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Electrochemiluminescence Determination of 2′,6′-Difluorophenyl 10-Methylacridan-9-carboxylate Robert Wilson,*,† Hashem Akhavan-Tafti,‡ Renuka DeSilva,‡ and A. Paul Schaap‡
Department of Chemistry, Liverpool University, Liverpool L69 7ZD, U.K., and Lumigen, Inc., 24485 West Ten Mile Road, Southfield, Michigan 48034
Electrochemical oxidation of the acridan 2′,6′-difluorophenyl 10-methylacridan-9-carboxylate produces the corresponding acridinium ester, which reacts with hydrogen peroxide forming a dioxetanone intermediate. Decomposition of the dioxetanone generates light at 430 nm when it relaxes to the ground state. The effect of pH and hydrogen peroxide concentration on this ECL reaction and on the stability of the acridan were investigated. At pH 8.0 and a hydrogen peroxide concentration of 10 mM, light emission from the ECL reaction was used to determine the acridan concentration with a detection limit of 54 pmol L-1. Results suggest that acridan esters could be used as labels in ECL immunoassays and nucleotide assays. Acridinium esters were discovered in 1964 as a result of investigations into the mechanism of firefly luciferase1 and subsequently developed as labels for immunoassays2,3 and nucleotide assays.4,5 The chemiluminescence (CL) reaction mechanism of these compounds is well known;6 it is shown in Scheme 1. In alkaline solution, nucleophillic attack of a peroxide anion (HOO-) on the 9-position of the acridinium nucleus is followed by internal cyclization leading to the formation of a metastable dioxetanone intermediate. This spontaneously decarboxylates to give the singlet excited state of N-methylacridone, which emits blue light at 430 nm when it relaxes to the ground state. The CL quantum yield is typically between 1 and 10%.7 Nucleophilic attack of peroxide anions on acridinium esters is extremely rapid, but in the absence of peroxide, other nucleophiles can form an adduct with the 9-position of the acridinium nucleus.8 When hydroxide †
Liverpool University. Lumigen, Inc. (1) Chemi- and Bioluminescence; Burr, J., Ed.; Marcel Dekker: New York, 1991. (2) Weeks, I. CL Immunoassay; Elsevier: Amsterdam, 1992; pp 225-255. (3) Weeks, I.; Sturgess, M.; Brown, R. C.; Woodhead, J. S. In Methods in Enzymology; DeLuca, M. A., McElroy, W. D., Eds.; Academic Press: London, 1986; Vol. 133, pp 366-387. (4) Nelson, N. C.; Hammond, P. W.; Wiese, W. A.; Arnold, L. J. In Luminescence Immunoassay and Molecular Applications; Van Dyke, K., Van Dyke, R., Eds.; CRC Press: Boca Raton, Florida, 1990; 293-309. (5) Arnold, L. J.; Hammond, P. W.; Wiese, W. A.; Nelson, N. C. Clin. Chem. 1989, 35, 1588-1594. (6) Pringle, M. J. In Advances in Clinical Chemistry; Spiegel, H. E., Ed.; Academic Press: London, 1993; Vol. 30, pp 89-183. (7) McCapra, F. In Progress in Organic Chemistry; Carruthers, W., Sutherland, J. K., Eds.; Butterworth: London, 1973; Vol. 8, pp 231-277. ‡
10.1021/ac000553s CCC: $20.00 Published on Web 01/18/2001
© 2001 American Chemical Society
Scheme 1. CL Reaction of 2,6-Difluorophenyl 10-Methylacridinium-9-carboxylate, the Acridinium Ester Corresponding to DMC
Scheme 2. pH-Dependent Equilibrium between an Acridinium Ester and Its Pseudobase
is the addend, the product is known as a pseudobase or carbinol (Scheme 2).9,10 Although this reacts with hydrogen peroxide to give N-methylacridone, structural constraints preclude the formation of a dioxetanone intermediate, and therefore, no light is emitted. The need to avoid this “dark reaction” determines the way in which the chemiluminescent reaction is initiated. Typically an immunoassay would start with acridinium esterlabeled antibodies or antigens in buffered solution at a pH suitable for the immune reaction; at this pH, the acridinium ester is (8) Ros, M. P.; Thomas, J.; Crovetto, G.; Llor, J. Can. J. Chem. 1996, 74, 365370. (9) Bunting, J. W.; Chew, V. S. F.; Abhyankar, S. B.; Goda, Y. Can. J. Chem. 1984, 62, 351-354. (10) Littig, J. S.; Nieman, T. A. J. Biolum. Chemilum. 1993, 8, 25-31.
