Anal. Chem. 1987, 59, 869-872
869
Chemiluminescence Analysis in Flowing Streams with Luminol Immobilized on Silica and Controlled-Pore Glass Kevin Hool and Timothy A. Nieman* Department of Chemistry, University of Illinois, 1209 West California Street, Urbana, Illinois 61801
An (am1noalkyl)sllane was bound to the partlcie and glutaraldehyde used to bridge between that amine group and the amine group on lumlnol. Due to lumlnol solubliity conslderatlons, the glutaraldehyde reaction was carried out in an ethanol/dlmethyl sulfoxide solvent mixture. Loadings obtained were 29 pM iumlnol/g of controlled-pore glass and 86 pM luminoi/g of silica. There Is no evidence of any degradation of the lumlnol to 3-amlnophthalate. The immobliired material was packed Into a flow cell and used In a flow injection system. HPLC analysis of flow cell effluent plus comparison of chemllumlnescence Intensities of lmmoblllzed lumlnol and lmmoblllzed lsolumlnol indicates that the lmmoblllred luminol Is hydrolyzed from the support prior to or during reaction and that the emission occurs in solution. I t Is estimated that over 500 peroxide assays can be done with a gram of silica containing lmmoblllzed lumlnol. For peroxide determinations, the working range and detection limits are essentially the same as those seen with use of dissolved reagents.
fluorophore whose fluorescence was subsequently measured. Other applications of immobilized CL reagents are those used to quantitate a species not involved in the CL reaction, but rather a species that quantitatively liberates the CL reagent from its support. A polysaccharide matrix binding a thiol-modified luminol derivative has been used as a detection scheme for thiols (9); a thiol-disulfide interchange releases the soluble luminol derivative which is subsequently washed from the gel and quantitated by CL with hydrogen peroxide and potassium ferricyanide. A sensitive assay for proteolytic enzymes has been developed by use of an immobilized isoluminol (IO). The isoluminol is linked to a gel with a peptide. The proteolytic action of the enzyme releases soluble isoluminol from the substrate. The CL reagent is then separated from the gel and quantitated a t high pH with peroxide and heamitin. This paper reports the first use of an immobilized CL reagent (luminol and isoluminol) to quantitate peroxide in a flowing stream without prior separation of the CL reagent.
To date, analytical chemiluminescence (CL) determinations have been implemented almost exclusively with dissolved reagents. This paper reports a new way of utilizing CL reagents (luminol here, although others are possible) by immobilizing them to solid supports. Because of the large surface area available on small particles, this offers a way to employ CL reagents at effective concentrations far in excess of their normal solubilities. Additionally, the method is a convenient way to package and contain CL reagents in large quantities suitable for flow through reactors (e.g., flow injection analysis or postcolumn detection for HPLC) intended for long-term use or small quantities intended for single-shot batch reactions. Immobilization of reagents to solid supports has found wide usage in analytical chemistry. Reagents have been immobilized for solid-phase reactions for derivatization in HPLC ( I ) , preconcentration of metal ions ( 2 , 3 ) ,and enzymatic processes (4, 5). In the past decade several approaches have been used to combine the advantages of immobilized reagents with CL reactions. One approach has been to immobilize the “catalyst” of the CL reaction to the solid support. Seitz and Freeman have developed a CL fiber-optic probe for hydrogen peroxide based on the luminol reaction using immobilized peroxidase (CL catalyst) (6). A second approach has been to immobilize the CL reagent which is used irreversibly to quantitate a species involved in the CL reaction. However, unlike the immobilized CL catalyst the reagent immobilized in this approach is a consumed reagent. Luminol-impregnated membranes coupled with peroxidase-glucose oxidase impregnated paper pads along with an instant photographic detection (Polaroid film) scheme have been used for the analysis of glucose (7).Peroxyoxalates have been utilized in a packed bed type reactor for the analysis of hydrogen peroxide in rainwater (8). In this case, the oxalate was not actually immobilized but rather dissolved from a solid form (packed bed) using nonaqueous solvents and upon oxidation with peroxide transferred its energy to an immobilized
