Y
-
+ td
-
Anal. Chem. 1008, 60, 2677-2674
Y,,
NOMENCLATURE
= the assignment operator vT = the transpose of the vector v vTv = the vector product of the vector v llvll = the norm of the vedor v is the square root of the vector product Registry No. Felodipine, 72509-76-3.
LITERATURE CITED (1) Ljung, B. Drugs 1985, 29(suppl 2), 46-58. (2) Mlller, James A. Pharm. Techno/. 1977, l(2). 19-42. (3) Munson. J. P. J . Pharm. Ebmed. Anal. 1988, 4 , 717-724. (4) Wold, H. Research Papers in Statlstics;DavM, F. N., Ed.; Wlley: New YO&. 1966; pp 411-444. (5) Naes, T.; Irgens, C.; Martens, H. J . R. Statist. SOC., C 1986, 35. 195-206.
2871
(6) Wold, S.; Albano, C.; Dunn. W. J., 111; Edlund, U.; Esbensen. K.; Geladi, P.; Hellberg, S.; Johansson, E.; Lindberg, W.; Sjbtrom, M. Chemomeffics. Mathematics and Staffsticsh ChemMy; Kowalski, B. R.. Ed.; D. Reidel: Dordrecht, 1984; pp 225-250. (7) Welser, W. E.; Pardue, H. L. Clin. Chem. (Winston-Sahsm, N . C . ) 1983. 2 9 , 1673-1677. (8) The United States Pharmcopsia, 21th rev.; U.S. Pharmacopeia1Convention, Inc.: Rockvllle. MD, 1985; p 1244. (9) Shenouda, L. S.;Adarns. K. A.; Alcorn, G. J.; Zogllo, M. A. Drug Dev. Ind. Pharm. 1986. 12, 1227-1239.
(IO) Sjostrom, M.; Wold, S.; Llndberg, W.; Persson, J. A.; Martens, H. Anal.
Chim. Acta 1983, 150, 61-70. ( 1 1 ) Persson, J. A.; Johansson, E.; Albano, C. Anal. Chem. 1986, 5 8 , 1173-1 178. (12) Geladl, P.; Kowalskl, B. Anal. Chim. Acta 1988, 185, 1-17. (13) Box, G. E. P.; Hunter, W. G.; Hunter, J. S. StaffsHCsforGper/mentm; Wlley: New York, 1978; pp 4-7. (14) Papas, A. N.; Alpert, M. Y.; Marchese, S. M.; Fitzgerald, J. W.; Deb ney, M. F. Anal. Chem. 1985, 5 7 , 1408-1411. (15) Ecksteln, R. J.; Owens, 0. D.; Balm, M. A.; Hudson, D. A. Anal. Chem. 1988, 5 8 , 2116-2320.
RECEIVED for review January 6,1988. Resubmitted June 17, 1988. Accepted August 31,1988.
Fiber-optic Sensor for the Determination of Glucose Using Micellar Enhanced ChemiIuminescence of the Peroxyoxalate Reaction Monzir S. Abdel-Latif a n d George G. Guilbault*
Department of Chemistry, University of New Orleans, New Orleans, Louisiana 70148
The use of cetyltrlmethylammonlum bromlde (CTAB) as a surfactant to enhance the chemllumlnescence (CL) generated from the reactlon ol bls(2,4,6trlchIorophenyl) oxalate (TCPO) with hydrogen peroxlde has been lnvestlgated In the presence of perylene with Incorporation of flber optics. I n the presence of 2 X M CTAB, problems of mlxlng, and hence reproduclblllty, Involved In the peroxyoxalate system were ellmlnated, and the CL Intensity Is llnearly proportlonal to the concentratlon of peroxlde In the range of 8 X lo4 to 8 X lo9 M H202with a llmit of detection equal to 2.5 X lo-' M H2OP The coefflclent of varlatlon (five measurements) Is 0.3% for lo-' M H202. Uslng glucose oxidase (GOx) lmmoblllred on an lmmunodyne membrane, glucose could be assayed by measurlng the concentratlon of the enzymatically generated peroxlde. The callbratlon curve was h e a r In the range of 3 X to 3 X lo-' All glucose and the llmlt of detection was 6 X lo-' M glucose. The coefflclent of varlatlon (five measurements) was 0.3% for lo-' M glucose.
