Anal. Chem. 1988, 60,834-837
034
Immobilized Luminol Chemiluminescence Reagent System for Hydrogen Peroxide Determinations in Flowing Streams Kevin Hool’ and Timothy A. Nieman*
Department of Chemistry, University of Illinois, 1209 West California Street, Urbana, Illinois 61801
Slllca particles are modified wlth an (amlnoalkyl)sllane to , and allow a glutaraldehyde attachment of lumkrd, horseradish peroxidase. These knmoblllzed chemHumInescent reagents are incorporated Into various flow lnjectlon schemes to allow sensitive detectlon of hydrogen peroxide. Additionally, a flow Injection scheme is described as uslng an electrode to replace the lmmoblllzed chemllumlnescent catalyst. I n all of these systems, no reagent solutions are necessary for flow Injection analysls detmlnatlon of hydrogen peroxlde. The detectlon lhntl for hydrogen peroxide Is approxknately 100 pmol.
The luminol reaction has long been known for its use in hydrogen peroxide determinations in flowing streams (1-3). Luminol is oxidized in an alkaline solution in the presence of a catalyst to form 3-aminophthalate in an excited state luminol
+ catalyst + peroxide
-
3-APA
+ light (425 nm)
The reaction can be made pseudo first order with respect to the peroxide concentration so that the emitted light intensity is directly proportional to the analyte (peroxide) concentration. Until recently, reagent solutions for a luminol reagent stream and a catalyst reagent stream were necessary to perform a peroxide determination via flow injection analysis (FIA). We have previously reported the first use of a covalently immobilized chemiluminescent (CL) reagent (luminol and isoluminol) ( 4 ) and electrogeneration of luminol CL (5) to quantitate peroxide in flowing streams. The importance of immobilized reagents for chemical analysis is well established with many analytical applications appearing recently in the literature. Reagents have been immobilized for enzymatic processes (6-8), preconcentration of metal ions (9, IO), and luminescence processes (11, 12). This paper reports the continuing development and characterization of immobilized CL reagents for use flow injection analysis. Specifically, our goal has been to achieve a detection scheme for peroxide based upon the luminol reaction and using entirely immobilized or “solid-state” reagents. In such a system all reagents are immobilized and no reagent solutions are needed. This paper reports the first use of a flow injection detection scheme based on luminol where all of the reagents involved in the CL reaction are in immobilized or solid-state format. EXPERIMENTAL SECTION Instrumentation. The flow injection systems used in this work are shown in Figures 1 and 2. A Rainin rabbit peristaltic pump was used to generate the flowing streams indicated in these figures. A Rheodyne Model 5020 sample injection valve (with 100- or 250-pL loop sizes) was used to inject the sample into the flowing stream. Teflon tubing (0.8 mm id.) was used between all components in the flow injection systems. The particles containing
Current address: Dow Chemical Co., Midland, MI.