EXPERIMENTAL SECTION Instrumentation. The flow injection system used in this work is shown in Figure 1. A Rainin rabbit peristaltic pump was used to pump two channels at equal rates to provide a flow rate of 1.5 mL/min at the cell exit. Channel one contained water as an eluent for the sample delivery system and channel two contained a solution of 5 pM hemin as the CL catalyst. The unbuffered hemin solution had a pH of 9.5. A Rheodyne Model 5020 sample injection loop was used to inject a 100 pL sample aliquot into the stream of water. The catalyst stream and sample stream converge at a mixing tee just prior to the cell containing the immobilized CL reagent. Teflon tubing (0.8 mm i.d.) was used between the injector and the cell. The cell was placed directly in front of a Hamamatsu Model 1P28 photomultiplier tube (PMT). The PMT 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 recorder. Figure 2 depicts the front and side views of the cell which contained the immobilized CL reagent. The cell was made from transparent Plexiglas with a white reflective Teflon backing added to improve sensitivity. The cell creates a 1.5 cm wide X 3.5 cm tall x 0.5 cm thick cavity and a volume of approximately 3 mL. The total cell cavity surface area is exposed to the PMT. The cell requires approximately 1.25 g of particles containing the CL reagent to fill the cell. The void volume is estimated to be 30% of the total cell volume or just under 1 mL. The cell contained fittings (with 1/4-28 threads) at either end to provide a convenient connection to Altex plumbing. The particles were packed into the cell by pipetting a wet slurry into the cell and allowing it to settle. The particles were retained in the cell by placing a disk of Whatman No. 1fiiter paper at each end of the cell. These paper frits were held in place with the Altex connection fittings and allowed the necessary flow without tearing. Solution flow was from bottom to top through the cell as shown. A Hewlett-Packard HP 8450A diode array spectrophotometer was used to assay, via UV-visible absorbance, the amount of luminol immobilized on the silica and controlled-poreglass (CPG) particles. An Altex HPLC system was used to evaluate the eluent flow from the cell containing the immobilized CL reagent. The system was comprised of a Model llOA Altex pump, a Model 210 Altex injector with a 20-pL injection loop, a Wescan anion exchange
I_
0003-2700/87/0359-0869$01.50/0 0 1987 American Chemical Society
870
-
ANALYTICAL CHEMISTRY, VOL. 59, NO. 6, MARCH 15. 1987 waste
-
Peristaltic mmu
Mixing Tee
pl]1;
m i ,
WSf.3,
t
BEFORE IMMOBIUUTION
Injection valve Photometer
flgure 1. Flow injection system using an immobilized CL reagent
detection scheme. Side View Teflon
Front View Plexiglas
WAVELENGTH h m l
Figwe 3. W-vislble absorbance spectrum of luminol. The deaease in absorbance is used to quantitate the amount of luminol immobilized.
1mmobili;ed CL Reagent Flgure 2. Flow cell to contain immobilized CL reagents. column, and a Model 153 AItex 254-nm UV-visible absorbance detector. The mobile phase was a 75 mM acetate buffer (pH 4.7) and flowed a t 1.1mL/min. Reagents. Silica gel (&ZOO mesh) obtained from Baker was sifted through wire mesh sieves and particles with a size of 1W250 pm were collected for use as the support for the immobilized CL reagent. This silica has a mean pore diameter of 60 A (11). Controlled-pore glass (CPG)was obtained from ElectmNudeonics in 80-120 mesh size (125-180 pm) with a mean pore diameter of 522 A. Luminol and isoluminol were obtained from Aldrich and used as received, Hemin was obtained from Sigma Chemical Co. as type I. Glutaraldehyde was obtained from Eastman Kcdak. The (3-aminopropyl)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 hy a Millipore Continental water purification system. Immobilization Procedure. The immobilization procedure used in this work is based on a similar procedure developed in this Laboratory (22)to hind enzyme to CPG. An (aminoalkyl)silnne is bound to the CPG and glutaraldehyde is used to bridge between the primary amine from the silane-CPG to the primary amine of the reagent. Because both luminol and isoluminol contain a primary amine functionality,the immobilization procedure used in ref 12 was used as a starting place. Because luminol has very low solubility at the optimal solution conditions (pH 7.0) the procedure had to he modified extensively. Additionally, the glutaraldehyde reaction is fairly slow in aqueous solution, so a nonaqueous method was developed. The reaction time for glutaraldehyde attachment has been deereased from 2-3 h to 15 min. The silica and CPG were cleaned in concentrated nitric acid under vacuum. The particles were washed with water and dried at 110 "C. The particles were then silanized with (3-aminopropyl)triethoxysilanein a 0.05 M acetate buffer (pH 5) at 90 OC for 2 h. The silanized particles were washed with water and ethanol consecutively and dried a t 100 OC. The particles were placed in ethanol at room temperature and sonicated (Ultra-sonic bath) to wet all pores. Glutaraldehyde was added directly to this solution with stirring. Appearance of a red color indicates the glutaraldehyde attachment to the silane. After a lbmin reaction time, the particles are washed with ethanol. The particleailaneglutaraldehyde product may be dried and stored indefmitely. This feature proved convenient since large amounts may he prepared and these "intermediate" particles may also he used for binding enzymes as well. The CL reagent immohihtion consisted of dissolving 3M) mg of luminol (or isoluminol) in 15 mL of a 50150 vol ?& mixture of ethanol and dimethyl sulfoxide (Me,SO). One
