-limiting step would be the diffusion of electrolytic reactants and products in the light path of the probe. No advantage could be realized by stirring the solution because it would actually replenish the DNA/ethidium complex to the small depleted area near the electrode/optic-probe end face. The DNA/ethidium bromide system is but one example of many bioanalytical procedures exploiting the ability of fluorescent dyes to interact selectively and reversibly with biological components (12). Examples can also be found in topics related to flow cytometry, immunoassay, histochemical procedures, and affinity labeling. Many of these fluorescent dyes are plausible candidates for the fiber-optic spectroelectrochemical scheme, and such an approach should be explored for biosensor development. Note that the probe can be used as a fiber-optic sensor or an amperometric sensor, separately and independently. The preliminary result here by no means represents detection limits achievable under more favorable instrumental conditions. For example, more sophisticated optics and a laser source would allow the use of a single fiber for both excitation and the collection of fluorescence, an arrangement yielding benefits both in sensitivity and miniaturization (6, 13). Miniaturization, in turn, would produce shorter electrochemical response times. Chemiluminescence. The optical fiber/electrode combination can also be applied to electrogeneration of chemiluminescence. Chemiluminescence is especially attractive for analytical methods based on fiber-optic probes because there is no light source as in fluorescence; stray light can be eliminated and there is no need for monochromation, both of which result in extremely high sensitivity. The previous work of one of the authors (D.A.V.) involved the application of electrogenerated chemiluminescence (ECL) of luminol to the detection of hydrogen peroxide using a transparently faced thin-layer cell in a flow injection analysis scheme (4, 5 ) . Kuwana et al. have studied the mechanism of electrogenerated chemiluminescence of luminol in some detail (15-1 7). Basically, the electrode is used in place of a chemical catalyst. Luminol is oxidized at the positively biased electrode, and chemiluminescence is generated in the presence of H2OP This scheme has been coupled to H202-producingenzymatic systems for the analysis of such compounds as glucose ( 4 , 5 , 1 8 ) . The fiber-optic spectroelectrochemical probe of Figure 2 was designed to accommodate the flow injection analysis (FIA) scheme. The hollow platinum tubing with small holes at the bottom formed a “channel” electrode for the electrooxidation of luminol in the flowing stream. The platinum wire cap and the surrounding wall served as a reflector to enhance the collection of light by the optical fiber bundle, which was centered just above the platinum cylinder. In the determination of H202,the platinum surface was electroplated with a thin film of gold, which was necessary for the elimination of Pt-catalyzed decomposition (in the absence of applied potential) of HzOz. The analyte was pumped through the probe via a short length of small-diameter Tygon tubing
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Table I. Electrogenerated Luminol Luminescence peroxide concn, M 10” 10-5 lo4 10-3
peak intens, nA
no. obsvn
Fiber-Optic/ElectrodeProbe 3.5 x 10-2 2 1.4 X IO-’ 4 5.5 x 10-1 4 7.3 x 10-1 2
re1 std dev, 70
20 6.9 2.7 7.8
Transparently Faced Thin-Layer Flow Cell 1.7 X IO-’ 3 14 7.9 x 10-1 3 0.7 7.7 3 1.7 52.2 2 17 56.6 2 0.5
10-7 10” 10-6 10-4 10-3