immobilized luminol were packed into short Plexiglas columns (13)with a volume of about 0.75 mL and holding 0.3-g particles. The observation cell used in FIA system I1 consisted of a Teflon spacer (providing approximately 175-pL volume) sandwiched between a Plexiglas front plate and a reflective Teflon backing plate. This cell has been used previously in this laboratory and is described in more detail elsewhere (14). The flow cell containing the gold electrode is a commercially available LCEC thin layer flow cell (BioanalyticalSystems TL-6A) which has been modified with a Plexiglas front plate to allow transmission of the luminescence and is described in more detail elsewhere (5). The flow cell(s) were placed directly in front of a Hamamatsu 1P28 photomultiplier tube (PMT) which was biased at -900 V for all light measurements. The PMT anode current was amplified by a Pacific Precision Model 126 photometer and output to a Pedersen Model 37 strip chart recorder. A Hewlett-Packard HP M50A diode array spectrophotometer was used to assay, via UV-vis absorbance, the amount of reagent immobilized on the silica and controlled-pore glass. A Bioanalytical Systems BAS-100 electrochemicalanalyzer was used to control the potential of the gold electrode. A AgJAgC1 reference electrode was used along with a stainless steel counter electrode. Reagents. Silica gel (60-200 mesh) obtained from Baker was sifted through wire mesh sieves, and particles with a size of 180-250 pm were collected for use as the support for the immobilized CL reagents. This silica has a pore diameter of 60 8, ( 4 ) and an estimated surface area of 200 m2/g. The controlled-pore glass (CPG) was obtained from Electro-Nucleonicsin 80-120 mesh size with a mean pore diameter of 250 8, and a surface area of 62 m2/g. Luminol and sodium cyanoborohydride were obtained from Aldrich and used as received. Hemin (type I), hemoglobin (bovine), and horseradish peroxidase (type I, EC 1.11.1.7)were obtained from Sigma Chemical Co. Glutaraldehyde was obtained from Eastman Kodak. The (3-aminipropy1)triethoxysilanewas purchased from Petrarch Systems. All other reagents used were reagent grade chemicals and used without further purification. All solutions were prepared with water purified by a MilliporeJContinental water purification system. Immobilization Procedures. The general immobilization scheme used in this work to bind the reagents t o a silica support is shown in Figure 3. A glutaraldehyde molecule is used to bridge from a primary amino group on the reagent (H2N-R) to a primary amine functionality on the silanized silica surface. The procedure for immobilizing luminol on silica and subsequent assay (via UV-vis absorbance) has been described in greater detail ( 4 ) . The luminol was immobilized in an amount of 15 mg of luminol/g of silica (86 pmol of luminol/g of silica). Silica particles containing luminol immobilized via the glutaraldehyde method were a deep red color. This color was characteristic of the amount of glutaraldehyde bound to the silica particles and not the bound luminol. Particles that were faint red or light orange indicated that only a little glutaraldehyde was bound and resulted in reduced luminol yields. The first attempt to immobilize the CL catalyst was to simply adsorb hemin on silica. Approximately 5 g of silica (acid washed and dried) were added to 30 mL of water and 5 mL of 1mM hemin for 24 h with occasional stirring. The amount of hemin adsorbed was determined by UV-vis absorbance (similar to the luminol assay). This procedure resulted in 0.125 mg of hemin adsorbed/g of silica. The silica particles changed from pure white to dark green in color. The product was filtered, washed with water, and
0003-2700/88/0360-0834$01.50/0t 2 1988 American Chemical Society
ANALYTICAL CHEMISTRY. VOL. 60. NO. 9, MAY 1, 1988 835 w**te
Flgure 1. Flow Injection system 11. The sample (hydrogen peroxidelbuffer)is injected Into a flowing stream of water which flows through a reservoir containing the immobilized luminol. The high pH of Vw sample plug h a v e s a small amount of knrrlnol from Vw support. The peroxidelbufferlluminol plug then combines with the CL catalyst (5 ~ l . 4 hemin in pH 11 phosphate buffer)at the mixing tee and enters into a small volume flow cell where the reaction takes place and the luminescence is measured with a PMT. wllate
Flow injectlon system 111. The sample (hydrogen peroxidelbuffer)is Injected into a flowlng stream of water and then flows through the reservoir where a small amount of luminol is cleaved from the support. This peroxidelbufferliumlnoi plug then enters into a cell which contalns the CL catalyst. This catalyst can be either an immobilized one (such as hemin, hemoglobin. OT tmrseradlsh peroxidase) or an electrode contained in the flow ceil. The luminol reaction proceeds and the luminescence is measured with a PMT. Figure 2.
0
&CH,NH~
+
0 .