milliliter of this solution was removed for assay purposes. Approximately 5 g of the glutaraldehyde-containing particles was placed into this solution at room temperature. The particles were again sonicated for 5 min to wet all pores. After 12 h another 1-mL sample of the luminol solution was removed to he used to determine (via UV-visible absorbance) the amount of luminol hound. The particles are then washed with generous portions of the ethanol/M&O mixture, ethanol, ethanolfwater, and water. This wash sequence will prevent any luminol not hound from precipitating out onto the particles. The particles with immobilized CL reagent are stored in water at room temperature with no apparent loss of CL reagent over months of storage. RESULTS AND DISCUSSION Amount of Luminol Immobilized. Several methods have been considered to determine the amount of luminol i m m o hilized. Although it is indirect, the most convenient is to determine the amount of luminol removed from solution at the time the immobilization is accomplished. The W-visihle absorbance spectrum of luminol is shown in Figure 3. The spectrum shows absorbance maxima at 298 and 360 nm. This figure shows the absorbance spectrum of a solution containing luminol before and after using that solution to immobilize luminol onto a batch of particles. Measurement of the difference in absorbance at 360 nm from the luminol solution prior to immobilization to the luminol solution after immobilization allows calculation of the amount of luminol removed from solution hy immobilization provided that no luminol degradation has occurred either in solution or on the particles. The absorbance peak at 298 nm is indicative of the 3aminophthalate (3-APA) portion of luminol. Because the ratio of the absorbance of light at 360 nm to that at 298 nm does not change after the immobilization. luminol is considered not to have degraded in solution. The reaction progress was monitored by taking a small portion of the luminol-containing solution at 12,24, and 36 h. No significant decrease in absorbance at 360 nm occurred after 12 h. Results of the assay show that 5.19 mg of luminol/g of CPG (29 pmol of luminol/g of CPG) and 15.25 mg of luminol/g of silica (86 pmol of luminol/g of silica) are immobilized. This large difference is likely due to the larger surface area available on silica. The CPG has a surface area of 40.7 m2/g (taken from manufacturer) while the silica has a surface area of 450 m2/g (22). This difference is also evident in the appearance of the glutaraldehyde-immobilized particles. The silica particles have a much deeper red color, indicating much more glutaraldehyde was immobilized. The loading we find for luminol on silica is similar to the reported loading of 185 e o 1 of Bquinolinol/g of the same variety of silica (11). Given the loading we determined, the packed particle density, and the void volume around packed particles, one can calculate the effective luminol 'concentration" to which an analyte molecule in the void volume is exposed. This is approximately
ANALYTICAL CHEMISTRY, VOL. 59, NO. 6, MARCH 15, 1987
40 mM for the CPG and 100 mM for the silica. These concentrations are much higher than the millimolar level solubility of luminol one normally encounters. Qualitative Observations. Initial observations with the immobilized material in the flow cell (Figure 1)involved using a 5 pM hemin solution (buffered at pH 10.5with 0.1 M borate) in one channel and making injections of peroxide into the water carrier stream flowing in the other channel. Both flow rates were 0.7 mL/min. It was immediately noted that the luminescence was the same blue color characteristic of solution phase luminol CL. It was also noted that the waste stream continued to give chemiluminescence. It appears that some (or all) of the luminol is being stripped from the particles prior to or at the time of the CL reaction. The high pH is believed to be responsible for the cleaving of luminol from the glutaraldehyde linkage. T o examine this possibility, a small amount of the particles containing luminol was placed into a beaker and mixed with 50 mL of the buffered hemin and 10 mL of 10 mM H202. This solution was observed visually in a photographic darkroom. The CL appeared to come entirely from the solution above the particles indicating luminol has been stripped. The particles (dark red in appearance) did not show any visual CL and actually blocked (absorbed) the light when viewed from the bottom of the beaker. HPLC was employed to examine quantitatively the postcell effluent. H P L C Results. T o determine the amount of luminol stripped from the particles (cell was filled with CPG containing luminol) by alkaline solutions, a series of 100-pL injections of NaOH (25 mM) were made into a water