Anal. Chem. 2004, 76, 1518-1523
Technical Notes
Metabolite-Sensitive Holographic Biosensors Alexander J. Marshall, Duncan S. Young, Jeff Blyth, Satyamoorthy Kabilan, and Christopher R. Lowe*
Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QT, U.K.
A new type of biosensor that combines the inexpensiveness and mass-produceability of reflection holograms with the selectivity and specificity of enzymes is described. pHsensitive holographic sensors were fabricated from ionizable monomers incorporated into thin, polymeric, hydrogel films which were transformed into volume holograms using a diffusion method coupled with holographic recording, using a frequency-doubled Nd:YAG laser (532 nm). These holograms were used as transducer systems to monitor the pH changes associated with specific enzymatic reactions to construct prototype urea- and penicillin-sensitive biosensors. The diffraction wavelength (color) of the holographic biosensors was used to characterize their shrinkage and swelling behavior as a function of analyte concentration. The potential of these sensors for the measurement of the clinically and industrially important metabolites urea and penicillin G is demonstrated. Enzymes are extensively used as biorecognition elements in biosensors, since their catalytic activity creates physicochemical changes that offer the possibility of detection. However, despite the fact that many different enzyme/transducer combinations have been described in the literature, their commercialization has been slow, due in part to the use of transducer technologies that are not readily amenable to mass-production.1 The catalytic action of some enzymes on their respective substrates often results in the generation of acidic or basic products that alter the environmental pH and, therefore, may be detected using pH-sensitive transducer systems. Conventionally, this type of biosensor consists of an enzyme attached to an ordinary glass pH electrode,2,3 although many types of pH sensors have been exploited for this purpose, including pH-sensitive ionselective field-effect transistors (pH-ISFETs),4,5 polymeric membrane electrodes,6 metal-metal oxide electrodes,7 and acid/base * To whom correspondence should be addressed. Phone: (+44) 1223 334160. Fax: (+44) 1223 334162. E-mail:
[email protected]. (1) Lowe, C. R. Curr. Opin. Chem. Biol. 1999, 3, 106-111. (2) Tor, R.; Freeman, A. Anal. Chem. 1986, 58, 1042-1046. (3) Koncki, R.; Leszczynski, P.; Hulanicki, A.; Glab, S. Anal. Chim. Acta 1992, 257, 67-72. (4) Pijanowska, D. G.; Torbicz, W. Sens. Actuators 1997, B44, 370-376. (5) Jimenez, C.; Bartrol, J.; deRooij, N. F.; Koudelkahep, M. Anal. Chim. Acta 1997, 351, 169-176. (6) Koncki, R.; Leszczynska, E.; Glab, S. Anal. Chim. Acta 1996, 321, 27-34. (7) Przybyt, M.; Sugier, H. Anal. Chim. Acta 1990, 239, 269-276.
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indicators.8-11 Of these, the optical pH transducer systems offer a number of inherent advantages over their electrochemical counterparts for the construction of enzyme-based biosensors, particularly with respect to the lack of electrical connections and immunity from electrical interference, absence of reference electrodes, feasibility of miniaturization, low cost/disposable construction, and the possibility of remote sensing and in vivo measurement.12 However, despite the widespread interest in optical pH transducers, the mass production and commercialization of an inexpensive generic optical pH sensor suitable for biosensor applications has not yet been accomplished. Recently, an optical system based on pH-sensitive reflection holograms has been described.13 When compared with other optical pH sensing methodologies, these holographic gratings display a number of advantages, including an unusually high sensitivity (milli-pH unit resolution) and the possibility of reproducible, low-cost, mass-manufacture. Moreover, holographic pH transducers have proven suitable for use in opaque and very turbid samples and are highly stable with respect to calibration, because unlike many optical pH sensors, indicator photobleaching is not a problem. These characteristics suggest that the holographic pH system may be highly suitable for the construction of simple, inexpensive, low-power, and remotely interrogatable pH-based metabolite biosensors. Holographic sensors13-19 are fabricated by embedding holographic fringes of silver within thin-polymer films using a diffusion method20 coupled with exposure to laser light. In the case of holographic pH transducers,13 the holograms are recorded within (8) Goldfinch, M. J.; Lowe, C. R. Anal. Biochem. 