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converted to the inactive pseudobase. After washing away unbound material, an acidic solution of hydrogen peroxide is added to release the label into solution and reactivate the acridinium ester by driving the equilibrium shown in Scheme 2 away from pseudobase formation. Immediately afterward, sufficient sodium hydroxide solution is added to overcome the buffering capacity of the original solution/acidic peroxide and initiate the chemiluminescent reaction. To ensure rapid and reproducible mixing of the solutions, it is usual to inject a large volume into a small volume, which decreases the CL signal and can lead to less precise results. In an attempt to simplify the conventional initiation procedure that is imposed by pseudobase formation, Littig and Nieman investigated the possibility of triggering the chemiluminescent reaction electrochemically.11 The acridinium ester was dissolved in 5 mM ammonium acetate that had a pH of 5.0 to minimize pseudobase formation over the time scale of the assay, but which is not particularly useful for immunoassays and nucleotide assays. This solution was injected into a flowing stream of pH 12 phosphate buffer and pumped into a flow cell. On the way to the flow cell, diffusional mixing at the interface between the two solutions created a narrow region where conditions were suitable for CL, which was triggered in the cell by reducing dissolved oxygen electrochemically. Although this method has some of the advantages normally associated with electrochemiluminescence (ECL), such as fine control over the time and position of the lightemitting reaction, the conditions are a compromise between those required for CL and oxygen reduction and those necessary to avoid pseudobase formation. It would also be necessary to control the concentration of dissolved oxygen to obtain precise results, and this cancels out the increase in simplicity obtained by initiating the chemiluminescent reaction electrochemically. Acridan esters are synthesized by reducing the corresponding acridinium ester with ammonium chloride and zinc.12-14 They do not form addition compounds with nucleophiles, and therefore, the prospect of pseudobase formation with hydroxide ions does not arise.14 A large number of acridans have been synthesized as substrates for the enzyme horseradish peroxidase, which catalyzes their reconversion to the corresponding acridinium ester;14,15 as little as 0.1 amol of this enzyme can be detected by measuring light from the subsequent chemiluminescent reaction. Recently it was shown that one of these acridans (2′,6′-difluorophenyl 10methylacridan-9-carboxylate) can also be reconverted to the acridinium ester electrochemically.16 In this paper, the ECL determination of an acridan ester is reported for the first time, and the potential of these compounds as labels for ECL immunoassays and nucleotide assays is discussed. (11) Littig, J. S.; Nieman, T. A. Anal. Chem. 1992, 64, 1140-1144. (12) Stolle´, R.; Bergdoll, R.; Luther, M.; Auerhahn, A.; Wacker, W. J. Prakt. Chem. 1922, 105, 137-148. (13) Zomer, G.; Stavenuiter, J.; Van Den Berg, R.; Jansen.; E. In Luminescence Techniques in Chemical and Biochemical Analysis; Baeyens, W., DeKeukeleire, D., Korkidis, K., Eds.; Marcel Dekker: New York, 1991; pp 505521. (14) Akhavan-Tafti, H.; DeSilva, R.; Arghavani, Z.; Eickholt, R. A.; Handley, R. S.; Schoenfelner, B. A.; Sugioka, K.; Sugioka, Y.; Schaap, A. P. J. Org. Chem. 1998, 63, 930-937. (15) Akhavan-Tafti, H.; Sugioka, K.; Arghavani, Z.; DeSilva, R.; Handley, R. S.; Sugioka, Y.; Eickholt, R. A.; Perkins, M. P.; Schaap, A. P. Clin. Chem. 1995, 41, 1368-1369. (16) Wilson, R.; Akhavan-Tafti, H.; DeSilva, R.; Schaap, A. P. Chem Commun. 2000, 2067-2068.
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Figure 1. Laminar flow cell used for ECL detection of DMC.
