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Anal. Chem. 1991, 63, 1766-1771
LITERATURE CITED (1) Pekarek, L., Ion Waves and Ionization Waves.
10th I n t m t k n e l 1971; Donald Parsons 8
Conterenoe on pheromene In Ionized Gases Co. Ltd.: Oxford, England, 1971;pp 385-403. (2) Franklin, R. N. plesma phenomene In Gas Dlschargas; Claredon Press: Oxford, England, 1976. (3) Oleson, N. L.; Cooper. A. W. Movlng Strlatlons. In Advences k, ElechonlCS end €&iron physlcs; Marton, L., Ed.; Academic Press: New York, 1988;Vol. 24. (4) Harley, J.; Pretorlus, V. Nature 1956, 178, 1244. ( 5 ) Pltkethly, R. C. Anal. Chem. 1958, 8 , 1309-1314. (6)Lovelock, L. E. Anal. Chem. 1961, 33, 182-178. (7) Maimstadt, H. V.; Enke, C. G.; Crouch, S. R. Ekpctronics end Instru-
mentetbn for SdenHSts;The Benjamin/Cummlngs Co., Inc.: Reading, MA. 1981;p 210. (8) Davld, D. J. Gas chrometograph/c Detectors; John Wiiey 8 Sons: New York, 1974; p 4. (9) Christlan, G. D.; ORellly, J. E. Insfrumentill Ana!mis. 2nd ed.; Ailyn and Bacon, Inc.: Newton, MA. 1986;p 749. (10) Locke, D. C.; Brardt, W. W. Reduced Pressure Gas Chromatography. In Gas chromatography; Fowler, L.. Ed.; Academic Press: New York,
1963. (11) Giddings, J. C. Anal. Chem. 1962, 5 1 , 314-319.
”R
for review March 11,1991. Accepted June 10,1991.
Micellar-Enhanced Aqueous Peroxyoxalate Chemiluminescence Niya Dan, Miu Ling Lau, and Mary Lynn Grayeski* Department of Chemistry, Seton Hall University, South Orange, New Jersey 07079
The aqueous peroxyoxalate (CL) reactbn of blr[N-[2-(N’-methyl-2‘-pyrldlnlumyl)ethyl~N-[(trHluoromethyl)sulfonyl]~xamlde(METQ) wlth hydrogen peroxtde In the presence of a fluorophor was characterlzed In dlfferent mlcellar and homogeneous media, and the effects of the various types of surfactants on chemiluminescence lntenelty and quantum efflclencles were assessed. The surfactants examlned Include catlonlc, anlonlc, neutral, and zwfflerionlc types. I n the presence of hexadecyltrlmethylammonlum chlorlde (CTAC), polyoxyethylene(23) dodecanol (Brl) 35), and 3-( N~decyCN,Ndlmethylammonlo)-l-propanecwHonk acid (SB-12) micelles, enhancement of the CL lntenslty by factors of 1.6-130 was observed relatlve to that In buffer. I t was found that In mlcellar medkm not onty were Huoresccmce efflclencles Improved but also the reaction excltatlon efflckncy. The v a r h experhmtal varlabks that Influence the magnltude of the CL enhancements were studied. Lastly, the mkellar CL reactlon systems were evaluated for the analysis of fluorophors and hydrogen peroxide, and analytkal flgures of merlt were generally Improved In micellar medlum.