carrier stream a t 3-min intervals (time required for each plug to clear the cell) and the postcell effluent was collected and analyzed by using HPLC. The HPLC procedure used here has previously been used to monitor the luminol CL reaction (13). Because no peroxide was injected into the system the postcell effluent should contain luminol intact if the hydroxide hydrolyzes the glutaraldehyde-luminol bond. A series of luminol standards were run through the HPLC to determine retention time for luminol and to provide a working curve for a luminol-concentration determination. Injection of the postcell effluent resulted in a single peak with the same retention time as the luminol standards. Finding only luminol in this effluent indicates that only luminol was immobilized on the particles, and none of the luminol was degraded to 3-APA by the immobilization procedure. Additionally, the observation of luminol in the effluent combined with the observation of postcell chemiluminescence strongly supports the hypothesis that the hydroxide required for the CL reaction strips the luminol from both silica and CPG. This makes the pH of the solution even more critical for an effective system utilizing immobilized CL reagents. If a buffered catalyst is continuously run through the cell, the CL reagent is continuously being stripped from the particles; this reduces the useful lifetime of the particles. To minimize the CL reagent loss, the hemin stream was run unbuffered in water with as low a pH (pH 9.5 required to keep hemin soluble) as possible. With a 1.2 mL/min flow rate through the cell the luminol is stripped at a rate of only 0.007 pmo1,imin a t this pH. The base was added to the peroxide solutions that were to be injected. In this way the CL reagent is being significantly stripped from the supports only while the analyte plug is present in the cell. Because of this restriction, the concentration of hydroxide within the injection volume will strongly affect the CL signal. The amount of luminol released per injection of 100 pL of 25 mM NaOH was 0.1352 pmol. Because the cell holds 1.25 g of particles and the particles contain 30 pmol of luminol/g of CPG and 90 pmol of luminol/g of silica, the cell has a maximum lifetime of 277 injections for CPG and 832 injections for silica.
871
Table I. Effect of [OH-] on the CL Signal for 100 pM H,Oz Using Luminol Immobilized on Silica [OH-], mM
CL intens, nA
[OH-], mM
CL intens, nA
1 5 10
4.60 14.0
25 50
63.7 76.0
27.6 I
A i
10-6
10 -5
[PEROXIDE]
10-4
10 -3
(M)
Flgure 4. Working curves for hydrogen peroxide using luminol and isoluminol immobilized on CPG.
The effect of hydroxide concentration on the CL intensity was also examined. Table I gives the CL intensity (nA) for injections of 100 pL of 100 pM HzOzwith hydroxide concentrations varying from 1 to 50 mM. Above 25 mM hydroxide, there is only slight increase in CL intensity with increasing hydroxide. Therefore, a concentration of 25 mM hydroxide was chosen as a good compromise between sensitivity and CL reagent preservation. It should also be noted that for large concentrations of peroxide a sufficiently large concentration of hydroxide will be required to extend the linear range of the working curve. Immobilized Isoluminol vs. Immobilized Luminol. It is well-documented that isoluminol(4-aminophthalhydrazide) shows less reduction in CL intensity from substitution at the amine group then does luminol(14). To compare the behavior of immobilized isoluminol and luminol, some of each immobilized CL reagent was prepared from the same batch of glutaraldehyde-bound CPG using identical quantities of CL reagent in the immobilization solutions. Working curves for peroxide were prepared with both immobilized luminol and immobilized isoluminol and are given in Figure 4. The slopes of these log-log working curves are very nearly identical (slope = 1.07). The absolute peak intensities for the luminol response to peroxide were on the average 8.5 times more intense than those for isoluminol. This ratio needs to be compared with that normally seen with luminol and isoluminol for the unsubstituted molecules and for molecules with substitution at the aromatic amino group. The luminol/isoluminol intensity ratio has been reported to be about 20 (14). This value might be expected to vary according to the experimental conditions used, and for conditions similar to those used with the immobilized material we observe a ratio of about 5. The important point is that substitution at the aromatic amino group decreases light output with luminol but generally increases it with isoluminol such that the luminol/isoluminol intensity ratio drops dramatically. For instance, with a -CHzCHOHCH2NH2 substitution at one amine hydrogen, the ratio has been reported to be only 0.5
872
ANALYTICAL CHEMISTRY, VOL. 59,
NO. 6, MARCH 15,
1987
1000 r--7----^--T-----
100
-
CPG
-*/
1 ,;A 0-
SILICA
,/
N’ IPEROXIDEI
(M)
Figure 5. Working curves for hydrogen peroxide using luminol imon silica and CPG.