Peroxyoxalate CL is known to be the most efficient nonenzymatic CL reaction with quantum yields of about 25% (1). Hydrogen peroxide and TCPO react to give an intermediate that is capable of transferring 105 kcal/mol of energy to a fluorescent acceptor (2). This energy transferred excites the fluorescent acceptor, which then emits light. The light emitted is proportional to the concentration of hydrogen peroxide. The reaction sequence can be represented by the following scheme: 0003-2700/88/0360-2671$01.50/0
0-0
I I
+ Fluorescer-
/F-f\ 0 0 *
(Fluorescer)
muorescer*) + 2 co,
-
Fluorescer + h v
The light emitted has a A, dependent on the type of the fluorescer used. The peroxyoxalate CL reaction has been used by Seitz (3) to detect hydrogen peroxide generated from the glucose/ glucose oxidase reaction. However, dissolution problems of organic solvents in aqueous solvents were encountered, and the reproducibility of the system limits its use to a static manner. Also, the stability of TCPO in ethyl acetate, when methanol is added to improve the mixing, was a significant problem. This may be attributed to the fact that most commercial ethyl acetate preparations have acetic acid as an impurity. This catalyzes a nucleophilic attack on the TCPO. For the above-mentioned reason, a t least in part, flow injection and high-performance liquid chromatography (HPLC) have been used to eliminate the problems associated with the reaction system. The CL of the peroxyoxalate systems has been used to quantify substrates of several enzymatic systems (4-11) by measuring the HzOz generated. The use of surfactants to improve the analytical performance of various spectroscopic technqiues has been reported 0 1988 American Chemical Society
2672
ANALYTICAL CHEMISTRY, VOL. 60, NO. 24, DECEMBER 15, 1988
f-
J
3
0
including ultraviolet-visible absorption (12,13), fluorescence (14-1 7), phosphorescence (14-21), as well as chemiluminescence (22-29). The analytical applications of the chemiluminescent systems studied were limited to lucigenin and luminol. Reversed micelles were used to overcome the pH mismatch of the luminol CL system (28,29) but detection limits were far away from that which can be obtained in aqueous solutions a t alkaline pH values. In this work, we report the first analytical application of the use of micelles to enhance the CL intensity and eliminate the problems of dissolution encountered in the peroxyoxalate CL system. Resulta demonstrate the potential of the micelles to enhance CL for the detection of hydrogen peroxide, as well as oxidase-generated hydrogen peroxide for the determination of glucose. The advantage of increased CL intensity of the TCPO reaction is used in combination with fiber optics, which has many advantages in the field of chemical sensing, including the possibility of analyzing samples at inaccessible locations. Optrodes also offer a great potential for miniaturization of the probe, which is a requirement for in vivo measurements based on spectroscopic techniques. Effects of various experimental parameters on the CL intensity will be discussed. EXPERIMENTAL SECTION Apparatus. A corning Model 110 digital pH meter was used for all pH measurements. All CL measurements were made with a Lumac Biocounter, Model 2010, which was modified to accommodate a fiber-opticarrangement. The fiber-opticcable and the cell were the same as those previously described (30). However, the common end of the cable was attached to the Lumac Biocounter via an adapter. The end of one of the two arms of the bifurcated cable was fixed to the cell cover while the other end was fixed to the side of the cell 1.5 cm above the bottom of the cuvette (Figure 1). In the glucose measurements, GOx was immobilized on an immunodyne membrane (Pall, New York) (31). The membrane was mounted around a 2 cm long glass spacer which, in turn, was held at the end of the fiber-optic cable fixed to the cell cover. Reagents and Solutions. All chemicals were used as purchased without further purification. All solutions were prepared with doubly distilled deionized water. The CL reagent contained 2.4 g of cetyltrimethylammonium bromide (Matheson Coleman and Bell), 0.65 g of TCPO (CTC Organics) and 0.4 g of perylene (Alrich Chemical Co., Inc.). These were dissolved in 100 mL of 4 0 % hexane (EM Science) in ACS grade chloroform (Eastman Kodak Co.). The mixture was shaken for 5 min, allowed to stand for 30 min, and then filtered to remove undissolved perylene. A phosphate buffer was made by using 0.1 M KH2P04and an appropriate volume of 0.1 M NaOH; the f i a l volume was adjusted so that 0.03 M phosphate buffer pH 7.2 was obtained as monitored by a pH meter.