INTRODUCTION The peroxyoxalate chemiluminescence (CL) reaction, involving the oxidation of an oxalic acid derivative by hydrogen peroxide in the presence of a suitable fluorophor (I+), has been used analytically for the measurement of dansylated amino acids (3,hydrogen peroxide (8), biological reducing agents such as glucose and uric acid (9),fluorophors or f l u e rophor-labeled compounds (10, 1I ) , and polynuclear aromatic hydrocarbons (12-14). However, in many of these applications, some major problems are encountered. Most oxalic acid derivatives are soluble and efficient only in organic solvents, causing irreproducible mixing when measuring aqueous samples. Although water-soluble derivatives have been reported (15),efficiencies are very low. In many cases, the rates of the chemiluminescence reaction in an aqueous system are very fast, resulting in imprecise measurements. Some approaches have been reported to overcome these difficulties. Microemulsions have been used with nonaqueous peroxyoxalate CL (16). In our laboratory, cyclodextrins were used to enhance the aqueous peroxyoxalate CL quantum yield (17). Here we report the study of micelle effects on the aqueous peroxyoxalate CL. Micelles have been demonstrated to influence the chemistry and photophysics of molecules by altering the microenviron0003-2700/91/0383-1768$02.50/0
ment in which the molecules reside. Micelles can change microviscosity, local pH, polarity, reaction pathway or rate, etc. Different aqueous micelles have been used to improve lucigenin (18,191 and luminol CL reactions (20). Surfactants at concentrations below their critical micelle concentrations (cmcs) have been used to increase the efficiency of aqueous peroxyoxalate CL by a factor of 5 (21). The effects of four different types of micelles (anionic, cationic, zwitterionic, and nonionic) on the aqueous peroxyoxalate CL reaction of bis[ N -[ 2-(N’-methyl-2’-pyridiniumyl)ethyl]-N[ (trifluoromethyl)sulfonyl]]oxamide (METQ) with hydrogen peroxide are reported here. The effects of micelles on the excitation efficiency and fluorescence efficiency are studied. The potential analytical implications of these effects will be dimmed.
EXPERIMENTAL SECTION Apparatus. All CL measurements were obtained by using a Turner Designs (Mountain View, CA) Model TD20-e photometer equipped with a manual injector; a Fisher strip-chart recorder Model 2000 printer was used to record the CL intensity-time profiles. Some data were collected and stored on disks by an Apple IIe and graphed on a Gemini printer. The absorption spectra for fluorescence quantum yield determination were carried out with a Varian 2200 spectrophotometer. The fluorescence spectra were recorded on a Spex Fluorolog 2+2 spectrofluorometer. Reagents. The following reagents were used: hexadecyltrimethylammonium chloride (CTAC;Eastman Kodak Company), polyoxyethylene(23) dodecanol (Brij 35; IC1 Americas Inc.), 3(N-dodecyl-N,N-dimethylammonio)-l-propanesulfonic acid (SB12; Hoechst Calbiochem Behring Diagnostics), sodium dodecyl sulfate (SDS; BDH Chemical Ltd.), polyoxyethylene(20) cetyl ether (Brij 58; Aldrich Chemical Company), polyoxyethylene(20) stearyl ether (Brij 78; Aldrich), polyoxyethylene(20)oleyl ether (Brij 99; Aldrich), 8-anilino-1-naphthalenesulfonicacid (ANS; Sigma Chemica Company), rhodamine B (RH B; Aldrich), 1(dimethylamino)naphthalene-5-sulfonyl chloride (dansyl chloride; Aldrich), avidin-rhodamine isothiocyanate (avidin-RH B Sigma), human albumin (Sigma),and 30 w t % hydrogen peroxide (Aldrich). Bis [N-[2-(N’-methyl-2’-pyridiiumyl)ethyl]-N-[ (trifluoromethyl)sulfonyl]]oxamide (METQ) was prepared by the method of Tseng and Rauhut (22). Albumindawyl chloride was prepared by Jacobsen’s method (23).HPLC grade acetonitrile(Fisher)was used as obtained. All fluorophors and surfactants were wed as received. Buffers were prepared from ACS reagent grade sodium phosphate. The surfactants were dissolved in 0.1 M phosphate buffer at pH 7.0. hEXQ solution was prepared by dissolving 75 mg of dried METQ in 5 mL of acetonitrile(0.017 M). Hydrogen peroxide was diluted to 0.1 M in distilled water. ANS, RH B, avidin-RH B, and albumin-dansyl chloride were dissolved in distilled water. 0 I991 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 63, NO. 17, SEPTEMBER 1, 1991
Dansyl chloride was dissolved in acetonitrile. Procedure. ChemiluminescenceExperiments. The general CL analysis procedure consists of using Gilson microliter pipets to add 25 pL of hydrogen peroxide solution, 12.5 p L of fluorophor solution, and 1 mL of buffer or surfactant solution into a polypropylene (12- X 75-mm) disposable culture tube, which was subsequently placed into the cuvette holder of the luminometer. Fifty microliters of METQ solution was then injected manually, and the output was read from the Turner photometer. A delay time of 0 s and run time of 10 s were utilized. The CL signal was collected as the integrated CL intensity and the peak CL intensity. Surfactant Concentration. In the study of the effect of different concentrationsof surfactants on chemiluminescence, ANS and RH B were used as fluorophors with a concentration of 1.1 X 10”’ M. Quantum Efficiency. Fluorescence spectra were obtained by exciting a mixture of 45 pL of ANS or RH B and 3 mL of surfactant solution at 365 or 320 nm. The emission spectra were scanned from 380 to 600 nm for ANS and 500 to 650 nm for RH B. The absorbances of ANS or RH B in the surfactant solution and buffer solution were measured at 365 or 320 nm. In the study of the effect of fluorophor concentration on CL, CTAC and Brij 35 were chosen as surfactants with concentrations of 2.65 X lo-‘ and 2.50 X lo-’ M, respectively. The fluorophors were ANS and RH B. Dansyl chloride, avidin-RH B and albumin-dansyl chloride were also used in the analytical studies. In the study of the effect of hydrogen peroxide concentrations, the effect of METQ concentrations, and the effect of protein matrix, the concentrations of ANS and RH B were both 1.1 X M. The surfactants were CTAC and Brij 35 with the same concentrations as above.