connected to the bottom portion of the probe. The peristatic pump, injection loop, switching valves, and mixing ports were all located upstream and arranged as described previously (4, 5 ) . In a different situation, such as using the probe as an in situ catheter-like sensor, one could also pull the analyte into the probe by using suction from a syringe. (The side port shown near the top of Figure 2 was actually made of a plastic needle guard, which accepted a standard gastight syringe.) The luminol solution (0.4 mM luminol, 0.2 M KNOB,0.04 M borate buffer a t pH 10.26) in channel A was mixed with water in channel B, which included a 500-pL injection loop a t a total flow rate of 3.3 mL/min (1.65 mL/min each channel). At an applied potential of +0.35 V, the P M T output from background ECL (dissolved oxygen) was about 0.15 nA. to lo4 M With the injections of 500 pL of Hz02in the region, well-defined FM peaks were recorded. The peak width (one-half maximum height) was about 0.6 min. The detection limit was lo4 M. The data in Table I represent a typical net ECL intensity (peak height) vs concentration profile for H202 obtained with the cage electrode/fiber-optic probe. For comparison purposes, the calibration data (obtained in this lab) for a transparently faced thin-layer electrode under the same experimental conditions are also included in Table I. The detection limit for this detector was M, as the amount of ECL was generally 1-2 orders of magnitude higher due to the much larger electrode area and optical window. The detectability is limited by the noise level of the background ECL derived from residual dissolved oxygen in the eluent. The calibration curve is nonlinear with the luminescence signal leveling off at approaching the M level, probably due to the exhaustion of the luminol reagent in the flowing stream. Freeman and Seitz constructed a chemiluminescence fiber-optic probe for hydrogen peroxide by immobilizing peroxidase in a polyacrylamide gel on the end of a fiber optic (14). Instead of an enzymatic catalyst, we used a platinum electrode as the catalyst for the generation of luminol chemiluminescence at the fiber-optic probe. The detection limits of peroxide are comparable for the two approaches. However, the electrode catalyst has an added advantage that the catalytic activity can be turned on or off by simply switching the potential on or off. This adds possibilities for implementing signal modulation techniques for rejection of stray light, and correction of background chemiluminescence from other sources. In summary, we have demonstrated the concept of immobilization of an electrochemical ”reagent” for fiber-optic fluorometric and electrogenerated chemiluminescent measurements. The two different probes presented here illustrate the flexibility of possible designs to accommodate different schemes and conditions. The DNA/ethidium bromide model represents an important approach to improving analytical selectivity in biosensor development by taking advantage of the biological recognition process. The concept of immobilized elecrochemical reagents on the end of a fiber optic is a useful
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addition to the field of chemical sensor and biosensor development. Registry No. Pt, 7440-06-4; H202, 7722-84-1; ethidium bromide, 1239-45-8;luminol, 521-31-3.
LITERATURE CITED (1) (2) (3) (4)
Seitz, W. R. Anal. Chem. 1984, 56, 16A-34A. Peterson, J. I.; Vurek, G. G. Science 1984, 224, 123-127. VanDyke, D. A'; Cheng, H.-Y. Anal. Chem. '9889 60, 1256-1260. VanDyke, D. A. Ph.D. Thesis, University of Illinois, 1986. (5) VanDyke, D. A.; Nieman, T. A., submitted for publication in Anal. Chem . ( 6 ) Coleman, J. T.; Eastham, J. F.; Sepaniak, M. J. Anal. Chem. 1984, 56,2249-2251. (7) Le Pecq, J. B.; Paoletti, C. Anal. Blochem. 1966. 1 7 , 100-107.
(8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18)
Karsten. U.; Wollenberger, A. Anal. Biochem. 1972, 4 6 , 135-148. Karsten, U.; Wollenberger, A. Anal. Blochem. 1977, 7 7 , 464-470. Boer, G. J. Anal. Blochem. 1975, 65,225-231. Holl, W. W.; Webb, R. L., Smith Kline and French Labs, personal communication, 1988. Modern Fluorescence Spectroscopy, Wehry, E. L.,Ed.; Plenum Press: New York, 1976; Vol. 2. Angel, S. M. Spectroscopy 1986, 2, 38-47. Freeman. T. M.; Seitz, W. R. Anal. Chem. 1978, 50, 1242-1246. Kuwana, T. J. Nectroanal. Chem. 1963, 6 , 164. Epstein, B.; Kuwana. T. fhotochem. Photobiol. 1965, 4 , 1157. Epstein, B,; Kuwana, T, J , Electrmna/, Chem, 1967, 16, 3899, Hool, K.; Nieman, T. A. Anal. Chem. 1988, 60,834-837.
RECEIVED for review August 25, 1988. Accepted November 22, 1988.