Materials. 2′,6′-Difluorophenyl 10-methylacridan-9-carboxylate (DMC) was synthesized and characterized as described previously.14 Stock solutions of DMC were prepared in 1:1 ethanol/ dioxane. All work was carried out in 10 mM TRIS buffer, pH 8.0, containing 0.1 M NaCl, 10 mM, 1 mM EDTA, and 0.025% Tween20 unless otherwise stated. Stock solutions of DMC were dissolved in buffer to give a final solvent concentration of 0.25%. Light was excluded from all DMC solutions. Equipment. Transparent electrodes were made from ITOcoated glass from Balzer Ltd. (Buckinghamshire, U.K.) that had a sheet resistance of 200 Ω/square. Linear sweep voltammetry was carried out in a three-electrode cell made of a cuvette that was placed in a Perkin-Elmer MPF-43 spectrofluorometer as described previously.17 The reference electrode was a silver chloride-coated silver wire immersed in the solution under study, and the counter electrode was a platinum wire; both electrodes were located in the cell behind an ITO working electrode that had an area of 1 cm2. The ITO surface faced the detector, which was set at 430 nm with a slit width of 20 nm. Potentials were controlled with an in-house-built potentiostat and a waveform generator (PPR1, Hi-Tek Instruments, Buckinghamshire, U.K.). All other work was carried out in an experimental arrangement that has been described previously,18,19 except a laminar flow cell (Figure 1) was used. Solutions were selected and pumped to the flow cell using an ASIA flow injection analyzer fitted with a variopump, six-way valve, and debubbler (Ismatec, Avon, U.K.). The entire system was controlled by a 486/33 PC programmed with Viewdac data acquisition software (Keithley Data Acquisition). The potentiostat and flow cell were made in-house. The body of the flow cell was made of PTFE and sealed to the 10cm2 ITO working electrode with damp-proof double-sided adhesive tape. The working electrode had an area of 1 cm2. Light generated in the flow cell was transmitted through the working electrode (17) Wilson, R.; Schiffrin, D. J. Anal. Chem. 1996, 68, 1254-1257. (18) Wilson, R.; Kremesko ¨tter, J.; Schiffrin, D. J.; Wilkinson, J. S. Biosens. Bioelectron. 1996, 11, 805-810. (19) Wilson, R.; Barker, M. H.; Schiffrin, D. J.; Abuknesha, R. Biosens. Bioelectron. 1997, 12, 277- 286.
electrode to a type 9558QA photomultiplier tube (PMT; Thorn EMI) connected to a model 475R power supply (Brandenburg, U.K.). The output from the PMT was amplified and converted to a voltage that was fed to the PC via a mains frequency filter. Linear Sweep Measurements. DMC was dissolved to a final concentration of 50 µM in buffer containing 10 mM H2O2. Light intensity at 430 nm and current were recorded as the potential was swept in an anodic direction at 10 mV s-1. Real-Time ECL Transients. DMC was dissolved to a final concentration of 5 nM in buffer containing 10 mM H2O2. Transients were obtained by pumping the solution into the thinlayer flow cell and recording the light intensity for a total of 180 s: from 0 to 30 s the applied potential was 0 V, from 30 to 60 s it was 1 V, and from 60 to 180 s it was 0 V. A second set of transients were obtained in the same way except that the time during which a potential of 1 V was applied was extended to a total of 180 s. Effect of Hydrogen Peroxide and pH on ECL. The effect of hydrogen peroxide was investigated by dissolving DMC to a final concentration of 10 nM in buffer containing H2O2 in the concentration range 0-50 mM. The effect of pH was investigated by dissolving DMC to a final concentration of 10 nM in buffer containing 10 mM AMP and 10 mM H2O2 in the pH range 7-10. Measurements were made in the thin-layer cell by integrating the light intensity for 30 s when the applied potential was 0 V and subtracting it from the integral obtained when the applied potential was 1 V for 30 s. Five measurements were made at each concentration/pH during a total time of 40 min. Stability Measurements. DMC (10 µM), in phosphatebuffered saline (PBS) containing 1 mM EDTA and 0.025% Tween20, was assayed for activity during a total time of 8 h, by diluting it to a final concentration of 100 nM with TRIS buffer containing 10 mM H2O2 and measuring the ECL signal as for