RESULTS The effects of micelles on chemiluminescenceefficiency and rate are studied by monitoring the intensity-time curve and the peak height. A comparison of integrated area under the intensity-time curves is indicative of relative chemiluminescence efficiency, &,, which is the product of the excitation efficiency of the reaction, I$,,, and fluorescence efficiency of the emitting species, c#+ The peak height generally increases with increasing rate of chemiluminescence reactions. Effects of Surfactants and Fluorophors. Four types of Surfactants are evaluated: anionic, cationic, nonionic, and zwitterionic. SDS, CTAC, Brij 35, and SB-12 were chosen because they have different charge groups but similar hydrophobic chain lengths. ANS and RH B, which have frequently been used as fluorophor labels (7),were chosen because they are water soluble with similar structures but different charges. The presence of surfactant causes an increase in chemiluminescenceintensity, but the effect of the increase is dependent on the nature of the fluorophor (Figures 1and 2). The enhancement of intensity depends on the type and concentration of surfactant. The greatest enhancement is obtained with Brij 35 a t about 2.5 X M 130-fold with ANS and 5.6-fold with RH B. With ANS, the increase in chemiluminescence intensity can also be observed in CTAC and SB-12 at concentrations above their cmcs, but the enhancement is less significant than that in Brij 35. In contrast, SDS has much less effect on the CL intensity. While using RH B, the greatest enhancement is observed with SB-12, followed by SDS and then CTAC. With respect to different fluorophors, hydrophobicity and charge differences affect the enhancement. In Brij 35, the hydrophobic association seems to be the major factor enhancing CL intensity. The effect of CTAC, SB-12, and SDS on ANS and RH B can be understood in terms of charge interaction. A t pH 7.0, ANS exists primarily as the anionic form, which interacts strongly with the cationic micelles of CTAC but less with the partially cationic micelles of SB-12. The anionic SDS repels the anionic form of ANS, causing a decrease in CL intensity. With a positive charge on RH B,
1787
ci al v)
o
800
I
-100 -7.0
I
I
I
- 1 .o
-5.0 -3.0 Log[surfactant](M)
Figwe 1. Effect of surfactant concentration on CL enhancement. ANS as fluorophor. (...e-.)Brij 35;(---e---) CTAC; (-e-) SB-12; (-e--) SDS; Concentrationsof reagents: MET0 = 0.017 M, fluorophore = 1 X lo4 M; hydrogen peroxide = 0.1 M.
750
I
600 v)
0
5
450
;
U
l
-7.5
- ’
I
I
I
-5.5
-3.5
-1.5
Log(surfactant](M) Figure 2. Effect of svfactant concentration on CL enhancement. RH B as fluorophor. (.-0.-) Brij 35; (---0---) CTAC; (-0-) SB-12; (- -0- -) SDS; Concentrations of reagents as Figure 1.