Hydrogen peroxide solution (3%) was obtained from EM Science and standardized against standard permanganate. Aqueous solutions of 10-'-104 M H202were made by subsequent dilutions. Glucose oxidase from Aspergillus niger, type X (100 Ujmg) was purchased from Sigma Chemical Co. The enzymatic membrane was prepared and stored as previously described (30). However, the membrane used in this study was 1 X 2.5 cm. Glucose standard solutions of 10-'-1O4 M glucose were prepared by subsequent dilutions of 1M glucose in 0.03 M phosphate buffer pH 7.2. Procedure. For the determination of H202the following reagents were used: 940 p L of 0.03 M phosphate buffer, pH 7.2, 30 pL of H202of the appropriate concentration, and 30 pL of the CL reagent. The cuvette was quickly shaken for 2 s and the CL intensity was recorded at the maximum. The background signal was checked by using water instead of H20z. The time required for the maximum intensity to be reached depends slightly on the H202concentration and is in the range of 4-10 s. For the application of the peroxyoxalate CL to glucose determination, 3 mL of 0.03 M phosphate buffer pH 7.2 was transferred into a cuvette and 100 pL of glucose of an appropriate concentration was added. The cell was closed and the contents were stirred for 3 min; then stirring is stopped and 100 pL of the CL reagent was added. Cell contents were stirred again and the CL intensity is recorded at the maximum. The maximum intensity was also achieved in 4-10 s. RESULTS AND DISCUSSION The first parameter that was investigated is the solvent system which should be used to dissolve TCPO and perylene. Potential solvents were ethyl acetate, chloroform, methyl acetate, dimethylformamide, dioxane, carbon tetrachloride, and, to a lesser degree, hexane. Chloroform was chosen as the solvent due to the likely stability of TCPO in this solvent. Addition of hexane to the solution decreased the background CL signal and 40% hexane in chloroform was optimum. The effect of concentration of TCPO on the CL intensity was also investigated. Results suggest that as the concentration of TCPO increases, the signal increases. However, at concentrations of TCPO above 6.5 mg/mL the increase in signal is no longer proportional to the increase in TCPO concentration, and a small increase in the signal is achieved. Therefore, a concentration of 6.5 mg/mL was used through this study. The study of the effect of perylene concentration on the CL intensity showed that as the concentration of perylene increases, the signal increases. The maximum amount of perylene is limited by its solubility in the solvent system and was about 3 mg/mL. The concentration of CTAB in the CL reagent was optimized by studying the effect of CTAB concentration on the CL intensity. The optimum concentration was found to be 0.06 M CTAB. However, concentrations above this value have very limited effect on the CL intensity. Buffer Concentration. The investigation of the effect of buffer concentration on the CL intensity was conducted in different buffer concentrations. Unlike the results that were previously reported ( 3 ) ,the effect of buffer concentration showed a different behavior probably due to the presence of the micelles. The use of other buffer systems also affects the CL behavior. When phosphate buffers of pH 6-8 are used, the CL intensity occurs at 0.03 M, then the signal decreased steeply as the buffer concentration decreased. However, when borax buffers of pH 9.6 and 10.2 were used, the CL intensity was not as much affected by the decrease in buffer concentration. It was also noted that the CL intensity of the peroxyoxalate is 4 times greater when phosphate-borax buffer is used a t pH 7.2 in place of just phosphate buffer. pH Effect. The pH of the buffer solution is an important parameter that affects the CL intensity and hence has to be optimized if very sensitive assay is required. The effect of pH on the CL intensity was investigated using 0.03 M
ANALYTICAL CHEMISTRY, VOL. 60, NO. 24, DECEMBER 15, 1988
Table I. Effect of pH on the Reproducibility of the CL Signal
PH
av CL intensity
re1 std dev (n = 5)
7.2 7.4 8.0 8.6 9.2
5230 5620 9320 14790 13220
1.5 2.5 3.0 3.3 11.4
2673
6 -
4 -
Results are the average of five measurements reported in arbitrary units.