~ II( c H ~ II ) ~+ c HH~ N - R
NaCNBH3 NHCH (CH ) CH2NH-R 2 2 3
~ l ~ u3.r ew r a b m immobnization mem~d.~n (am+makylhilane is used to modify a silica surface. A glutaraldehyde molecule is then uwK( to tha amhw soup from tha silane to an amino group from the reagent to be immobilized. The resun is two Imine bonds formed (Schiffbase reaction) which can be hydrolyzed whh base. This is the final product in the case of the immobilization of luminol. However, to prevent the hydrolysis of the reagent from the support. the imine bonds can be reduced with sodium cyanoborohydride as shown. stored dry. When the material was to be used, the particles were placed into water and sonicated (ultrasonic bath) for about 5 min to wet all pores. Hemoglobin was immobilized to CPG via the glutaraldehyde method. CPG particles containing the silane and glutaraldehyde additions were used as the support for the immobilized hemoglobin. A 50-mg amount of hemoglobin was dissolved in 50 mL of 0.1 M phosphate buffer (pH 7.5). The CPG particles were added to thissolution and allowed to stand for 24 h with occasional
stirring. The particles were filtered, washed, and stored in a pH 7 buffer until used. The UV-vis absorbance assay procedure indicated immobilization of 10.3 mg of hemoglobin/g of CPG. Horseradish peroxidase (HRP) was immobilized to CPG via the glutaraldehyde method with subsequent cyanoborohydride (CBH) reduction of the imine bond as shown in Figure 3. The glutaraldehyde containing CPG (20 g) was added to 100 mL of 0.05 M phosphate buffer (pH 7.5) containing 320 mg of HRP and allowed to sit for 24 h with occasional stirring. The product was washed, filtered, and stored in a pH 7 buffer. The UV-vis assay showed immobilization of 3.2 mg of HRP/g of CPG. A portion of this product was then treated with 10 mg of sodium cyanoborohydride/g of particle. Approximately 24 h was allowed for the reduction to occur. The product (slightly lighter in color) was washed, filtered, and stored in a pH 7 buffer until further use. A similar batch of reduced product was done by using 65 mg of CBH/g of CPG. RESULTS AND DISCUSSION Previous Work. In our previous work with immobilized luminol(4) a single flow cell served as both the reservoir to contain the immobilized luminol and the location where CL emiasion was monitored. That approach will be denoted FIA system I to facilitate comparisons with the work reported here. Because with FIA I the luminescence measurement was made in the presence of the dark (ahsorbing) and light scattering medium of the silica-bound luminol, losses in sensitivity were likely. It has been shown (4)that luminol is cleaved from this support (likely via hydrolysis of the imine bond shown in Figure 3) by the base required in the CL reaction. Therefore, the CL reaction need not take place within this absorbing and scattering medium. Rather the flow injection scheme can he modified to take advantage of luminol being cleaved from the support prior to the CL reaction. Flow Injection System 11. With FIA I1 the immobilized luminol was contained in a small flow-throughcell (which acta as a reagent reservoir) upstream from the flow cell where the luminescent measurement was made. In this approach, the base necessary for the CL reaction and cleavage of luminol was added along with the hydrogen peroxide to be injected. Passing a continuow stream of alkaline solution through the luminol reservoir would continually cleave luminol from the support and deplete the reagent rapidly. It is only neceasary to have luminol significantly cleaved from the support while the analyte is present in the flowing stream. Because some of the hase in the injection volume is consumed (in hydrolysis of luminol) an addition (to the peroxide solutions) of a high-capacity buffer (0.2 M phosphate, pH 11.0) was selected instead of simply NaOH. The buffer was to ensure that the pH would remain a t an optimal level for luminol CL and hopefully improve precision. The plug of luminol/peroxide/buffer then combines with the catalyst a t a mixing tee and flows into the observation cell where the CL reaction takes place and the light is measured. An additional advantage to be realized with this system is that the observation cell, having smaller cell dimensions as compared to the cell used in FIA system I will present a better collection geometry to the PMT; as a result approximately 2.5 times more light is measured for FIA system I1 at a given hydrogen peroxide concentration. Precision and detection limits for the two systems are essentially the same (approximately 5% relative standard deviation and l rM peroxide, respectively). Immobilized Catalyst. In order to eliminate yet another flowing stream from the previous flow injection system(s), and thus achieve a totally "solid-state" detection scheme (FIA system 111) based on the luminol reaction, i t was necessary to immobilize the catalyst and incorporate it into the system. It was noticed in previous work (4) that silica particles adsorbed the hemin catalyst (which was then continually flowing through the cell containing the silica). Therefore, in one immobilized catalyst approach, hemin was adsorbed to silica