the opposite effect is observed, as expected. Effect of pH. Phosphate buffer was used because, in a previous report studying the effects of buffers on cyclodextrin-enhanced METQ-HZOZCL, the highest intensity was observed in phosphate (17). The increase of pH results in the increase of both CL intensity and peak height observed for 2 min (Table I). At pH 8.0, the reaction is too fast to be monitored in CTAC, SB-12, and Brij 35 systems. These results are consistent with the fact that the reaction with METQ is base catalyzed and the efficiency increases because the chemiluminescence reaction is favored over hydrolysis. Effect of Micelles on Fluorescence. The fluorescence emission spectra of both fluorophors were compared in micelles and solvents of varying polarity (Table 11). The ANS spectra in buffer and SDS micelles are similar; both have two emission peaks with the same maximum emission wavelength. However, in Brij 35, CTAC, and SB-12 micellar media, the shorter wavelength emission peak is decreased, while the longer wavelength peak is greatly enhanced. The maximum emission wavelengths of enhanced peaks are blue shifted to 489 nm relative to 517 nm observed in buffer and SDS. The spectra of RH B in buffer and SDS are similar, with a strong emission peak at 578 nm and a second very small peak at 636 nm. The fluorescence intensity of the peak at 578 nm is greatly enhanced in SDS. In Brij 35, CTAC, and SB-12, this peak is also increased but less than that in SDS. In contrast to ANS, the second emission peak at 636 nm is significantly
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 17, SEPTEMBER 1, 1991 ""
Table I. Effect of pH on CL Intensity
40 -
CL intensitv. AU" integrated" peak
micellar s o h
pH 4.0 597 f 26 7f2 122 f 34 342 f 41 1.5 f 0.3
CTAC SDS Brij 35
SB-12 buffer
pH 6.0 1018 f 101 33 14 417 20 744 f 57 22 f 5
CTAC SDS Brij 35 SB-12 buffer
*
pH 8.0 b 14 i 8 b b 0.6 f 0.3
CTAC SDS Brij 35 SB-12 buffer
50
a
743 f 20 15 f 5 151 f 44 434 f 54 9.3 f 4
-E
Y
30-
v
8332 f 1005 100 f 44 3810 f 58 9213 f 648 212 f 16
20
-
- .
b 187.4 f 34.6 b b 70.0 f 19.0
"Arbitrary units. bZc, has not been measured for CTAC, Brij 35, and SB-12at pH 8.0 because the reactions were too fast to be recorded under these condition. Concentrations of surfactants are CTAC, 2.6 X M; SDS, 1.6 X M Brij 35, 2.0 X lo4 M and SB-12,7.2 X M, using ANS as fluorophor. Others as in Figure 1.
enhanced in Brij 35, SB-12, and CTAC. The spectral changes in the two peaks, which are indicative of different electronic transitions, are due to the differences in the microenvironment in the various systems. The longer wavelength emission peak of ANS is probably due to a AT* transition. The nonpolar environment will stabilize a x orbital, resulting in a blue shift as observed in the solvents less polar than water in Table 11. The wavelengths in Brij 35, CTAC, and SB-12 are about the same as in acetonitrile and methanol, which is consistent with the expectation that most ANS molecules are probably in the Stern layer, which reportedly has a polarity similar to that of methanol (24). Within the micellar media of SDS, however, the repulsion between acidic ANS and anionic SDS keeps ANS molecules in the bulk homogeneous solution. As expected, the spectrum in SDS is similar to that in the aqueous system. The shorter wavelength emission peak of ANS might correspond to the n-x* electron transition from a nonbonding electron on nitrogen to a higher antibonding orbital. This process appears to be less affected by the polarity changes in the environment. Table 11. Effect of Solvent Polarity on Fluorescence ANS dielectric constant
solvent water NaH2P0, buffer CTAC Brij 35 SB-12
87.74 (0 "C)
-
37
SDS acetonitrile methanol ethanol 2-propanol tetrahydrofuran chloroform
37.5 (20 "C) 32.70 (25 "C) 24.30 (25 "C) 19.92 (25 "C) 7.58 (25 "C) 4.81 (20 "C)
hem, nm 411 411 411a 411" 411" 411 411" 411" 411" 411a 411" 408"
peak 1 intensity, AU 0.15
0.14
0.14
RHB
&em,
nm
517 517 489 489 489 513 487 490 480 481 473 484
peak 2 intensity, AU 0.07 0.06 58.5 82.6 3.28 0.08 2.23 3.43 2.25 3.01 3.57 0.02
Xlsmr
nm
572 572 570 572 574 572 563 567 564 563 563 555
peak 1 intensity, AU
hem, nm
1.20 1.19 2.00 2.21 1.86 2.82 3.98 6.07 6.77 6.66 0.32 6.89
636" 636" 635 635 635 636" 636 636 636 636 636 636
peak 2, intensity, AU
0.47 2.35 0.82 0.71 0.76 0.78 0.65 0.28 0.82
"Fluorescence intensity of the peak was very small. Dielectric constant of Brij 35 was from ref 19. Concentrations of reagents as in Figures 1 and 3.