Reproducible Nuclear Magnetic Resonance Surface Coil Fabrication by Combining Computer-Aided Design and a Photoresist Process Teresa W.-M. Fan*%' Nuclear Magnetic Resonance Facility, University of California, Davis, California 95616
Richard M. Higashi University of California Bodega Marine Laboratory, Bodega Bay, California 94923
INTRODUCTION The vigorous development of in vivo NMR spectroscopy and imaging has generated great interest for applications in biology and medicine. While improved electronics and sophisticated pulse sequences are being developed, the success of nuclear spin manipulation in in vivo NMR experimentation will depend, in part, on the efficiency of NMR probes in delivering the desired pulses. Thus, probe design needs to be optimized and constructed accurately for a given application. Winding copper wire has been the method of choice , tailored copper foil has for making surface probes ( l ) while been commonly used for high-resolution NMR probes. Generally, copper foil should be superior to wire as the coil material in probes due to the following: (a) lower inductance, which makes it easier to tune to the higher frequencies that are increasingly common in in vivo applications; (b) larger conductive surface, giving rise to lower radio frequency (rf) impedance and a better Q factor (2);(c) better rf homogeneity, leading to better line shape. Although both wire and foil tape can be used for simple coil designs without much difficulty, they require specialized skills and tools when one is dealing with even slightly more complicated coil designs such as spirals or when they must be tailored to the shape of the sample. In this note, we report an alternative probe coil fabrication method that exploits recent low-cost advances in personal computers, rudimentary computer-aided design (CAD) programs, and high-quality graphics printers and combines them with the proven photoresistive etching process for printed circuit (PC) boards. This method is easy to implement, uses only commonly available materials and equipment, and offers the flexibility and precision needed for the construction of considerably more complex coil designs. Although constructing NMR surface coils from PC boards by etching (3)and machining (2)has been reported, these two methods were limited to available templates or special skills and tools. The combination of CAD and photoetching of PC boards eliminates these technical requirements and provides the following advantages in addition to the inherent advantages of copper foil:
' Current address: Department of Environmental Toxicology, University of California, Davis, CA 95616.
(a) Coils of any shape and complexity can be readily fabricated to scale for a given application. (b) Theoretical designs can be rapidly and accurately made into real probes for testing. (c) Coils can be made with high precision such that even slightly different coil designs can be fabricated with ease for empirical optimization of performance. (d) Reproducible coils can be made by photocopying a design directly from the pages of a journal such as this. (e) The tight binding of copper on plated boards reduces microphonics or other mechanical ringing that can occur with free-standing wire coils (4).
EXPERIMENTAL SECTION The procedure for fabricating photoetched coils was straightforward. Coils were drawn with Cricket Draw program (Cricket Software, Malvern, PA) running on a Macintosh Plus computer (Apple Computer,Inc., Cupertino,CA) and printed onto transparencies by a 300 dots-per-in. (dpi) resolution Laserwriter Plus (Apple). There were no freehand drafting skills involved; in this case, spiral coils were created by using the starburst and radial grate tools of Cricket Draw. If a high-resolution graphics printer is not available, coil designs could be printed oversized with a lower resolution printer and reduction-copied onto transparencies by using a photocopier,the entire process being scaled such that the resulting transparency is 300 dpi or better. Photosensitized single-sided PC boards (35.6 wm thick, 3 in. X 4 in. or 3 in. X 6 in., positive type, GC Electronics, Rockford, IL) were then exposed under the transparencies according to the manufacturer's recommendation. We found that an exposure time of 20 min at a distance of 6-7 in. from a General Electric (GE) Fl5BL ultraviolet lamp was sufficient. After removal of the photoexposed material with the developer (GC Electronics) according to the manufacturer's instruction, the boards were etched with potassium persulfate in deionized H20at 40-50 OC with stirring. Although we avoided the more commonly employed FeC1, etchant because of possible paramagnetic contamination, we experienced no consistent difficulty with using it. After etching was complete, the unexposed photoresist coating was removed with stripping solution (GC Electronics) or ethanol. The coils were then turned to desired frequencies and matched to 50-R impedance to complete the probe construction. We have chosen the balanced matching circuitry approach ( 5 ) for better Q factor and performance on conductive samples. The variable capacitors used were 1-10- and 1-30-pF nonmagnetic air piston
0003-2700/89/0361-0636$01.50/0 1989 American Chemical Society