2 -
I 0
I
I
I
I
I
1
2
3
4
5
-log[glucose]
(M)
I
6
I
7
Figure 3. log-log calibration plot for standard glucose: 3 mL of 0.03 M phosphate buffer, pH 7.2, 100 pL of glucose, and after 3 min is added 100 pL of the CL reagent.
0
1
2
3
4
5
6
7
8
9
10
- l ~ g [ H l O l ] (MI Figure 2. log-log callbration plot for standard H20,: 940 pL of 0.03 M phosphate buffer, pH 7.2, 30 pL of H202, and 30 pL of the CL reagent.
phosphate buffer in the pH range of 6-10. The optimum pH was obtained at about pH 8.5. It can also be concluded that the CL intensity decreases slightly at pH values higher than 9. Due to the nature of the glucose oxidase reaction with glucose, the pH of the buffer was maintained at pH 7.2 where the enzymatic reaction is optimum. Further study (Table I) showed that the best precision was achieved at this pH, and consequently all analyses were performed a t pH 7.2. Assay of HzOz. A log-log calibration plot for the determination of HzOz(Figure 2) shows that the response was linear in the range of 8 X lo4 to 8 X M HzOP The coefficient of variation for five replicates of M HzOz was found to be 0.3%. These results suggest that the use of micelles to enhance the CL intensity of the peroxyoxalate reaction is of great potential and can be used for very sensitive assay of hydrogen peroxide. The detection limits were lowered by almost 2 orders of magnitude, dissolution problems were eliminated with a homogeneous solution, and reproducible results are obtained. It should also be mentioned that the background CL signal was very small and close to zero. Another project involved the use of aqueous buffer solution, containing 0.1 M CTAB, to dissolve TCPO and perylene, and hence all measurements can be made in aqueous solutions. While the dissolution process seemed to be efficient, the CL intensity was very weak. Therefore, it seems necessary to have an organic solvent so that maximum CL can be observed. No further study on the CL of aqueous TCPO and perylene was carried out. Assay of Glucose. Figure 3 shows the log-log calibration plot for glucose standards. The response is linear in the range of 3 x to 3 x l0-B M glucose with a limit of detection equal to 6 X M glucose. The coefficient of variation for five measurements of lo4 M glucose was 0.3%. The determination of oxidase-generated HzOzby the peroxyoxalate CL reaction is significant because it has been reported (3) that uric acid does not interfere in glucose determination using this system at pH 6. This makes the analysis for glucose using TCPO a
favorable alternative, especially when the problems associated with the system were eliminated by the incoporation of micelles. GOx immobilized on immunodyne membranes showed significant stability, as evidenced by the high activity of a GOx membrane prepared 8 months before and used for over a 100 assays. The peroxyoxalate enhanced CL reaction has been used to analyze for glucose in Gatorade Orange Drink. The same procedure for the analysis of standard glucose was used except that the standard glucose solution was replaced by the Gatorade Orange Drink. Very good results were obtained where the coefficient of variation for six replicate measurements was 0.20% and the relative error was 4%. The results were confirmed by comparison to colorimetric assay results using the GOx/POD enzymatic reaction and 2,2’-azinobis(3-ethylbenzthiazolinesulfonicacid) as the dye (coefficient of variation was 0.36%). These results indicate that better precisions can be obtained with the peroxyoxalate CL system. The potential of reversed micelles was explored as a method for the determination of glucose or glucose oxidase using luminol (29). Since reversed micelles cannot usually be used with immobilized enzymes, the use of normal micelles to enhance the CL assay of enzymatic reactions is advantageous because the enzyme used, in most cases, i s expensive and therefore is immobilized on a solid support so that it can be reused instead of using it on a one time basis. In our laboratory, encouraging results were obtained for the analysis of oxidase-generated peroxide using normal micelles at the physiological pH with luminol CL as the detection system.