ANALYTICAL CHEMISTRY, VOL. 63, NO. 17, SEPTEMBER 1, 1991
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Table IV. Effect of Chain Length of Nonionic Surfactant
surfactanta Brij 35 Brij 58 Brij 78 Brij 99
201 0 0.00
structure
C12H2S(OCH2CH~)m0H96.7 10.6 780 949 ClsH~(0CH2CH&,0H 97 8 1050 C18H3,(OCHzCHz)mOH 106 24 1580 C18H,(OCH2CH2)m0H 161 f 63
*
I
1
0.05
0.10
I-
Table V. Effect of Cations
0.1 5
cation
Figure 4. Effect of hydrogen peroxide concentration. (0)ANS in Brij 35; (0)ANS in CTAC; (+) RH B in Brij 35; (A) RH B in CTAC. Concentrations of reagents as Figure 3.
Na+
K+ NH4+
CL intensity, AU integrated peak BufferO 10.42 1.96 7.9 f 2.3 17.1 f 1.9
Table 111. Relative Efficiency Enhancement in Micelles
micellar soh
ICL(m)/ICL(b?
CTAC
51.2 f 8.0 0.65 0.12 39.6 2.8 130 f 29
SB-12 Brij 35
IFL(m)/IFLb?
Iex(m)/zex(bf
3.39 1.97 26.98 25.18
15.2 0.33 1.47 5.16
1.25 2.19 0.65 0.49
1.28 1.35 3.37 11.4
ANS
*
RH B CTAC SDS SB-12
Brij 35
* 6762 206 * 583
All surfactants are dissolved in 0.1 M sodium phosphate buffer M. Other reagents conat pH 7.0. Concentrations are 2.5 X centrations as in Figure 1.
t
[H202]
SDS
CL intensity, AU integrated peak
1.60 0.20 2.95 f 0.61 2.19 0.27 5.60 f 0.98
*
ORatio of CL efficiency in micelle to that in buffer. bRatio of fluorescence efficiency in micelle to that in buffer. Ratio of excitation efficiency in micelle to that in buffer. where A represents the absorbance at the excitation wavelength and F the area under the fluorescence emission curve. Buffer and micelle are respectively b and m. As shown in Table 111, the fluorescence efficiency of ANS is increased by 27-fold in SB-12, followed by Brij 35, CTAC, and finally SDS. In contrast, the fluorescence efficiency of RH B is increased only 2 times in SDS and 1.25 times in CTAC. SB-12 and Brij 35 cause the decrease in fluorescence efficiency. For several systems, such as ANS with Brij 35, the observed enhancement of the fluorescence efficiency is much less than that of the CL efficiency, while for some other systems, RH B with Brij 35, for example, the CL efficiency is enhanced but the fluorescence efficiency is actually dehanced. These results indicate that only a fraction of CL enhancement is due to the changes of the fluorescence efficiency. The remaining enhancement can be attributed to the increases in the excitation efficiency, which is calculated as the ratio of excitation efficiencies in the micellar to the buffered media (Table 111). The greatest enhancement of excitation efficiency of ANS is observed in CTAC, followed by Brij 35 and then SB-12. SDS causes a reduction of excitation efficiency. With R H B, the greatest enhancement is obtained in Brij 35, followed by SB-12 and then SDS. CTAC decreases the excitation efficiency. Effect of Surfactant Chain Length. The increase of hydrocarbon chain length of surfactant results in the increase of both chemiluminescence intensity and peak height (Table IV). If fluorophors and METQ associate with the micelles, hydroperoxide anions must penetrate into the hydrophobic chain of micelles in order to react. Varying the length of the
*
67.71 12.12 55.0 8.3 107.4 f 11.4
Brij 35b Na+ K+ NH4'
394 9 838 f 28 1386 37
*
3707 f 168 7107 331 9206 93
*