the investigation of hydrogen peroxide concentration and pH. Detection Limits. DMC in the concentration range 0-10 nM was added to buffer containing 10 mM H2O2. Five measurements were made at each concentration in the same way as for the investigation of hydrogen peroxide concentration and pH. RESULTS AND DISCUSSION Although ECL reactions have been known for many years, efforts to exploit their potential as an analytical technique have only been begun recently.20,21 To date, the most successful development is a combination of ECL and paramagnetic bead technology which has made it possible to carry out highthroughput immunoassays22,23 and nucleotide assays.24,25 The immunoassays are carried out by mixing the sample with ruthenium chelate-labeled haptens or antibodies and paramagnetic beads coated with complementary antibodies. After allowing time for the antibody binding reaction to take place, the solution is pumped into a flow cell where material bound to the paramagnetic (20) Knight, A. W.; Greenway, G. M. Analyst 1994, 119, 879-890. (21) Lee, W. Y. Mikrochim. Acta 1997, 127, 19-39. (22) Deaver, D. R. Nature 1995, 377, 758-760. (23) Yu, H. J. Immunol. Methods 1996, 192, 63-71. (24) Zhao, S.; Consoli, U.; Arceci, R.; Pfeifer, J.; Dalton, W. S.; Andreeff, M. BioTechniques 1996, 21, 726-731. (25) Blackburn, G. F.; Shah, H. P.; Kenten, J. H.; Leland, J.; Kamin, R. A.; Link, J.; Peterman, J.; Powell, M. J.; Shah, A.; Talley, D. B.; Tyagi, S. K.; Wilkins, E.; Wu, T, G.; Massey, R. J. Clin. Chem. 1991, 37, 1534-1539.
Figure 2. Dependence of ECL on potential for 50 µM DMC in 10 mM Tris buffer, pH 8.0, 0.1 M NaCl, 10 mM hydrogen peroxide, 1 mM EDTA, and 0.025% Tween-20. Sweep rate 10 mV s-1.
beads is concentrated on an electrode magnetically. ECL is initiated by applying a positive potential to the electrode either before or after washing away unbound material. These pioneering assays clearly illustrate the advantages of ECL as an analytical technique, including speed, sensitivity, automation, and detection over a wide range of concentrations. Until recently, the only practical alternative to ruthenium chelate labels for ECL were labels based on luminol ECL.26,27 The CL of luminol derivatives such as N-(4-aminobutyl)ethylisoluminol can be triggered by oxidizing them electrochemically in the presence of hydrogen peroxide,28,29 but the reduction in CL quantum yield (sometimes more than 99%) that occurs when these luminol derivatives are used as labels is a well-known limitation.30,31 By contrast, the quantum yield of acridinium esters is higher than luminol and does not decrease when they are used as labels, probably because of the dissociative mechanism.2,32 The main purpose of the current investigation was to find out if acridan esters could be used to harness the potential of acridinium ester CL for ECL assays similar to those currently carried out using ruthenium chelates. Although DMC is the subject of the current investigation, it should be understood that a large number of related acridans have been synthesized and some of these may be more suitable for ECL assays. Acridan esters are stable in solid form with no detectable change in chemiluminescent activity at ambient temperature for more than two years provided light is excluded. DMC was stable in PBS for 8 h, and unlike the corresponding acridinium ester, it is not prone to pseudobase formation. Therefore, labels would remain active during the period when antibody or nucleotide binding reactions were taking place. At the end of this time, (26) Haapakka, K. E.; Kankare, J. J. Anal. Chim. Acta 1982, 138, 263-275. (27) Kremesko ¨tter, J. Ph.D. Thesis, University of Liverpool, U.K., 1995. (28) Gilman, S. D.; Silverman, C. E.; Ewing, A. G. J. Microcolumn. Sep. 1994, 6, 97-106. (29) Sato, M.; Yamada, T.; Horikawa, M. Denki Kagaku 1983, 51, 111-112. (30) Schroeder, H. R.; Hines, C. M.; Osborn, D. D.; Moore, R. P.; Hurtle, R. L.; Wogoman, F. F.; Rogers, R. W.; Vogelhut, P. O. Clin. Chem. 1981, 27, 1378-1384. (31) Simpson, J. S. A.; Cambell, A. K.; Ryall, M. E. T.; Woodhead, J. S. Nature 1979, 279, 646-647. (32) Weeks, I.; Beheshti, I.; McCapra, F.; Cambell, A. K.; Woodhead, J. S. Clin. Chem. 1983, 29, 1474-1479.
