Holographic Enzyme Inhibition Assays for Drug Discovery

Hologram construction was based on the principle of the diffusion method as outlined by Blyth et al.(18) pH-sensitive polymer films were synthesized a...
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Anal. Chem. 2009, 81, 7579–7589

Holographic Enzyme Inhibition Assays for Drug Discovery Eu Vian Tan and Christopher R. Lowe* Institute of Biotechnology, Department of Chemical Engineering and Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QT, U.K. Optical sensors are widely utilized in drug discovery to analyze biomolecular interactions in vitro. Aside from additional time and cost demands, other issues associated with labeled screening methods include signal interference that can arise from the label per se and/or the screened compounds. This report describes an enzyme inhibition-based holographic sensor as a potential labelfree detection system, using acetylcholinesterase (acetylcholine acetylhydrolase; EC 3.1.1.7; abbreviated herein AChE) as the model enzyme. pH-responsive reflection holograms, incorporated into “smart” hydrogel films bearing ionizable monomers, were used to monitor the pH change resulting from acetic acid produced by the hydrolysis of the substrate acetylcholine. The enzyme was immobilized on the sensor by an entrapment and in situ cross-linking method; no chemical modification and/or prelabeling of the enzyme (or the substrate) was required. The fully reversible sensor exhibited good operational and storage stability, allowing relatively short assay times and repeated use of a single sensor. Apparent inhibition parameters for several drug inhibitors of the enzyme were determined. The feasibility of adapting these sensors into an array format for prospective high-throughput screening, without compromising their intrinsic optical and functional properties, was also demonstrated. Biological screening forms an essential aspect of the drug discovery process, whereby batches of putative therapeutic compounds are tested for binding or biological activity against target molecules.1-3 Most screens are designed around the microtiter plate format, incorporating a variety of labeled assays (such as radioactivity, fluorescence, or absorbance) to report an interaction of a compound with the target enzyme/receptor.4-6 Fluorescence-based measurements are the most widely implemented detection modality and offer unprecedented sensitivity and flexibility for screening assays. However, several drawbacks of * To whom correspondence should be addressed. Phone: (+44) 1223 334160. Fax: (+44) 1223 334162. E-mail: [email protected]. (1) Bartfai, T.; Lees, G. V. Drug Discovery (from Bedside to Wall Street); Academic Press: Oxford, U.K., 2006. (2) Go´mez-Hens, A.; Aguilar-Caballos, M. P. Trends Anal. Chem. 2007, 26, 171–182. (3) Inglese, J.; Johnson, R. L.; Simeonov, A.; Xia, M.; Zheng, W.; Austin, C. P.; Auld, D. S. Nat. Chem. Biol. 2007, 3, 466–478. (4) Hertzberg, R. P.; Pope, A. J. Curr. Opin. Chem. Biol. 2000, 4, 445–451. (5) Sundberg, S. A. Curr. Opin. Biotechnol. 2000, 11, 47–53. (6) Gribbon, P.; Sewing, A. Drug Discovery Today 2003, 8, 1035–1043. 10.1021/ac9008989 CCC: $40.75  2009 American Chemical Society Published on Web 08/14/2009

fluorescence have been highlighted, including interference with the emission signal from the fluorophore itself which could lead to false positives or negatives.6 Interference can be additive (for example, autofluorescence) or subtractive (for example, selfquenching), and the degree of either interference is dependent on the concentration of the fluorophore used and the excitation and emission wavelengths. Moreover, signal interference could also arise from the screened compounds themselves, which confounds the interpretation of any intrinsic biological activity. The interference by the screened compounds is further exacerbated by the common use of fluorophores at concentrations within the nanomolar range in many assays, whereas test compounds are usually screened in micromolar concentrations. Alternative label-free screening methods have thus emerged, some of which are based on optical sensing technology.7,8 Although initially developed for clinical diagnosis9 and environmental monitoring,10,11 advances in experimental design, fabrication, and instrumentation have fuelled the increasing use of optical sensors throughout the drug discovery process.7,12,13 Most of the well-established optical sensors exploit surface plasmon resonance (SPR), which measures changes in refractive index associated with the binding of molecules in solution to surface-immobilized receptors.7,14 Despite their current dominance in the label-free screening market, these sensors have not yet established a foothold in the landscape of drug discovery technology due to various reasons, particularly