aAllbuffers are adjusted to pH 7.0 with concentration of 0.1 M. M. Others as in Figure 1. bConcentration of Brij 35 is 2.5 X hydrocarbon chain of surfactants changes the size of the micelles; thus, the depth of penetration is different. Four nonionic surfactantswith different chain lengths are evaluated. Increasing the chain length of surfactants results in increased chemiluminescence signal. Micelles might be enlarged by the increase of hydrocarbon chain length, and thus, the hydrophobic part is getting looser so that the penetration of hydroperoxide anions into micelles becomes easier. Besides the hydroperoxide anions, the hydroxide anions can also penetrate into the micelles, but the hydrated OH- anions are larger than hydrated OOH- anions. The penetration of OH- would be slower and more difficult than that of OOH-, which in turn decreases METQ hydrolysis and enhances CL intensity. However, it should be noted that the increased intensity in Brij 99 cannot be explained by chain length only since it actually differs from Brij 78 by only two hydrogen atoms. It is possible that the double bond in Brij 99 decreases ita hydrophobicity, which may be responsible for the increased CL. Effect of Cations. Three phosphate buffers are studied, potassium phosphate, sodium phosphate, and ammonium phosphate, to evaluate the effect of different cations on the CL intensity (Table V). The hydroperoxide anions penetrate into nonionic micelles. The interaction between the polar surface of micelles and buffer cations provides an electrostatic attraction for the hydroperoxide anions. Ammonium phosphate buffer shows the greatest enhancement in both homogeneous buffer solution and Brij 35 micellar solution. Ammonium ion is a good nucleophilic reagent, which should be a more effective catalyst than hydroxide. In a buffered system, the behavior of the potassium cation is similar to the sodium cation. However, with Brij 35, the exihancement in potassium phosphate buffer is twice as large as in sodium phosphate buffer. It was suggested that the degree of counterion binding, defined as the ratio of counterions and amphiphile ions in a micelle, follows the effective radius of the hydrated ions (25). For alkali ions, the degree of binding usually follows the sequence Li < Na < K < Rb < Cs (26). Therefore, the greater enhancement with potassium phosphate-Brij 35 can be explained by the stronger electrostatic attraction between hydroperoxide anions and potassium cations.
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 17, SEPTEMBER 1, 1991
Table VI. Analytical Parameters
RSD, % analyte
medium”
dansyl chloride
buffer
Brij 35 CTAC
RH B
buffer Brij 35 CTAC
buffer Brij 35
ANS
albumin-dansyl chloride avidin-RH B H202-ANS
H202-RH B
CTAC buffer Brij 35 CTAC
buffer Brij 35 CTAC buffer Brij 35 CTAC buffer Brij 35 CTAC
linear range,*M (6.0-100) X 10” (0.6-3.0) X IO” (3.0-10) X 10“ (0.055-0.55) X 10” (0.055-110) X 10“ (0.055-55) X 10” (0.11-5.5) X 10“ (0.055-55) X 10” (0.055-55) X 10” (0.07-1.7) X 10” (0.07-8.7) X 10” (0.07-8.7) X 10“ (0.072-72) X 10“ (0.36-36) X 10” (0.36-36) X 10” (1.1-11) x 10-3 (1.1-55) X (0.055-55) X (0.055-5.5) X (0.055-55) X (0.055-55) X
U.C.’
3.43 2.55 2.24 8.26 6.40 4.75 2.92 0.46 1.74 5.41 5.86 1.87 9.15 0.20 3.30 5.91 2.54 1.32 1.16 2.35 3.42
1.c.’
slope*
15.2 2.75 5.05 14.3 11.5 14.5 8.48 6.10 2.26 7.39 6.84 2.05 11.7 17.4 5.93 6.54 2.75 6.46 6.44 4.06 5.22
0.01 0.15 2.31 0.54 4.11 1.13 0.29 9.94 3.74 3.34 3.80 14.2 0.60 0.62 0.36 0.13 4.42 1.80 0.48 2.27 5.56
detection limit? M 1x 4x 2x 9x 2x 4x
Ix
3x 2x 2x 6X 2x 2x 4x 2x 9x 3x 4x 5x 2x 2x
10-8 10-3 10-3 10-3 10-3 10-3 10-3 10-3 10-3 10-3
IO” 10-3 10-3 10-3 10-3 10-3 10-3 10-3 10-3 10-3 10-3
‘Buffer: 0.1 M sodium phosphate at pH 7.0. Brij 35, 2.5 X M; CTAC, 2.7 X M. bAs obtained from calibration curves (log integrated intensity vs log concentration). ‘u.c., upper concentration of linear range; I.c., low concentration of linear range (N= 3). dLimit of detection given in molarity of original analyte sample, taken as blank signal + 3RSD of blank signal.
