Anal. Chem. 2007, 79, 8578-8583
8-Hydroxyquinolinyl Azo Dyes: A Class of Surface-Enhanced Resonance Raman Scattering-Based Probes for Ultrasensitive Monitoring of Enzymatic Activity Andrew Ingram, Robert J. Stokes, Julie Redden, Kirsty Gibson, Barry Moore, Karen Faulds, and Duncan Graham*
Centre for Molecular Nanometrology, WestCHEM, University of Strathclyde, 295 Cathedral Street, Glasgow, UK
A series of surface-enhanced resonance Raman scattering (SERRS) based probes for the detection of lipase activity are reported. A number of novel SERRS-active 8-hydroxylquinolinyl azo dyes have been prepared and via synthetic esterification or subsequent enzymatic hydrolysis at the 8-hydroxyl position the SERRS signal can be “switched” on or off. In the first instance, the technique has been demonstrated for the successful detection of lipase from Pseudomonas cepacia, and these new compounds offer a limit of detection of 0.2 ng mL-1 enzyme, up to a 100fold lower limit than observed for benzotriazolyl dyes used in previous studies. The chemical synthesis is straightforward and allows for facile introduction of a wide range of different masking groups, using commonly known synthetic methodologies. The potential for multiplexing analysis of enzyme activity using this technology is presented within. The power of surface-enhanced resonance Raman scattering (SERRS) as an analytical and bioanalytical tool has been well documented, with limits of sensitivity to rival fluorescence techniques,1-4 and an increased capacity for multiplexing.5,6 Pesticide detection,7 explosives detection,8 protein analysis,9 and numerous examples of DNA detection10-13 are just some of the varied applications where SERRS has proven its usefulness. * To whom correspondence should be asddressed. E-mail: Duncan.Graham@ strath.ac.uk. (1) Hildebrandt, P.; Stockburger, M. J. Phys. Chem. 1984, 88, 5935-5944. (2) Kneipp, K.; Wang, Y.; Dasari, R. R.; Feld, M. S. Appl. Spectrosc. 1995, 49, 780-784. (3) Kneipp, K.; Wang, Y.; Kneipp, H.; Perelman, L. T.; Itzkan, I.; Dasari, R. R.; Feld, M. S. Phys. Rev. Lett. 1997, 78, 1667-1670. (4) Rodger, C.; Smith, W. E.; Dent, G.; Edmondson, M. J. Chem. Soc., Dalton Trans. 1996, 791-799. (5) Munro, C. H.; Smith, W. E.; Armstrong, D. R.; White, P. C. J. Phys. Chem. 1995, 99, 879-885. (6) Faulds, K.; McKenzie, F.; Smith, W. E.; Graham, D. Angew. Chem., Int. Ed. 2007, 46, 1829-1831. (7) Alak, A.; Vo-Dinh, T. Anal. Chem. 1987, 59, 2149-2153. (8) McHugh, C. J.; Keir, R.; Graham, D.; Smith, W. E. Chem. Commun. 2002, 580-581. (9) Broderick, J.; Natan, M.; O’Halloran, T.; Van, Duyne, R. Biochemistry 1993, 32, 13771-13776. (10) Graham, D.; Mallinder, B. J.; Smith, W. E. Angew. Chem., Int. Ed. 2000, 39, 1061-1063. (11) Graham, D.; Fruk, L.; Smith, W. E. Analyst 2003, 128, 692-699. (12) Graham, D.; Brown, R.; Smith, W. E. Chem. Commun. 2001, 1002-1003.