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 9, MAY 1, 1988
as described and used as the CL catalyst. The adsorbed hemin was incorporated into the flow injection system shown in Figure 2. The sample of peroxide and buffer was injected into a flowing stream of water where it was carried into the reservoir containing the immobilized luminol, cleaved a small amount of reagent, and entered into the cell (in this case the same cell used in FIA I) containing the adsorbed hemin. The CL reaction takes place and the luminescence is measured with a PMT. Multiple injections of a single concentration of hydrogen peroxide resulted in a monotonic decrease in the measured CL signal. Apparently, the adsorbed hemin washes away from the support in substantial amounts with each injection. This is a problem primarily because (unlike immobilized luminol) very small molar amounts (0.19 pmol/g of silica) of hemin were adsorbed; loss of a substantial amount with each injection depletes the catalyst rapidly. Because we could not produce controlled release of the adsorbed hemin (as we have with the immobilized luminol), it was necessary to find another method of immobilizing the CL catalyst. Glutaraldehyde-BoundCatalyst. Because the glutaraldehyde-bound luminol resulted in an immobilized reagent which can be hydrolyzed from the silica in a controlled fashion, the glutaraldehyde method was selected as the method of immobilization for the CL catalyst. Because hemin has no available primary amino groups (necessary for glutaraldehyde method of immobilization) other catalysts were selected for immobilization. Both hemoglobin and horseradish peroxidase (HRP) are effective catalysts for luminol chemiluminescence (15, 16) and contain free amino groups for use in immobilization. The flow injection system used was the same one used for the adsorbed hemin. Multiple injections of a single concentration of hydrogen peroxide/buffer still resulted in a monotonic decrease in the measured CL signal, although it was a much slower decrease injection to injection than that experienced with the adsorbed hemin. In fact, an entire peroxide working curve can be generated with good correlation (R = 0.99) and only slightly degraded precision (as compared with FIA 11). Again, because of low catalyst loading (10 mg of hemoglobin/g of CPG),it is likely that a significant amount of catalyst was being consumed (hydrolyzed from the support) and its loss was responsible for the monotonic decrease observed. Both hemoglobin and HRP were immobilized to silica supports in identical fashion and used as the CL catalyst with aqueous luminol. The same monotonic decrease was noted with both immobilized reagents; however, the signals from the immobilized HRP resulted in slightly larger CL signals as compared to the immobilized hemoglobin. In order to attain a successful immobilized CL catalyst, it was necessary to either immobilize the catalyst in much larger amounts (at least comparable to bound luminol) and/or find a way of retaining the catalyst within the support. Because of the unlikely possibility of drastically increasing the amounts of catalyst immobilized, we examined methods for retaining the catalyst. One possible solution is presented in Figure 3. The imine bond formed as a result of the Schiff-base reaction is likely the bond being hydrolyzed by base and releasing the reagent into the flowing stream. By reducing the imine bond with sodium cyanoborohydride (CBH), we are left with a bond not easily hydrolyzed with base. Sodium cyanoborohydride has previously been used as a stabilizing reagent for immobilization of enzymes via the glutaraldehyde method (17). Ideally, the CL catalyst would remain intact within the flow cell and retain its good catalytic ability. When the immobilized HRP (with reduced imine bond) was used as the catalyst, the familiar monotonic decrease in CL signal appeared for only the first few injections of peroxide
Table I. Comparison of Immobilized Catalyst Systems signal for 80 W MHzOz
system hemoglobin HRP/CBH electrode
intensity, nA
precision, % RSD
6.8 1.6 1.0
18 11, 2” 4
linear range, pM
40-600 40-600
4-600
detection limit, p M 10 2“ 1
“10 mg of CBH/g of CPG yielded 11% RSD and 65 mg of CBH/g of CPG yielded 2% RSD. The two different amounts of CBH yielded the same intensity and linear range. The detection limit listed is for 65 mg of CBH/g of CPG.