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Figure 3. Light and current transients for a potential step from 0 to 1 V for 5 nM DMC in 10 mM Tris buffer, pH 8.0, 0.1 M NaCl, 10 mM hydrogen peroxide, 1 mM EDTA, and 0.025% Tween-20. (A) 1.0 V applied from 30 to 60 s and (B) 1.0 V applied from 30 to 180 s.
the sample would be pumped into a flow cell where acridan ester label bound to a solid phase in suspension would be separated from the rest of the sample and concentrated on an electrode. Then the flow cell would be filled with buffered hydrogen peroxide solution containing EDTA to curtail trace metal ion contaminantcatalyzed oxidation of the acridan and a nonionic detergent (Tween-20) to enhance light emission.14 Linear sweep voltammetry showed (Figure 2) that peak ECL occurred at a potential of 0.75 V, which corresponds to the twoelectron oxidation of DMC to the acridinium ester (Scheme 3) Scheme 3. Electrochemical Oxidation of the Acridan Ester DMC to the Acridinium Ester 2,6-Difluorophenyl 10-Methylacridinium9-carboxylate
followed by the chemiluminescent reaction of the acridinium ester with hydrogen peroxide (Scheme 1). This implies that a potential in excess of 0.75 V would be suitable for analytical work, and the real-time transients shown in Figure 3 record how light intensity varied with time when a potential of 1.0 V was applied to nanomolar concentrations of DMC in the laminar flow cell; most of the current is due to the oxidation of EDTA, which has no effect on the light-emitting reaction. Pseudobase does not form because the acridinium ester is produced in the presence of hydrogen peroxide, which immediately reacts with it at a rate that is ∼104 766
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Figure 4. Dependence of ECL on pH for 10 nM DMC in 10 mM Tris/AMP buffer, 0.1 M NaCl, 10 mM hydrogen peroxide, 1 mM EDTA, and 0.025% Tween-20. Five measurements, at 7-min intervals, were made at each concentration by integrating the light intensity for 30 s after a potential step from 0 to 1 V.
times faster than the rate at which hydroxide ions form an adduct.33 Investigation of the effect that pH (Figure 4) and hydrogen peroxide concentration (Figure 5) had on the ECL of DMC showed that it was stable at pH 8.0 in the presence of 10 mM hydrogen peroxide for at least 40 min. This was considerably longer than the time required to fill the flow cell with solution and even exceeds the time required for most binding reactions, suggesting that it would be possible to carry out separation-free assays provided other reagents and the analyte are unaffected by 10 mM hydrogen peroxide. The pH of the ECL solution (8.0) is close to that at which binding reactions would be carried out (typically 7.5), and therefore, dissociation of bound antibodies or nucleotide duplexes would be unlikely to occur while the flow cell was being filled. A plot of the integrated light intensity against concentration for DMC in the range in 0-10 nM was linear with (33) Weeks, I. CL Immunoassay; Elsevier: Amsterdam, 1992; p 38.
analytical reactions that are carried out using immunoassays,34 even without prior concentration of the acridan ester label on an electrode. It compares favorably with the lower limit of 100 pM reported for luminol ECL35 and is close enough to the 0.2 pM detection limit reported for ruthenium chelates to suggest that related acridans, which produce more intense ECL, are practical alternatives to existing labels.
Figure 5. Dependence of ECL on hydrogen peroxide concentration for 10 nM DMC in 10 mM Tris buffer, pH 8.0, 0.1 M NaCl, 1 mM EDTA, and 0.025% Tween-20. Five measurements, at 7-min intervals, were made at each concentration by integrating the light intensity for 30 s after a potential step from 0 to 1 V.
a slope of 0.51 au/nM. The limit of detection, calculated as the concentration equivalent to mean + 2.5 × SD of the zero calibrator (n ) 9), was 54 pM. This figure meets the requirements of many
CONCLUSIONS Electrochemical oxidation of the acridan DMC converts it to the corresponding acridinium ester, which reacts chemiluminescently with hydrogen peroxide. The electrochemical step simplifies the procedure required to initiate acridinium ester CL and allows the well-known advantages of these compounds to be harnessed for sensitive ECL assays.
Received for review May 15, 2000. Accepted October 26, 2000. AC000553S (34) Cambell, A. K. CL; VCH: Cambridge, U.K., 1988; p 249. (35) Nieman, T. A. J. Res. Natl. Inst. Stand. 1988, 93, 501-502.
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