because of their low throughput and inadequate sensitivity.14-16 Under extremely well-controlled conditions, however, label-free detection has been perceived as a promising analytical tool, which confers high flexibility and efficiency to assay design, with potentially fewer artifacts, and facilitates successful integration with other technologies. At present, the use of label-free detection for “Yes/No” end-point primary screening is still minimal in comparison to that for kinetic and affinity analyses during the secondary screening and lead optimization stages of drug discovery. (7) Cooper, M. A. Nat. Rev. Drug Discovery 2002, 1, 515–528. (8) Chan, L. L.; Pineda, M.; Heeres, J. T.; Hergenrother, P. J.; Cunningham, B. T. Chem. Biol. 2008, 3, 437–448. (9) Frost, M. C.; Meyerhoff, M. E. Curr. Opin. Chem. Biol. 2002, 6, 633–641. (10) Amine, A.; Mohammadi, H.; Bourais, I.; Palleschi, G. Biosens. Bioelectron. 2006, 21, 1405–1423. (11) Rogers, K. R. Anal. Chim. Acta 2006, 568, 222–231. (12) Lowe, C. R. Curr. Opin. Chem. Biol. 1999, 3, 106–111. (13) Keusgen, M. Naturwissenschaften 2002, 89, 443–444. (14) Cooper, M. A. Anal. Bioanal. Chem. 2003, 377, 834–842. (15) Cooper, M. A.; Whalen, C. Drug Discovery Today: Technol. 2005, 2, 241– 245. (16) Cooper, M. A. Drug Discovery Today 2006, 11, 1061–1067.

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This report presents the concept of a simple reflection hologram as a potential label-free detection modality for primary screening. Holographic diffraction gratings are generated within photosensitive polymer-silver halide photographic emulsions upon exposure to a single collimated laser beam, which passes through the polymer film and is then reflected back by a mirror.17-19 Interference between the mutually coherent incident and reflected beams generates a standing wave pattern which, after development and fixing, creates a virtual three-dimensional pattern comprising fringes of ultrafine metallic silver grains embedded within the thickness (∼10 µm) of the polymer film. These silver fringes lie in planes parallel to the substrate surface and are separated by a distance of approximately half the wavelength of the laser beam used to generate them. Under white light illumination, the holographic fringes reflect light of a specific narrow band of wavelengths, hence acting as a sensitive wavelength filter and recreating a monochromatic image of the original mirror used in their construction. Constructive interference between partial reflections from each fringe plane produces a distinctive spectral peak with a wavelength governed by the Bragg equation (λmax ) 2nd cos θ). Any physical, chemical, or biological reaction that alters the fringe spacing (d) or the refractive index (n) will generate visible changes in the wavelength (color) or intensity (brightness) of the reflection hologram. The intensity of holographic diffraction is also determined by the number of fringe planes and the modulation depth of the refractive index. Swelling of the holographic film increases the distance between fringes producing a red-shift in the wavelength of reflected light, whereas film contraction results in a blue-shifted light. In essence, the holographic gratings act as a reporter, whose optical properties are dictated by the physical characteristics of the holographic film. Reflection holograms have proven advantageous in many aspects, including the simplicity bestowed by the holographic element providing both the analyte-sensitive matrix and the optical transducer. This generic technology allows fabrication of holograms in a wide range of natural, synthetic, and rationally designed hydrogels that can be tailored to enable selectivity for different target analytes.20 In addition to an inexpensive fabrication procedure and amenability to mass production, holographic sensors can adopt multiple configurations making them suitable for miniaturization and multiplexing formats. As reflection holograms generate a perceptible color change across the visible spectrum, they offer direct visual readout. These features are general prerequisites for the development of array optical sensors, presenting an opportunity for high-throughput analysis and multianalyte detection. Another promising attribute of this sensor technology is that prelabeling of either the (bio)recognition component or the analyte of interest is not required to track a (bio)chemical event; hence, the system is essentially label-free. (17) Blyth, J.; Millington, R. B.; Mayes, A. G.; Frears, E. R.; Lowe, C. R. Anal. Chem. 1996, 68, 1089–1094. (18) Blyth, J. Imaging Sci. J. 1999, 47, 87–91. (19) Marshall, A. J.; Kabilan, S.; Blyth, J.; Lowe, C. R. J. Phys.: Condens. Matter 2006, 18, S619–626. (20) Mayes, A. G.; Blyth, J.; Millington, R. B.; Lowe, C. R. J. Mol. Recognit. 1998, 11, 168–174.