CONCLUSION This work demonstrates the potential of micelles to enhance the CL intensity of the peroxyoxalate system. No problems were encountered in the determination of oxidase-generated HzOzand, subsequently, the quantification of glucose. Hence, this method can be used to assay for glucose in the micromolar level with increased sensitivity and reproducibility. The use of normal micelles to enhance the CL of the peroxyoxalate reaction for the determination of enzymatically generated Hz02is feasible and can be utilized especially when immobilized enzymes are involved. This reduces the cost and improves both sensitivity and precision. It is worth mentioning that the highly enhanced CL yields were obtained without the use of any catalyst or cooxidant. Finally, the incorporation of these advantages in the peroxyoxalate CL system with fiber optics makes it feasible to develop a miniaturized self-contained fiber-optic sensor for enzymatically generated HzOz. The performance characteristics of micelle-mediated luminol CL for the determination of oxidase-generated HzOz
Anal. Chem. 1988, 60, 2674-2679
2874
by using fiber optics is now under investigation in our laboratory and will be discussed in a later report.
LITERATURE CITED Rauhut, M. M.; Bollyky, L. J.; Roberts, E. G.; Loy, M.; Whltman, R. H.; Iannota, A. M.; Semsei, A. M.; Clarke, R. A. J . Am. Chem. SOC. 1967, 89, 6515. Lechtken, P.; Turro, N. J. Mol. fhotochem. 1974, 6 , 95. Wllliams, D. C.; Huff, G. F.; Seltz, W. R. Anal. Chem. 1978, 48, 1003. Zoonen, P.; Herder, I.; GoolJer,C.; Velthorst, N. H.; Frei, R . W. Anal. Left. 1988, 19, 1949. Rlgln, V. I.J . Anal. Chem. USSR (Engl. Transl.) 1981, 36, 111. Rigin, V. I. J. Anal. Chem. USSR (Engl. Transl.) 1983, 38, 1265. Rigin, V. 1. J. Anel. Chem. USSR (Engl. Transl.) 1983, 38, 1328. Scott, G.; Seitz, W. R.; Ambrose, J. Anal. Chim. Acta 1980, 115, 221. Williams, D. C.; Huff, G. F.; Seltz, W. R. Anal. Chem. 1078, 48, 1478. Rigin, V. I.J . Anal. Chem. USSR(Engl. Transl.) 1979, 3 4 , 619. Zoonen, P.; Kamminga, D. A.; Gwljer, G.; Veithorst, N. H.; Frei, R. W. Anal. Chlm. Acta 1085, 167, 249. Diaz-Garcia, M. E.; Sanz-Medel, A. Talante 1988, 33, 255-275. Spurlln. S.; Hlnze, W. L.; Armstrong, D. W. Anal. Left. 1977, 10, a a 7 - .-"-. inn~ Hinze, W. L. SoluilOn Chemlstry of Surfactants; Mittal, K. L., Ed.: Pienum: New York, 1979; Vol. 1, pp 79-127. Hinze, W. L.; Slngh, H. N.; Baba, Y; Harvey, N. G. TrAC, Trends Anal. Chem. (Pers. Ed.) 1984, 3 , 193-199.
"".
(16) Singh, H. N.; Hinze, W. L. Anawst (London) 1982, 107, 1073-1080. (17) Sanz-Medel, A.; Aionso, J. I.Anal. Chim. Acta 1984, 165, 159-169. (18) Cline Love, L. J.; Harbarta, J. G.; Dorsey, J. G. Anal. Chem. 1984, 5 6 , 1132A-1148A. Cline Love, L. J.; Grayeskl, M. L.; Noroski, J.; Weinberger, R. Anal. Chim. Acta 1985, 170. 3-12. Cline Love, L. J.; Skrilec, M.; Habarta, J. Anal. Chem. 1980, 52, 754-759. Armstrong, D. W.; Hinze, W. L.; Bui, K. H.; Slngh, H. N. Anal. Lett. 1081, 14; 1659-1667. Thompson, R. B.; McBee, S. E. S. Langmuir 1988, 4, 106-110. Malehorn, C. L.; Riehl, T. E.; Hinze, W. L. Analyst (London) 1988, 11 1 , 941-947. .. . .. . . Yamada, M.; Suzuki, S. Anal Left. 1984, 17, 251-263. Kato, M.; Yamada. M.; Suzuki, S. Anal. Chem. 1984, 56.2529-2534. Hinze. W. L.; Riehi, T. E.; Slngh, H. N.; Baba, Y. Anal. Chem. 1984, 56, 2180-2191. Klopf, L. L.; Nieman, T. A. Anal. Chem. 1084, 56, 1539-1542. Hoshino, H.; Hinze, W. L. Anal. Chem. 1987, 59, 496. Igarashl, S.; Hinze, W. L. Anal. Chem. 1988. 60, 446. Abdel-Latif, M. S.; Sulelman, A. A.; Gullbault, G. G. Anal. Left. 1988, 21, 943. Assolant-Vinet. C. H.; Couiet, P. R. Anal. Left. 1988, 19, 875.