DISCUSSION The observed enhancements in CL intensity are due to a combination of changea in fluorescence efficiency, excitation efficiency, and reaction rate. For maximum efficiency, a high concentration of the intermediates must be produced and complexed with the fluorophor for maximum energy transfer. Both fluorescence efficiency and excitation efficiency changes show that more than one species are associated with the micelles. There are several possible species involved. Possible micellar association includes METQ or some intermediates or product derivatives of METQ. This is indicated by the variation of CL enhancement with varying METQ concentration. The association of METQ with the micelles might increase the local concentration of METQ and protect it from the hydrolysis, thus enhancing the excitation efficiency. If METQ is associated with the micelles, hydrogen peroxide anions must penetrate to react. This process is favored by longer chain surfactants, which form looser micelles. It is also possible that the micelles stabilize products or reaction intermediates. Fluorophors are also associated with micelles, as shown by the changea of fluorescenceemission spectra. The fluorescence efficiency of ANS is enhanced with all kinds of micelles, but the fluorescence efficiency of RH B is enhanced only with micelles of SDS and CTAC. The greatest CL enhancement is observed for ANS in Brij 35 in which the polyethylene chain provides the most favorable environment for penetration and association of all relevant species.
ANALYTICAL IMPLICATIONS Determination of Fluorophors, Fluorophor-Labeled, Proteins, and Hydrogen Peroxide. Although there are many analytical applications of the peroxyoxalate CL reaction in mixed solvent systems to measure hydrogen peroxide and fluorophor compounds, there are few analyses using aqueous peroxyoxalate CL. Analytical figures of merit are evaluated for the analysis of fluorophors and hydrogen peroxide. The analytical parameters are generally improved with the introduction of micelles (Table VI). The detection limits of dansyl chloride are improved by factors of 2.5 and 5 in the
micellar media of Brij 35 and CTAC. In Brij 35, the detection limit of ANS is improved 3 times. The dynamic linear ranges of RH B and ANS are increased by 2-3 orders of magnitude. Precision is generally improved in micellar media. However, although the slopes of calibration curves, which represent the sensitivities, are increased with micelles, the background signal is also increased,which restricts the improvement of detection limits. The detection limits of albumin-dansyl chloride and avidin-RH B are not improved with micellar medium. By using ANS as a fluorophor, the detection limits of hydrogen peroxide are improved by factors of 3 and 22 in Brij 35 and CTAC micelles. With RH B, only 2-fold improvement of the detection limit is observed in CTAC micelles. Despite improved sensitivity, the high background limits detection of hydrogen peroxide. The dynamic linear ranges are extended by 1-2 orders of magnitude due to the formation of micelles. Micelles enable improved detection of fluorophors and hydrogen peroxide but not fluorophor-labeled proteins. The large proteins probably inhibit the association of fluorophors with micelles. Therefore, the choice of labeled analytes must allow for inclusion with the micelles for the maximum sensitivity. The high background signal, which was caused by nonrepeatable pipeting reactants and mixing by static system, appears to be the major limitation for the further improvement of detection limits, despite the increased sensitivities in micelles. It is expected that using a constant background technique such as a flow injection analysis, which can provide reproducible sample introduction and mixing, may further improve both precision and detection limits. Effects Due to Protein. CL measurements are ideally suited to the assay of aqueous clinical samples but suffer from imprecise mixing and efficiency problems of mixed solvent systems. An aqueous CL measurement system would be beneficial in alleviating these difficulties. However, matrix effects due to proteins must be considered since it has been reported that proteins also enhance peroxyoxalate CL signals (27). To evaluated these potential interferences, the effect of a protein matrix is also studied. The effects of protein albumin on this micelle-enhanced CL system are evaluated (Figure 5), and CL enhancement is observed. In this study, with RH B, the presence of albumin
ANAL.TICAL CHEMISTRY, VOL. 63, NO. 17, SEPTEMBER 1, 1991
40
-
a a 0
--e.
a
30-
. 20 -
a
-0 E
a
0 -
v
10
0
+
:
+ a
A
A . I
+ a
A
a
+
*
* *
a .
I
A
I
c
.