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To date, holographic sensors of varying specificities have been developed,21-29 including pH sensors which have also been utilized to create enzyme sensors for detecting metabolites such as urea and penicillin.24,25 Reversible and visually perceptible color changes as a function of pH can be observed on either side of an apparent dissociation constant (pKa) and are dictated by the nature of the ionizable monomer integrated into the hydrogel. By selection of ionizable monomers with appropriate pKa values, it is possible to adjust the holographic response to the pHsensing range of interest. Here, a similar concept is applied for constructing an enzyme inhibition-based holographic sensor, with acetylcholinesterase (acetylcholine acetylhydrolase; EC 3.1.1.7; AChE) as the model enzyme. The pH change as a result of acetic acid production upon the hydrolysis of the substrate acetylcholine by AChE is quantified by a pH-sensitive hologram, which has been incorporated into a poly(2-hydroxyethyl methacrylate) (polyHEMA) backbone comprising methacrylic acid (MAA) and ethyleneglycol dimethacrylate (EDMA). MAA is a weak acid (pKa∼ 6.0) that imparts pH sensitivity, while EDMA maintains the structural and spatial integrity of the hydrogel network required for incorporation of a holographic grating. This particular composition is selected as MAA-based holograms have an ionizable function that is active in the pH range expected to be associated with AChE activity.24 As one of the most extensively studied enzymes, the major biological function of AChE is the termination of impulse transmission at cholinergic synapses by fast hydrolysis of the neurotransmitter acetylcholine.30,31 Elucidation of the structure and catalytic mechanism of the enzyme at the molecular level is crucial for understanding the effects of mechanism-based AChE inhibitors that are widely used as therapeutic agents in several diseases such as Alzheimer’s disease.32-34 Given the pharmaceutical implications of AChE and its inhibitors, the enzyme is thus deemed a suitable model system for this study. This paper reports the initial studies on the development of an AChE inhibition-based holographic sensor. Detailed here is the preliminary characterization of the sensor, and the subsequent application in screening of test compounds to determine pharmaceutically relevant parameters such as IC50 and Ki. The IC50 value is a practical readout of relative effects of different inhibitors on a target enzyme, representing the inhibitor concentration required to inhibit (21) Millington, R. B.; Mayes, A. G.; Blyth, J.; Lowe, C. R. Anal. Chem. 1995, 67, 4429–4233. (22) Mayes, A. G.; Blyth, J.; Kyro ¨la¨inen-Reay, M.; Millington, R. B.; Lowe, C. R. Anal. Chem. 1999, 71, 3390–3396. (23) Mayes, A. G.; Blyth, J.; Millington, R. B.; Lowe, C. R. Anal. Chem. 2002, 74, 3649–3657. (24) Marshall, A. J.; Blyth, J.; Davidson, C. A.; Lowe, C. R. Anal. Chem. 2003, 75, 4423–4431. (25) Marshall, A. J.; Young, D. S.; Blyth, J.; Kabilan, S.; Lowe, C. R. Anal. Chem. 2004, 76, 1518–1523. (26) Marshall, A. J.; Young, D. S.; Kabilan, S.; Hussain, A.; Blyth, J.; Lowe, C. R. Anal. Chim. Acta 2004, 527, 13–20. (27) Lee, M. C.; Kabilan, S.; Hussain, A.; Yang, X.; Blyth, J.; Lowe, C. R. Anal. Chem. 2004, 76, 5748–5755. (28) Sartain, F. K.; Yang, X.; Lowe, C. R. Anal. Chem. 2006, 78, 5664–5670. (29) Bhatta, D.; Christie, G.; Madrigal Gonza´lez, B.; Blyth, J.; Lowe, C. R. Biosens. Bioelectron. 2007, 23, 520–527. (30) Sussman, J. L.; Harel, M.; Frolow, F.; Oefner, C.; Goldman, A.; Toker, L.; Silman, I. Science 1991, 253, 872–879. (31) To ˜ugu, V. Curr. Med. Chem.: CNS Agents 2001, 1, 155–170. (32) Cummings, J. L. Am. J. Psychiatry 2000, 157, 4–15. (33) Bourne, Y.; Taylor, P.; Radic, Z.; Marchot, P. EMBO J. 2003, 22, 1–12. (34) Houghton, P. J.; Ren, Y.; Howes, M. J. Nat. Prod. Rep. 2006, 23, 181–199.