mobilized
( 1 4 ) . Thus, our observation of a ratio as large as 8.5 supports the idea that the CL reagent is being stripped from the support concurrent with or prior to the CL reaction. We are investigating other immobilization schemes which may result in the CL reagent being retained during luminescence and should thus improve the sensitivity for the isoluminol chemiluminescence reaction. However, for use of luminol, maximum emission intensity (due to maximum quantum efficiency) would result by controlled release of luminol from the immobilized material at the time needed; this is the approach that we use. Analytical Performance. Figure 5 shows the log-log working curves for 1 pM to 1 mM hydrogen peroxide using luminol immobilized on silica and CPG, respectively; 5 pM hemin and 25 mM hydroxide were used as before. The slopes of both curves were approximately 1. For peroxide log-log working curves over the concentration range of 20-600 pM least-squares analysis yields a correlation coefficient of 0.998 and a standard error estimate of 0.047. Measurements of replicate injections (three to five injections per data point) over the entire concentration range gave a relative standard deviation of 3% for silica and 4% for CPG. The presence of the bed of particles containing immobilized CL reagent results in somewhat degraded precision. If the particles are removed and dissolved luminol is used, the relative standard deviation is less than 1 %. Also, the observed peaks are 80% wider in the presence of the particles. Very irreproducible results were obtained if the cell contained an air bubble or the packed particles developed cracks or channels through which most of the flow was directed. In-run reproducibility was also of concern since the CL reagent is continuously being used. Figure 6 shows two sets of replicate injections for 100 WM hydrogen peroxide with luminol on silica and 25 mM OH-. All conditions for these two sets of injections were identical. However, between these two sets of injections over 3 elapsed. During that time the immobilized CL material and system
‘
t_
5 min
jIO”*
Flgure 6. Demonstration of in-run reproducibility for injections of 100 p M hydrogen peroxide using immobilized luminol.
were in continuous use with injections being made for a variety of peroxide concentrations between 100 and lo00 pM and with OH- concentrations between 5 and 50 mM. This in-run reproducibility was poorer for the CPG immobilized luminol since it contained less total luminol. A 1-mL sample loop was also tried and, as expected, gave much better signal intensity, 11 vs. 0.6 nA (100-pL loop) for 1 pM peroxide. However the smaller loop was used in the interest of reagent conservation. Detection of submicromolar concentrations of peroxide should be readily obtainable with the use of the larger sample loop size. Another problem encountered with the use of glutaraldehyde as the linkage for luminol is the likely possibility of the particles absorbing a significant amount of the chemiluminescence. Other immobilization schemes are currently being devised which may alleviate this problem and will be addressed in a future publication. Cell design is also being reexamined to take advantage of the luminol cleavage and hopefully improve reproducibility.
LITERATURE CITED (1) Xie, K. H.; Coglan, S.;Krull, I. S. J . Liq. Chromatogr. 1983, 6 , 125-15 1. (2) Marshall, M. A.; Mottola, H. A. Anal. Chem. 1983, 55, 2089-2093. (3) Leyden, D. E.; Luttrell, G. H.; Nonidez, W. K.; Werho, D. B. Anal. Chem. 1978, 4 8 , 67-70. (4) Weetall, H. H. Anal. Chem. 1974, 4 6 , 602A-615A. (5) Bowers, L. D.; Carr, P. W. Anal. Chern. 1976, 4 8 , 544A. (6) Freeman, T. M.; Seitz, W. R. Anal. Chem. 1978, 5 0 , 1242-1246. (7) Carter, T. J. N.; Whitehead, T. P.; Kricka, L. J. Talanta 1972, 29, 529. (8) VanZoonen, P.; Karnrninqa, D. A.; Gooljer, C.; Velthorst, N. H.; Frei, R. N. Anal. Chim. Acta 1985, 174, 151-161. (9) Lipprnan. R. D. Anal. Chirn. Acta 1980, 116, 181-184. (10) Branchini, 6. R.; Salituro. F. G.;Herrnes, J. D.; Post, N. G. Blochem. Biophys. Res. Commun. 1980, 9 7 , 334. (11) Marshall, M. A,; Mottola, H. H. Anal. Chim. Acta 1984, 158, 369-373. (12) Klopf, L. L.; Nieman, T. A. Anal. Chem. 1985, 5 7 , 46-51. (13) Nekirnken H. L. Ph.D. Thesis, University of Illinois, Urbana, IL, 1986. (14) Kricka, L. J.; Carter, T. J. N. Clinical and Biochemical Luminescence; Marcel Dekker: New York, 1982; Chapter 8, p 153.
RECEIVED for review June 24,1986. Accepted November 18, 1986. This research was supported in part by a grant from Dow Chemical Co.