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Figure 1. SERRS-inactive BCIP, which upon exposure to alkaline phosphatase is transformed into a colored, SERRS-active, compound.15
Probes for spectroscopic detection of enzyme activity have traditionally been reliant on a fluorescent or colorimetric response,14 but a number of recent publications have reported the potential use of SERRS in this field.15-17 Spectroscopic probes for enzyme activity are used for applications such as detection of diseases, histochemical analysis, or immunoassay techniques. Invariably fluorescence and colorimetry both provide broad electronic spectra, and as such, identification of multiple probes within a sample is not straightforward. As such, multiplexing analysis of enzyme activity is limited for these techniques. Often when multiplexing has been successfully performed, it is used in conjunction with a second technique, for example, capillary electrophoresis,18,19 microarray platforms,20-25 or time-resolved fluorescence,26-29 all of which increase the time and expense, (13) Faulds, K.; Smith, W. E.; Graham, D. Anal. Chem. 2004, 76, 412-417. (14) Invitrogen. Molecular Probes Handbook, 2006. (15) Ruan, C. M.; Wang, W.; Gu, B. H. Anal. Chem. 2006, 78, 3379-3384. (16) Moore, B. D.; Stevenson, L.; Watt, A.; Flitsch, S.; Turner, N. J.; Cassidy, C.; Graham, D. Nat. Biotechnol. 2004, 22, 1133-1138. (17) Ingram, A.; Stirling, K.; Faulds, K.; Moore, B.; Graham, D. Org. Biomol. Chem. 2006, 4, 2869-2873. (18) Xue, Q. F.; Wainright, A.; Gangakhedkar, S.; Gibbons, I. Electrophoresis 2001, 22, 4000-4007. (19) Ma, L. J.; Gong, X. Y.; Yeung, E. S. Anal. Chem. 2000, 72, 3383-3387. (20) Davies, H.; Lomas, L.; Austen, B. Biotechniques 1999, 27, 1258-1261. (21) Mendoza, L. G.; McQuary, P.; Mongan, A.; Gangadharan, R.; Brignac, S.; Eggers, M. Biotechniques 1999, 27, 778. (22) Wiese, R.; Belosludtsev, Y.; Powdrill, T.; Thompson, P.; Hogan, M. Clin. Chem. 2001, 47, 1451-1457. (23) Ekins, R.; Chu, F. W. Trends. Biotechnol. 1999, 17, 217-218. (24) Ekins, R. P. Clin. Chem. 1998, 44, 2015-2030. (25) Ekins, R. P.; Chu, F. W. Clin. Chem. 1991, 37, 1955-1967. (26) Kakabakos, S. E.; Christopoulos, T. K.; Diamandis, E. P. Clin. Chem. 1992, 38, 338-342. (27) Xu, Y. Y.; Pettersson, K.; Blomberg, K.; Hemmila, I.; Mikola, H.; Lovgren, T. Clin. Chem. 1992, 38, 2038-2043. 10.1021/ac071409a CCC: $37.00
© 2007 American Chemical Society Published on Web 10/17/2007
Figure 2. SERRS masked benzotriazolyl dyes and their enzymatic reaction products. For (a) 2H benzotriazolyl alkyl esters and (b) electrondeficient 1H esters.
Scheme 1a
Reagents and conditions: i) Hatu, (S) 3-phenyl butyric acid, DIPEA, DMF.
which is ultimately counterproductive to the rationale for multiplexing analysis. SERRS offers the potential for multiplexing analysis in a simple and rapid fashion with relatively inexpensive equipment, and often analytes with differing spectra can be identified by the naked eye. Ruan et al. reported that 5-bromo-4-chloro-3-indolyl phosphate (BCIP) forms a SERRS-active species upon reaction with alkaline phosphatase.15 BCIP is traditionally used for colorimetric detection, and it is likely it is the indigo dye product that is SERRS active. Generation of a colored compound allows for greater resonance enhancement of the Raman scattering when using a laser of 633-nm excitation frequency, hence increasing the SERRS response. Ruan reported it is possible to detect alkaline phosphatase at 4 × 10-15 M using this technique. Our own previous work utilized a technique where the surfaceseeking moiety of a good SERRS-active dye was covalently alkylated, thus preventing binding to the metal surface and surface enhancement of the Raman scattering was limited.16 By careful design of the covalent mask, it can be recognized and selectively cleaved by specific enzymes, thus regenerating a surface-seeking species and accordingly an increase in SERRS response that can be quantified. In the previous study (28) Luo, L. Y.; Diamandis, E. P. Luminescence 2000, 15, 409-413. (29) Scorilas, A.; Bjartell, A.; Lilja, H.; Moller, C.; Diamandis, E. P. Clin. Chem. 2000, 46, 1450-1455.