enzyme activity by 50% under specific assay conditions; whereas Ki is the true dissociation constant for the enzyme-inhibitor complex.35,36 Configuration of the system as an array of holographic sensors, in which responses are recorded in parallel and in real time at various inhibitor concentrations, is also established. EXPERIMENTAL SECTION Materials. All biologicals and chemicals were of analytical grade unless otherwise stated. Acetylcholine chloride (ACh), acetylcholinesterase (acetylcholine acetylhydrolase, AChE; EC 3.1.1.7; from Electrophorus electricus), ascorbic acid, bovine serum albumin (BSA), 1,1′-diethyl-2,2-cyanine iodide (QBS, photosensitizing dye), dextran sulfate sodium salt (from Leuconostoc spp.), 2,2′dimethoxy-2-phenyl acetophenone (DMPA), edrophonium chloride, eserine, ethyleneglycol dimethacrylate (EDMA), formaldehyde solution (37%, w/v), galanthamine hydrobromide, gelatin (type A; from porcine skin), hydroquinone, 2-hydroxyethyl methacrylate (HEMA), HEPES, D-lactitol monohydrate, lithium bromide, methacrylic acid (MAA), neostigmine bromide, pyridine-2aldoxime methochloride (2-PAM), potassium phosphate (monobasic, anhydrous), silver perchlorate, sodium ascorbate, sodium hydroxide, sodium thiosulphate (HYPO), and tacrine (9-amino-1,2,3,4tetrahydroacridine) were purchased from Sigma Aldrich, U.K. Tris(hydroxymethyl)amino-methane (Tris base) was purchased from Fisher Scientific Ltd., U.K. 2-(Cyclohexylamino)ethanesulphonic acid (CHES), 3-cyclohexylamino-1-propanesulphonic acid (CAPS) and 2-(N-morpholino) ethanesulphonic acid hydrate (MES) were purchased from Acros Organics, Belgium. Silicone elastomer (polydimethylsiloxane, PDMS) and curing agent were purchased from Sylgard, U.K. The Amplex Red Acetylcholinesterase Assay Kit was purchased from Invitrogen, U.K., comprising 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red reagent, AmpR), choline oxidase (choline:oxygen 1-oxidoreductase, ChO; EC 1.1.3.17; from Alcaligenes spp.), hydrogen peroxide (H2O2), and horseradish peroxidase (donor:hydrogen-peroxide oxidoreductase, HRP; EC 1.11.1.7). Equipment. Microscope slides (Super Premium, 1-1.2 mm thick) were purchased from BDH (Merck) Ltd., U.K. Aluminized 100 µm polyester film (grade MET401) was purchased from HiFi Industrial Film Ltd., U.K. PVC electrical insulating tape was purchased from Maplin Electronics, U.K. A UV exposure unit (UV Stratalinker 2400) was purchased from Stratagene, U.K. A vacuum oven was purchased from Gallenkamp, U.K. A standard benchtop pH meter, a pH microelectrode, electrode cleaning solution, and calibration buffers were purchased from Jencons-PLS, U.K. Instrumentation. A frequency-doubled Nd:YAG laser (350 mJ, 532 nm, Brilliant B, Quantel, France) was used for hologram construction. Holograms were analyzed using a spectrophotometer (LOT-ORIEL MS127i model 77480) in single-channel mode with a 256 × 1024 pixels InstaSpecIV charge-coupled device (CCD) detector and a tungsten halogen light source. Spectrophotometer calibration was carried out using a spectral calibration lamp (374405) purchased from Ealing Electro Optics Plc., U.K. The setup used is similar as described in Mayes et al.23 A customized rig (Knight Photonics, U.K.) was built to analyze array holograms, (35) Copeland, R. A. Enzymes: A Practical Introduction to Structure, Mechanism and Data Analysis; Wiley-VCH: New York, 2000. (36) Macarro´n, R.; Hertzberg, R. P. Design and Implementation of High Throughput Screening Assays; Humana Press Inc.: Totowa, NJ, 2002.