and buffer. The precision thereafter was essentially identical with that observed with FIA system I1 (approximately 5 % relative standard deviation). The initial decrease in measured CL intensity for injections of a single concentration of peroxide is likely due to bound HRP that did not have its linkage reduced with CBH being cleaved from the support. In this case, the monotonic decrease will continue until essentially all of the catalyst with unreduced linkages is hydrolyzed from the support. The different amounts of CBH (10 mg/g vs 65 mg/g of particles) used in the reduction phase of the immobilization resulted in very little change in the results. In each case essentially the same amounts of light were measured for injections of peroxide and buffer. However, when compared to the solution phase catalyst (FIA 11),over 1 order of magnitude less light was detected. The linear working range for hydrogen peroxide (40-600pM) was reduced by nearly one order of magnitude as compared to FIA system 11. However, the peroxide detection limit remained the same at approximately 1 pM. Use of an Electrode as the CL Catalyst. The use of an electrode to catalyze the oxidation of luminol has been studied extensively (5). Essentially, the electrode performs a oneelectron oxidation of luminol, and this intermediate can undergo further oxidation with a solution-phase oxidant such as oxygen or hydrogen peroxide to produce the excited-state species. Because oxygen is normally present in aqueous solutions, the hydrogen peroxide (analyte) generated CL is measured above a background. The oxidation of luminol at a gold electrode occurs at approximately +0.25 V (vs Ag/ AgC1). The emission is confined near to the surface of the electrode. The electrode can replace the immobilized solution phase catalysts in the totally “solid-state”detection scheme (i.e. FIA 111). Hydrogen peroxide is injected into a flowing stream of 0.2 M KNOB(supporting electrolyte) along with buffer and flows into the reservoir containing luminol, where a small amount is cleaved and flows into the flow cell containing the electrode. The flow cell containing the electrode maintains all the advantages of the flow cells used in FIA I1 and FIA I11 (with immobilized catalyst). Collection geometry is different with the electrode cell since the luminescence is confined to the area of the electrode and the electrode surface is a different distance from the PMT than the solutions in the flow cells of FIA I1 and FIA I11 are. Experimentally we find that lower intensities are detected with the electrocatalyzed chemiluminescence. A comparison of various aspeds of peroxide detection using three different solid-state catalysts in an FIA I11 approach is given in Table I. It should be remembered that the immobilized hemoglobin and HRP used the same flow cell, but the electrode results were obtained in a different flow cell. As a result, one can say that use of immobilized HRP results in lower intensities than use of immobilized hemoglobin; however, one cannot easily compare the intensity observed with the electrode to the other intensities. The highest concentration
ANALYTICAL CHEMISTRY, VOL. 60, NO. 9, MAY 1, 1988
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We have operated these FIA systems for over 10 h of continuous injections. Over that time we observe no degradation in peak width or precision. For example, injections of the same solution 4 h apart yielded peak heights within 1.4% of each other.
CONCLUSIONS
40
Flgure 4. Recorded CL intensities for replicate injections of hydrogen peroxlde (concentratkns given in micromoles per Mer) via F I A I11 with the electrode as catalyst.