N-alkylated benzotriazoles were used (Figure 2), as alkylation of the benzotriazole moiety prevents effective binding to the silver surface used for enhancement. The alkyl ester employed as a masking group was chosen as it was known to be a substrate for the lipase from Pseudomonas cepacia. Upon treatment with lipase, the masking group was hydrolyzed and the SERRS-active species generated. Such is the sensitivity of the technique, it was estimated that as little as 500 enzyme molecules were providing the signal. In our first study, it was found that only 2H isomers would fully mask the SERRS effect under the experimental conditions employed. It was hypothesised that 1H and 3H regioisomers could bind, albeit weakly, to the silver colloid and thus some SERRS was obtained. In a second study, it was found that, by introduction of an electron-withdrawing group to benzotriazole, 1H and 3H isomers would then mask SERRS as efficiently as 2H compounds as shown in Figure 2.17 It was proposed that the reduced SERRS from electron-poor benzotriazol-1/3-yl alkyl esters was because of the inductive effect of the nitro group. The basicity of the triazole ring is reduced, which prevents the binding to the silver surface, and subsequently prevents SERRS to a greater extent than the denitro analogue. Benzotriazole was chosen for its known complexing ability, but as discussed above, it can give problems due to the different stereoisomers possible. An alternative to benzotriazole is Analytical Chemistry, Vol. 79, No. 22, November 15, 2007
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Figure 3. Superimposed SERRS spectra for (a) 1 and 3 together, (b) 1, and (c) 3.
8-hydroxyquinoline, which is also known to strongly chelate to silver,30 and its aryl azo derivatives make excellent SERRS dyes.31 Through O-acylation of the 8-hydroxy moiety, it was found that surface complexation could be prevented and thus the SERRS effect minimized in a manner analogous to that of benzotriazole discussed above. This paper compares the simpler 8-hydroxyquinolinyl azo derivatives with the analogous benzotriazolyl azos in terms of their analytical SERRS performance. RESULTS AND DISCUSSION Changing from benzotriazole-based substrates to 8-hydroxyquinoline substrates has greatly simplified the chemical synthesis of these enzymatic probes. First, there is no potential isomerism in the reaction, so separation and characterization is more straightforward. The number of synthetic steps from commercial starting materials is reduced, and also, the “masking” step requires phenolic acylation of which there is greater breadth of information in the literature than that for benzotriazole alkylation. The latter point may be of use in the future when other masking groups, targeted to other enzymes substrates, will be considered. Fluorescein, coumarin, indolyl, and umbelliferyl enzymatic probes, which are among the most common spectroscopic probes, all employ enzyme cleavable masking groups via phenolic attachment.14 The synthesis of the SERRS-active component of the probe is a simple one-step reaction from commercial precursors,31 and the introduction of the masking group was via esterification using HATU. To exemplify the multiplexing potential of SERRS, two separate dyes 1 and 3 (Scheme 1) with distinguishable SERRS spectra were prepared, as illustrated in Figure 3. An assignment of the key SERRS band from each of these compounds is presented in Table 1. In the first instance, the same masking group has been used for both dyes, but incorporation of different masking groups is facile. Through a variation of both chromophore and masking group, one-pot analysis of multiple masked substrates targeted to different enzymes will be possible. (30) Phillips, J. Chem. Rev. 1956, 56, 271-297. (31) McHugh, C. J.; Docherty, F. T.; Graham, D.; Smith, W. E. Analyst 2004, 129, 69-72.