comprising an adjustable stage and two adjustable assemblies to which the fiber optics were attached. An AvaSpec 2048 multichannel spectrometer (with a 2048 pixel CCD detector operating in the 300-1000 nm range) and an AvaLight HAL tungsten halogen light source were purchased from Avantes, The Netherlands. The fiber optic cables (FC-UV600-2; 600 µm core diameter; 2 m long) and the adjustable collimators were also purchased from Avantes. Construction of pH-Sensitive Holograms. Hologram construction was based on the principle of the diffusion method as outlined by Blyth et al.18 pH-sensitive polymer films were synthesized as described by Marshall et al.24 from a monomer solution comprising HEMA with 6 mol % MAA, 5 mol % EDMA, and 1% (w/v) DMPA as the free radical initiator. Aliquots (100 µL) of the resulting solution were pipetted on the conducting side of an aluminized polyester sheet. Pretreated glass slides were placed silanized-side down onto the solution. Films were polymerized by UV-initiated free-radical reaction for 45 min at room temperature, after which they were carefully removed and rinsed with deionized water before being air-dried. Under red-safe lighting, the polymer film was immersed face-down into 0.3 M silver perchlorate in 80% (v/v) isopropanol/water (400 µL per slide) for 5 min. Excess solution was wicked off, and the film was dried briefly under a stream of warm air, after which the entire slide was placed polymer-side up in a solution comprising 40 mL of 3% (w/v) lithium bromide in 3:2 (v/v) methanol/water and 2 mL of 0.2% (w/v) QBS in methanol for 45 s under agitation. The slide was rinsed thoroughly in deionized water before being immersed polymer-side down into the hologram exposure bath, containing 0.1 M sodium ascorbate pH 5.0, for ∼10 min. The slide was then exposed to a single 10 ns pulse from a frequency-doubled (532 nm) Nd:YAG laser. After exposure, the slide was placed in a developer solution (20 mL of 5% (w/v) sodium hydroxide in 95% (v/v) methanol and 20 mL of 5% (w/v) hydroquinone in 50% (v/v) methanol) for ∼15 s. The slide was rinsed in deionized water, placed in a “stop” solution (5% (w/v) acetic acid) for ∼1 min, and further rinsed in deionized water before being immersed in 10% (w/v) sodium thiosulphate for ∼5 min to remove any undeveloped silver. Finally, the slide was rinsed in 50% (v/v) ethanol and then deionized water. Hologram sections (8-9 mm width) were cut from the processed slide for subsequent interrogation. Construction of Array Holograms. A contact printing method was used to produce regular, circular polymers as outlined by Dobson.37 A mask was created using PVC electrical insulating tape (0.14 mm thick). Four equally spaced holes of identical diameters (8 mm) were punched and the resulting mask adhered to the silanized side of a glass microscope slide. The same monomer solution was pipetted both onto the conducting side of an aluminized polyester sheet with the spacing corresponding to that of the mask (∼10 µL) and also into the recesses in the mask (∼10 µL). The entire slide was placed onto the polymer spots, and the polymerization was carried out under similar conditions as described earlier. Upon removal of the mask tape, the spot polymers were rinsed in deionized water and gently dried with tissue paper. Hologram recording was also achieved as described (37) Dobson, C. The Design, Fabrication and Characterization of Holographic Optical Elements. Ph.D. Dissertation, University of Cambridge, Cambridge, U.K., 2008.