RECEIVED for review July 19,1988. Accepted September 14, 1988* This work 'Onduckd with assistance from the Louisiana Education Quality Support Fund.
Ultrasensitive Photothermal Deflection Spectrometry Using an Analyzer Etalon Stephen E. Bialkowski* and Zhi Fang He
Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322-0300
The theory and experlmentat scheme for uslng an analyzer etalon to detect photothermal deflecth slgnals is developed. First, a theory for photothermal deflection spectrometry Is developed, whkh describes the observed signal decay In terms of the characteristic thermal decay tlme constant and whkh accounts for a flnlte probe laser beam walst radius. Second, a theory for the angular response for an analyzer etalon Is described. The analyzer etalon Is found to be extremely sensltlve to beam angle varlatlons and dramatlcally Increases the sensltlvlty of photothermal beam deflection measurements. A theoretkal enhancement over conventional deflectlon angle detectlon schemes of 100 Is calculated. Although the experlmental enhancement Is calculated to be only 0.4 of theoretlcal, a slngte laser pulse detectlon llmlt of 0.7 ppm (v/v) of CFC-12 In argon Is obtained by uslng a carbon dloxMe laser operatlng at 933 cm-' wlth a pulse energy of 1 mJ. This constitutes a slgnnkant Improvement over previously determlned detection Ilmlts. Ensemble averaglng can be used to decrease this llmlt In systems where analysls tlme Is not critical. The problems encountered In uslng this detectlon scheme are also due to the extreme angle sensltlvlty. The apparatus Is very susceptible to environmental factors such as air currents, laboratory temperature varlatlons, and vlbratlons.
Reported here is a new deflection angle detection scheme for photothermal spectroscopy. Photothermal deflection spectroscopy (PDS) is an ultrasensitive technique used for the measurement of small optical absorbance (1-4). Deflection of a probe laser beam path arises from a change in refractive
index (RI) that occurs when optical radiation from a second, pump laser light source is absorbed by the sample and not lost through subsequent emission of radiation. The energy is deposited in the sample in a finite volume and so results in a spatially anisotropic RI perturbation. To a first approximation, the linear gradient part of this RI perturbation is what is responsible for the deflection of the probe laser beam used to monitor the RI change in the sample. For the most part, experimental implementations of PDS have been based on position measurement of the probe laser beam spot at some distance past the sample cell. The two main methods for performing these position measurements have been to use a linear aperture that bisects the beam image, followed by a photodetector, or to use one of the bi-cell or lateral position sensors. Both of these methods rely on the fact that a deflection of the probe beam in the sample cell will result in a linear displacement proportional to the distance that the aperture or detector is positioned past the sample cell. But, since the probe laser beam, which is normally focused at the position where the RI perturbation occurs, is also diverging, the sensitivity of these position-sensitive schemes cannot be arbitrarily increased by moving the aperture or detector far from the sample cell. In the end analysis, there is no advantage to placing the detector at a distance beyond the Rayleigh range of the probe laser (1,5 ) . One exception to the beam-position-sensitive detection schemes is moire deflectometry (6). In this scheme, it is the beam angle itself that is measured by the change in probe laser intensity past a pair of matched ronchi rulings placed some distance apart. An advantage to this scheme is that the whole RI perturbation can be determined through the apparent spatially resolved deflection angles. This detection scheme is not dependent on the distance between the deflectometer
0003-2700/88/0360-2674$01.50/00 1988 American Chemical Society