A I
enhances the CL intensity, and the highest intensity is achieved (10 times) at an albumin concentration of 5 X 10" M. Within the albumin concentration range of (0.5-500) X 10" M, CL is enhanced by the protein in both Brij 35 and CTAC media. In contrast, the chemiluminescencewith ANS is decreased by the presence of albumin. In a normal serum sample, the concentration of albumin is between 44 and 66 mM; standard additions are necessary for calibration to avoid matrix effects.
ACKNOWLEDGMENT We thank A. Mohan and R. Schulze for their help with the METQ synthesis.
LITERATURE CITED (1) Rauhut, M. M. A m . Chem. Res. 1909, 2 , 80. (2) Chandross, E. A. Telreheaon Lett. 1903, 761.
1771
(3) Rauhut, M. M.; Bollyky, L. J.; Roberts, B. G.;Lay, M.; Whitman, R. H.; Iannotta, A. V.; Semsei, A. M.; Clarke, R. A. J . Am. Chem. Soc. 1967, 89, 6515. Bdlyky, L. J.; Whitman, R. H.; Roberts, B. G.; Rauhut, M. M. J . Am. Chem. Soc.1967, 80, 6523. Tseng, S. S.; Mohan, A. 0.; Halnes, L. 0.;Vizcarra, L. S.; Rauhut, M. M. J . Org. Chem. 1979, U , 4113. Givens. R. S.; Schowen, R. L. In chmhnkwcsnce and photochamlcal Reaction DetecMon In Chrometogra@~y;Birks. J. W.; Ed.; VCH: New York. 1989: Chaoter 5. (7) Curtis, T. G.;Sek.W: R. J . Chfomtogr. 1979, 734, 343. (8) Seitz, W. R. I n Mfhods in En2ymdogy; Deiuca, M. A., Ed.; Academic Press: New York, 1978 Voi. 57, p 445. (9) Scott, G.; Seltz, W. R.; Ambrose. J. Anal. Chim. Acte 1960, 715, 221. (10) Mlyaguchi, K.; Honda. K.; Imai, K. J . Chfomatogr. 1964, 316, 501. (11) Kobayashi, K.; Sekino, J.; Honda, K.; Imai, K. Anal. Blochem. 1961, 112, 99. (12) Sigvardson, K. W.; Bkks, J. W. Anal. Chem. 1963, 55, 432. (13) Siardson. K. W.; Kennish, J. M.; Birks, J. W. Anal. Chem. 1984, 58, 1096. (14) Mellbin, G.; Smith, 8. E. F. J . Chfomatogr. 1964. 312. 203. (15) Mohan, A. 0.;et ai. Aqueous Peroxyoxalete ChemMmlnescence, AD121396; Defense Technical Information Center, Cameron Station: Alexanbla, VA, 1982. (16) Thompson, R. 8.; McBee, S. E. S. kingmuif 1968, 4 , 106. (17) Wooif, E. J.; @ayeski, M. L. J . Lumlnesc. 1987, 39. 19. (18) Hlnze, W. L.; Riehi, T. E.; Singh, H. N.; Baba, Y. Anal. Chem. 1964, 56, 2180. (19) R M i , T. E.; Malehorn, C. L.; Hinze, W. L: Analyst 1986, 7 1 1 , 931. (20) Lasovsky, J.; eambal, F. Bioelectfod". Bhnerg. 1966, 15, 95. (21) Cohen. M. L.; Arthen, F. J., Jr.; Tseng, S. S. European Patent Application Publication 0096749, Dec 28. 1983. (22) Tseng, S. S.; Rauhut, M. M. U.S. Patent 4,282,357, 1981. (23) Jacobsen. C.; Jacobsen. J. Bkxhem. J . 1979, 181, 252. (24) Mukerjee, P. I n Micellkatlon, Sdubillzation, and Mlcroemulslons ; Mittal, K. L.. Ed.; Plenum: New York, 1977; Voi. I, p 241. (25) Mukerjee, P.; Mysels, K. J.; Kapauan, P. J . Phys. Chem. 1967, 71, 4166. (26) Kamenka, N.; Lindman, B.; Fontell, K.; Chorro, M.; Brun, B. C . R . Hew. Seances Aced. Sci., Ser. C 1977, 284, 403. (27) Woolf, E. J. Ph.D. Thesis, Seton Hall University, South Orange, NJ, 1986.
R E Cfor~review March 25,1991. Accepted June 10,1991. This work was supported in part by a grant from the donors of the Petroleum Research Fund, administered by the American Chemical Society.