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earlier, with a shorter time needed in the exposure bath (∼2 min) and the developer solution (∼5 s). Enzyme Immobilization. The final volume of immobilization mixture used was 50 µL per hologram section (10 µL per spot hologram). Unless otherwise stated, final concentrations of 50 U/mL AChE (predissolved in 10 mM phosphate buffer, pH 7.0 containing 1 mg/mL BSA), 8% (w/v) lactitol, and 0.1% (w/v) dextran sulfate were added to a warm solution of 2.5% (w/v) gelatin and mixed. The resulting mixture was coated on the hologram surface and left to dry at room temperature. Additional cross-linking was established by incubating samples in a sealed chamber enriched with formaldehyde vapor (created from a 37% (w/v) formalin reservoir) for 45 min at room temperature, after which they were washed and stored in assay buffer (1 mM TrisHCl, 154 mM NaCl, pH 8.0) at 4 °C prior to use. Purity Check of Inhibitors. The test inhibitors used in this study were edrophonium, galanthamine, neostigmine, tacrine, and eserine. Following dissolution in neat methanol to give a concentration of 0.5 mg/mL, all inhibitor samples were subjected to LC-MS analysis. The column (4.6 mm × 50 mm) comprised a stationary phase particle size of 3 µm, using two solvent systems: aqueous (water and 0.05% (v/v) formic acid) and organic (acetonitrile and 0.05% (v/v) formic acid). With the use of an injection volume of 5 µL, a standard methodology was initiated, comprising a total run time of 5 min, a flow rate of 3 mL/min, column temperature at 30 °C, and UV detection range of 220-330 nm. Fluorescence Microplate-Based Enzyme Assay. The method was adapted from Protocol A12217 (Invitrogen). The assay works on the basis of monitoring AChE activity indirectly using AmpR, a sensitive fluorophore for H2O2. AChE first converts ACh to choline, which is then oxidized to betaine and H2O2 in the presence of ChO. H2O2, in the presence of HRP, reacts with AmpR in a 1:1 stoichiometry to produce resorufin (absorption and emission maxima at 571 and 585 nm, respectively). All stock solutions were prepared in assay buffer (5 mM Tris-HCl, pH 8.0), except for AmpR and H2O2 that were made up in dimethylsulfoxide (DMSO) and deionized water, respectively. The total assay volume used per well was 200 µL, with a working solution comprising final concentrations of 50 µM AmpR, 1 U/mL HRP, 0.1 U/mL ChO, and 25 µM ACh in assay buffer. For IC50 experiments, inhibitor stock solutions were prepared at 1 mM in 10% (v/v) DMSO, after which a 10-point 1/3 serial dilution of all samples was executed. Each inhibitor (2 µL) was transferred to the corresponding wells of a black, flatbottomed 96-well plate where the assays were carried out, providing a maximum concentration of 10 µM inhibitor in 0.1% (v/v) DMSO. To all sample and positive control wells, 100 µL of 0.2 U/mL AChE solution was added to give a final concentration of 0.1 U/mL; 100 µL of assay buffer without AChE was added to the negative control wells. Following a 10 min preincubation at room temperature, 100 µL of the AmpR/ HRP/ChO/ACh working solution was added to each well to initiate the reaction. An end-point readout of fluorescence intensity was performed, with a preceding shake protocol (for ∼5 s) and a 15 min delay set in the microplate reader to allow incubation of the assay reactants. 7582

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For Ki experiments, different maximum concentrations were used for a 5-point 1/3 serial dilution in duplicate; nonetheless, all inhibitor samples were reconstituted in 10% (v/v) DMSO to give a 0.1% (v/v) final concentration. The components of the working solution were added to samples in a two-step procedure; a separate solution of 4 U/mL HRP and 0.4 U/mL ChO was first prepared. A series of AmpR/ACh working solutions was then prepared in a separate plate, with 200 µM AmpR and a concentration range of ACh (0-1 mM) added to each well, followed by a brief mixing. Each inhibitor (2 µL) was transferred to the corresponding wells of the assay plate. DMSO (10% (v/v); 2 µL) was also