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Table 1. Assignment of Selected SERRS Bands from Compounds 1 and 3 dye 1 (cm-1)
dye 3 (cm-1)
assignment
1413 1384
1593 1415 1384
quadrant stretch phenyl ring azo stretch NdN NdN and C-N bond to upper and lower ring systems
1371 1218 1245 1192
1371 1219 1269 1194
1138
1141
in-plane C-H bending 8-HQ ring in-plane stretching modes
An assignment of the SERS spectrum from 8-hydroxyquinoline, adsorbed onto a silver hydrosol has been reported previously.32 The resonance-enhanced spectra (SERRS) is often simpler than would be expected from SERS as only selected vibronic states are enhanced. This is desirable in the case of a multiplexed sample in a real biological matrix as only (narrow) SERRS lines that conform to Raman, resonance, and surface selection rules will be observed. The spectra obtained from dyes 1 and 3 are dominated by strongly allowed resonance-enhanced lines associated with the chromophore and the respective auxochromes. Although some contribution from 8-hydroxyquinoline ring stretching modes is expected in the region around 1360 cm-1, it is the lines that correspond to the azo (∼1415 cm-1) and the ring modes associated with this that dominate (1384 and 1371 cm-1). There is evidence in the spectra of the effect of surface selection rules. The normal Raman spectrum of 8-hydroxyquinoline includes a band at 1095 cm-1 that corresponds to the C-OH stretching. However, this band is absent or is shifted significantly in the SERRS spectra due to coordination to the silver surface.33 The synthesis of these reporter molecules demonstrates the importance of the resonance effect in generating strong Raman lines that are easily identifiable above the other molecules in the solution to be analyzed. In the study of enzymatic unmasking, substrates 2 and 4 were treated with the lipase extracted from P. cepacia. The metal (32) Chowdhury, J.; Ghosh, M.; Misra, T. N. J. Colloid Interface Sci. 2000, 228, 372-378. (33) Marchon, B.; Bokobza, L.; Cote, G. Spectrochim. Acta A 1986, 42, 537542.
Figure 4. SERRS spectrum recorded (a) immediately before and (b) 15 min after addition of lipase for substrate 2 and 4, respectively.
Figure 5. (a)Monitoring the emergence of the peak of maximum intensity over time, for each of the substrates. Inset is the structure of the masked benzotriazolyl substrate 5. Each line represents an average of three repetitions. Lipase from P. cepacia was added at 60 s. (b) is an enlargement of time period 60-200 s. Mean RSDs are 20, 22, and 25 for 2, 4, and 5, respectively.
substrate for SERRS in this study was colloidal silver, and the enzyme was added to this media, allowing for in situ reaction monitoring. A weak SERRS response was observed for the masked dyes 2 and 4, but upon reaction with lipase, a significant increase in signal was observed, which was attributed to the generation of unmasked dyes 1 and 3 (Figure 4). This emergence of product was also monitored by observing the increase in SERRS of the peak of maximum intensity over time. A comparative study between 8-hydroxyquinolyl masked substrates and benzotriazolyl masked substrates was conducted. From Figure 5 it is clear that the initial rate of hydrolysis
for 3 and 4 is greater than for 5. The rate of hydrolysis for a phenolic ester is widely reported to be faster than that for an alkyl ester, for a wide range of conditions,34,35 and the data would support this hypothesis. A higher SERRS response for 5 was observed over time, which was anticipated as the benzotriazolyl dye is perceived to be a more powerful SERRS dye than 1 or 3. The limits of detection between 8-hydroxyquinolinyl species 4 and benzotriazolyl substrate 5 were determined and compared. (34) Mata, E. G.; Mascaretti, O. A. Tetrahedron Lett. 1988, 29, 6893-6896. (35) Greene, T.; Wuts, P.G.M. Protective Groups in Organic Synthesis, 2nd ed.; John Wiley and Sons: New York, 1991.