tested was 600 rM, and none of the working curves had yet shown deviation from linearity at that concentration. Both the glutaraldehyde-bound HRP (reduced with CBH) and the electrode yield acceptable detection limits, working ranges, and precision. Use of the electrode does yield the best detection limit (approximately 100 pmol) and working range. The FIA I11 approach with immobilized luminol and an electrode as the catalyst yields the best hydrogen peroxide working range and detection limit that we have obtained to date for various approaches using immobilized luminol. Figure 4 shows replicate injections for hydrogen peroxide at various concentrations using an electrode as the catalyst. The analyte signal is measured above background which is established by injecting the buffer solution (blank). The blank signal is due to oxidation of the luminol intermediate (generated with the electrode) by dissolved oxygen. An injection of 100 MLtakes approximately 3 min to clear the system. System Ruggedness. Because the hydroxide in the sample volume is responsible for release of luminol from the immobilized material, it is necessary to consider the effect of the pH of the buffer that is added to the sample prior to injection. We have used borate and phosphate buffers to span the region from pH 9 to pH 11.5. The observed CL intensity increases with increasing pH. For FIA 11, the CL intensity increases by only 15-20% per pH unit. For FIA I11 with electrogenerated CL, the net CL intensity increases W 5 5 % per pH unit. With FIA I1 the sample pH influences only the amount of luminol released because the catalyst solution is buffered to control the CL reaction conditions. The greater sensitivity to sample pH in FIA I11 arises because the pH influences both the CL reaction and the amount of luminol released in this single-channel system.
837
This hydrogen peroxide detection approach, based on immobilized CL reagents, is attractive for extension to determination of analytes which are substrates in enzyme reactions that yield hydrogen peroxide as a product. In these cases, an immobilized enzyme reactor would be included in-line prior to the immobilized luminol. The initial work in this laboratory with immobilized CL reagents has focused primarily on use in flow injection analysis. However, use of immobilized CL reagents is not limited to FIA. The CL reagents could be immobilized?m other surfaces in quantities appropriate for single analyte determinations. Registry No. Hydrogen peroxide, 7722-84-1; gold, 7440-57-5; hemin, 16009-13-5.
LITERATURE CITED Seitz, W. R. CRC Crlt. Rev. Anal. Chem. 1981, 73, 1-58. Krlcka, L. J.; Thorpe, 0. H. 0. Analyst (London) 1983, 108, 1274-1296. Kricka, L. J.; Carter, T. J. N. I n Cllnlcal and Blochemlcal Lumlnescence; Krlcka, L. J., Carter, T. J. N., Eds.; Marcel Dekker: New York, 1 9 8 2 Chapter 8, pp 153-178. Hooi, K.; Niernan, T. A. Anal. Chem. 1987, 59, 869-872. VanDyke, D. A.; Nieman, T. A., submltted for publication in Anal. Chem Bowers, L. D.;C a r , P. W. Anal. Chem. 1978, 48, 544A-559A. Gray, D. N.; Keyes, M. H.; Watson, B. Anal. Chem. 1977, 49, 1070A-1078A. Johansson, G.; Ogren, L.; Olson, B. Anal. Chlm. Acta 1983, 745, 71-85. Marshall, M. A.; Mottola, H. A. Anal. Chem. 1983, 55, 2089-2093. Luhrmann, M.; Steiter, N.; Kettrup, A. Fresenlus’ Z . Anal. Chem. 1985. 322, 47-52. Miller, J. N. Pure Appl. Chem. 1985, 57, 515-522. Gubitz, G.; VanZoonen, P.; GooiJer, C.; Veithorst, N. H.; Frei, R. W. Anal. Chem. 1985, 5 7 , 2071-2074. Koerner, C. A.; Nleman, T. A. Anal. Chem. 1988, 58, 116-119. Swindlehurst, C. A. (Koerner); Nieman, T. A. Anal. Chim. Acta, in press. Neufeld, H. A.; Conkiin, J.; Towner. R. D.Anal. Blochem. 1985, 12, 303-308. Nekimken, H. L. Ph.D. Thesis, Unlversity of Illinois, Urbana, IL. 1986. Shoup, R. E. Bioanaiytlcai Systems, W. Lafayette, IN, personal communicatlon, August 1986.
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RECEIVED for review August 3,1987. Accepted December 22, 1987.