1984, 138, 430-438. (9) Fuh, M.; Burgess, L. W.; Hirschfeld, T.; Christian, G. D.; Wang, F. Analyst 1987, 112, 1159-1163. (10) Healey, B. G.; Walt, D. R. Anal. Chem. 1995, 67, 4471-4476. (11) Knocki, R.; Mohr, G. J.; Wolfbeis, O. S. Biosens. Bioelectron. 1995, 10, 653-659. (12) Wolfbeis, O. S. Anal. Chem. 2002, 74, 2663-2678. (13) Marshall, A. J.; Blyth, J.; Davidson, C. A. B.; Lowe, C. R. Anal. Chem. 2003, 75, 4423-4431. (14) Millington, R. B.; Mayes, A. G.; Blyth, J.; Lowe, C. R. Anal. Chem. 1995, 67, 4229-4233. (15) Millington, R. B.; Mayes, A. G.; Blyth, J.; Lowe, C. R. Sens. Actuators 1996, B33, 55-59. (16) Blyth, J.; Mayes, A. G.; Frears, E. R.; Millington, R. B.; Lowe, C. R. Anal. Chem. 1996, 68, 1089-1094. (17) Mayes, A. G.; Blyth, J.; Millington, R. B.; Lowe, C. R. J. Mol. Recognit. 1998, 11, 168-174. (18) Mayes, A. G.; Blyth, J.; Kyrolainen-Reay, M.; Millington, R. B.; Lowe, C. R. Anal. Chem. 1999, 71, 3390-3396. (19) Mayes, A. G.; Blyth, J.; Millington, R. B.; Lowe, C. R. Anal. Chem. 2002, 74, 3649-3657. 10.1021/ac030357w CCC: $27.50
© 2004 American Chemical Society Published on Web 01/28/2004
hydrogels containing acidic or basic monomers within the polymeric backbone. Ionization of the pendant functional groups causes the grating to swell as a result of electrostatic and osmotic forces that draw in or expel counterions and water into or out of the gel phase. This increases the fringe separation and causes longer wavelengths to be selected for reflection from the holographic mirror,16 and hence, the diffraction wavelength of the sensing hologram is dependent on the pH of the bathing medium. To date, a series of holographic pH sensors have been constructed in two different hydrogel systems, poly(hydroxyethyl methacrylate) and poly(acrylamide). Copolymerization with monomers bearing ionizable groups and suitable cross-linkers has been shown to confer a characteristic pH sensitivity dependent on the individual functional monomer incorporated.13 Reversible and visually perceptible color changes as a function of pH occur either side of an apparent dissociation constant (pKa) and are governed by the nature of the ionisable monomer incorporated into the holographic matrix. Through selection of acidic or basic monomers with appropriate pKa values, it is possible to tune the response of the resultant hologram to the pH range of interest for a particular application. In this report, holographic pH transducers are applied to monitor the pH changes associated with specific enzymatic reactions to construct biosensors for the clinically and industrially important analytes, urea, and penicillin. The successful union of holograms and enzymes for the detection of these analytes is demonstrated. EXPERIMENTAL SECTION Materials. All chemicals were of analytical grade unless otherwise stated. Bicine, dimethyl sulfoxide (DMSO), centrifuge filter devices (10 kDa), glutaraldehyde (pentane-1,5-dial; 25% (w/v) aqueous solution), penicillin G (sodium salt), S-acetylthioacetate (SATA), phenol red, sodium acetate, urea, bovine serum albumin (BSA), phosphate-buffered saline tablets (PBS; pH 7.4, 10 mM phosphate, 137 mM NaCl, 2.7 mM KCl), penicillinase [(EC 3.5.2.6) type I from Bacillus cereus; stated activity 2230 U/mg] and urease [(EC 3.5.1.5) type IX from Jack Beans; 100 U/mg] were purchased from the Sigma Chemical Company, Fancy Road, Poole, Dorest, U.K. 2-(N-Morpholino) ethanesulfonic acid (hydrate) (MES), 3-{tris(hydroxymethyl)methyl]amino}ethanesulfonic acid (TES), chloroacetate, cysteine, 5,5′-dithio-bis(2-nitrobenzoic acid) (Ellman’s reagent), ethylenediaminetetraacetic acid disodium salt (EDTA), hydroxylamine hydrochloride, and piperazine were purchased from Acros Organics, Janssens, Pharmaceuticalaan, 3A, 2440, Geel, Belgium. Sodium thiosulfate (hypo) and acetic acid (glacial) were purchased from Fisher Scientific Ltd., Bishop Meadow Road, Loughborough, Leicestershire, LE11 5RG, U.K. Hologram Construction. Holographic sensors were fabricated as described previously.13 All photosensitization, exposure, and development work was carried out under red safe lighting. A frequency-doubled Nd:YAG laser (350 mJ, 532 nm, Brilliant B, Quantel, France) was used in hologram construction. High-energy class IV pulsed lasers such as this are potentially dangerous, and the laser was located and used according to local safety regulations. (20) Blyth, J.; Millington, R. B.; Mayes, A. G.; Lowe, C. R. Imaging Sci. J. 1999, 47, 87-91.