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Figure 6. SERRS recorded from peak of maximum intensity for (a) 8-hydroxyquinolinyl substrate 4 and (b) benzotriazolyl substrate 5. Each inset is an enlargement of the lower concentration range. For each sample, the substrate is present at a concentration of 1 × 10-7 M.
The lower limit of detection for the 8-hydroxyquinolinyl substrate was ∼ 0.2 ng mL-1, whereas for the benzotriazolyl substrate, it was found to be between 2 and 20 ng mL-1, as seen in Figure 6. Given the greater SERRS intensity of unmasked benzotriazoles in comparison to 8-hydroxyquinolinyl azo dyes, this may be considered surprising. It is suggested that as the 8-hydroxyquinolinyl dyes appear to be unmasked more rapidly (Figure 5) and as such, over the 30-min reaction time used for this LOD study, benzotriazolyl substrate 5 had not been sufficiently unmasked to allow for a detectable response under the experimental conditions. Had the enzymatic reaction been allowed to proceed for a longer time, a lower limit of detection may be achieved. However, this indicates that 8-hydroxyquinolinyl azo dyes are excellent substrates for following enzyme activity by SERRS and allow faster accumulation of data. CONCLUSION A novel class of reporters of enzyme activity using SERRS detection has been prepared. This has simplified synthesis of such substrates and will allow for a greater breadth of masking groups to be introduced, in a facile manner, to the SERRS dye. In the first instance, a lipase-sensitive masking group has been demonstrated, but the introduction of other masking groups specific to different enzyme classes should be possible. Furthermore, these lipase-sensitive substrates appear to be unmasked more rapidly than benzotriazolyl masked substrates and give a detectable response within 30 min at up to 100-fold lower lipase concentration than for benzotriazolyl substrates previously reported. 8582 Analytical Chemistry, Vol. 79, No. 22, November 15, 2007
EXPERIMENTAL SECTION Solvents and Reagents. Solvents used for the preparation of SERRS samples were of spectroscopic grade. Lipase was obtained from the Sigma-Aldrich Chemical Co. Water used in preparation of silver colloid and all solutions was deionized using a water purification system. Chemical Analysis and Spectroscopy. Raman and SERRS analyses were performed on a Renishaw Mark III probe system with excitation by a Spectra Physics model 163 air-cooled argon laser producing an output of 15 mW at 514.5 nm. Citrate-reduced silver colloid was prepared by a modified version of the method described by Lee and Meisel.36 All colloids were characterized by UV spectroscopy. Synthesis of 1-4 can be found in the Supporting Information. Compound 5 was made as previously reported.17 SERRS Analysis. Stock solutions of the dyes to be analyzed were prepared at 10-4 M in anhydrous acetonitrile. Subsequent dilutions were carried out in water and discarded after 24 h. Unless otherwise stated, all SERRS analytes were studied in colloid at a concentration of 10-7 M. SERRS reaction monitoring was carried out in disposable plastic cuvettes, with in situ analysis. With the exception of the limit of detection study, the procedure employed was as follows: 900 µL of silver colloid 50% aqueous dilution and 20 µL of 0.001% poly(L-lysine) were added to the cuvette and left to aggregate for 15 min. A solution of lipase from P. cepacia and a 10-7 M solution of substrate were then added to the colloid and the spectra (36) Lee, P. C.; Meisel, D. J. Phys. Chem. 1982, 86, 3391-3395.
accumulated. The spectral acquisition time was 1 s, with the frequency between acquisitions variable. For the limit of detection study, a 10-7 M concentration of the appropriate masked substrate was placed in 900 µL of silver colloid 50% aqueous dilution, and then lipase from P. cepacia was added. The reaction was left for 30 min, then 40 µL of 1 M NaCl was added, and the SERRS spectrum was recorded. The spectral acquisition time was 1 s.
SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review July 3, 2007. Accepted September 12, 2007. AC071409A
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