Preparation of Urease-Modified Holograms. Single poly[HEMA-co-EDMA(5 mol %)-co-DMAEM(6 mol %)] holograms were first agitated in a solution of 10% (w/v) sodium thiosulfate at room temperature for 30 min. They were subsequently removed to a stream of tap water, washed in phosphate buffered saline (PBS; pH 7.4, 10 mM phosphate, 137 mM NaCl, 2.7 mM KCl) and dried, and then 100 µL of urease solution (100 U/mg; 25 mg/ mL in PBS) was pipetted on top such that the entire polymer surface was covered with enzyme solution. This was allowed to evaporate in a fume hood for 2 h to concentrate the enzyme on the surface of the hologram. Immobilization was achieved by addition of 100 µL of glutaraldehyde solution (25% (v/v) solution in water, diluted to 1.25% (v/v) with PBS), which was pipetted over the entire hologram surface and left for 2 h. Control holograms were also constructed by immobilizing bovine serum albumin (100 µL, 25 mg/ml in PBS) to a sister grating using the same procedure. Thiolation of Penicillinase with N-Succinimidyl S-Acetylthioacetate. Thiolation of pencillinase was achieved with Nsuccinimidyl S-acetylthioacetate (SATA) according to a protocol described in Hermanson21 with some modification. A stock solution of penicillinase was prepared in nitrogen-flushed buffer (0.9 mg/mL penicillinase in 50 mM phosphate buffer, pH 7.58, 5 mM EDTA). To 1 mL of this, 10 µL of freshly made SATA solution (65 mM in DMSO) was added, and the solution was mixed briefly and left to react for 30 min at room temperature. SATA-treated penicillinase was separated from unreacted SATA and reaction byproducts using centrifugal filter devices (molecular weight limit of 10 kDa) by transferring 400 µL of each enzyme preparation into separate centrifuge filter tubes, which were spun for 4 min at 13000g. The samples were then resuspended in sodium phosphate buffer (50 mM, pH 7.58, 5 mM EDTA) to a final volume of 400 µL and spun again. This process was repeated three times. The determination of sulfydryl groups in SATA-treated penicillinase samples was achieved using Ellman’s assay,22 and the effect of modification on the activity of penicillinase was determined using a standard phenol red-based colorimetric assay.23 Preparation of Penicillinase-Modified Holographic pH Sensors. SATA-treated penicillinase (400 µL of 2000 U/mL in 50 mM phosphate buffer, pH 7.58, 5 mM EDTA) was reacted with 40 µL of 0.5 M hydroxylamine in phosphate buffer (50 mM, pH 7.5, 25 mM EDTA) to deprotect the acetylated sulfydryl groups. This solution was vortexed briefly, and 200 µL was immediately pipetted onto individual poly[HEMA-co-EDMA(5 mol %)-co-MAA(6 mol %)] holograms such that the entire polymer surface was covered and allowed to react overnight at room temperature. Hologram Interrogation and Testing. Holographic devices were interrogated using an in-house-built reflection spectrophotometer as described previously.18,19 After each assay, holograms were washed in assay buffer to return them to their original diffraction wavelength prior to the addition of substrate before the next assay was performed. Enzyme-modified holograms were kept in PBS at 4 °C when not in use. (21) Hermanson, G. T. Bioconjugate Techniques; Academic Press Inc: London, 1996. (22) Ellman, G. L. Arch. Biochem. Biophys. 1959, 82, 70-77. (23) Saz, A. K.; Lowery, D. L.; Jackson, L. J. J. Bacteriol. 1961, 82, 298-304.
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Calibration of Holographic pH Transducers. Holographic pH sensors were tested using the following pH buffer systems: chloroacetate (pH 3), acetate (pH 4-5), MES (pH 5.5-6.5), MOPS (pH 7-8), and Bicine (pH 9). In each case, the concentration of buffer component was 20 mM, and the total ionic strength (I) was fixed to 200 mM using NaCl. The apparent pKa values of the holographic sensor pH-response curves were determined from the point of inflection of curves fit using SigmaPlot (version 8; SPSS Science, Woking, Surrey, U.K.). Urea Assays. Urease-modified holograms were incubated in 950 µL of pH 7 MES buffer 30 °C until a stable diffraction wavelength was obtained. To this, 50 mL of urea solution (in deionized water) was added to give the required final concentration of urea and the diffraction wavelength monitored for the duration of the experiment. Control experiments were preformed as above but using BSA-modified holograms. Penicillin G Assays. Penicillin-modified holograms were incubated in 900 µL piperazine buffer (10 mM, pH 7.0). Upon attainment of a stable diffraction wavelength, 100 mL of freshly prepared penicillin G solution (in piperazine buffer) was added. Control experiments were performed in the absence of penicillinase. RESULTS AND DISCUSSION Holographic pH Transducers. A series of pH-sensitive holographic sensors have been constructed, characterized, and shown to be sensitive over defined pH ranges.13 Such holograms are fabricated by coating a thin layer (∼10 µm thick) of unsensitized polymer film on a silanized glass slide and then immersing the slide sequentially in solutions of silver perchlorate and lithium bromide containing a photosensitizing dye, 1,1′-diethyl-2,2′-cyanine iodide. Following exposure of the film to laser light19 and a conventional photographic development step, an interference fringe pattern comprising layers of ultrafine (