added to the control wells. AChE (0.2 U/mL; 100 µL) was added to all sample and positive control wells and 100 µL of assay buffer without AChE to the negative control well. After a 10-min preincubation at room temperature, 50 µL of HRP/ChO solution was added to each well and then 50 µL of ACh/AmpR solution. Two end-point fluorescence readouts were performed at t ) 3 and t ) 5 min, using a similar setup as described for IC50 experiments. Monitoring Holographic Responses. Sensor holograms were interrogated with an in-house built spectrophotometer as described in Mayes et al.23 The sensor was placed with the biological- (and/or polymer-) side inward in a 4 mL cuvette, into which the assay solution was added. Unless otherwise stated, holograms were monitored using 2 mL of assay solution at 25 °C under agitation with a magnetic microflea/stirrer arrangement. Washing of sensors and exchanging of solutions were only carried out after a stable baseline was achieved (i.e., variation of 5% (v/v). While the diffusion of DMSO molecules across the matrix and accessibility to the enzyme might be restricted, the gelatin support per se and the added enzyme stabilizers might provide additional protection, hence showing an apparent improved tolerance of the sensor to DMSO. However, the rate of enzyme inactivation by DMSO might also be timedependent, the effect of which might not be obvious given the 10 min preincubation set in this experiment. For comparison purposes, a final concentration at 0.1% (v/v) DMSO was used. Control experiments were conducted to ensure that any pH change and holographic response observed were associated with enzyme activity only and to avoid potential signal interference by nonreacting components. As each inhibitor was incubated with the sensor prior to assay, the stability of the signal baseline within that interval was also monitored by equilibrating each sensor with the inhibitor at a concentration which showed >90% inhibition. No significant shifts in holographic response were observed, indicating little or negligible nonenzymatic interactions with the immobilization matrix. The apparent low sensitivity of the holographic system to DMSO could potentially overcome one of the key shortcomings associated with most optical label-free methods; these systems often suffer from large bulk refractive index shifts due to the disparate properties between DMSO and the assay buffer and thus require extensive calibration programs to correct for any bulk effects that may mask a specific binding signal.7,15 The immobilized enzyme appeared to deviate from MichaelisMenten behavior, producing a sigmoid dependence of initial velocity of enzyme reaction on substrate concentration. The data were fitted using a modified Hill equation (vi ) Vmax(app)[S]h/ KM(app)+ [S]h). The immobilized enzyme also seemed to exhibit positive cooperativity, although this is unlikely as the free enzyme has been found to obey Michaelis-Menten characteristics (also shown with the fluorescence assay).50,51 As such, the Hill constant, h, was assumed to be an arbitrary unit to reflect the apparent sigmoidicity of the data and to allow estimation of (56) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Plenum Press: New York, 1999.

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Figure 3. Concentration-response curves for AChE inhibitors using the holographic sensor prototype. The IC50(app) of the inhibitors were extrapolated from the curves: edrophonium (9), galanthamine (0), eserine (2), tacrine (b), and neostigmine (O). All inhibitors were preincubated with the sensor for 10 min prior to addition of 5 mM acetylcholine at 25 °C. Error bars represent the standard deviation from the mean (n ) 3).

KM(app) (∼3.4 mM). Several factors could account for the nonMichaelis-Menten behavior of the immobilized enzyme, including a substrate partitioning effect (the partition coefficient, F, estimated herein as 0.02), a diffusion limitation and an effect of pH. However, it was postulated that the most probable cause was the inherent buffering capacity of the sensor, either imparted by the gelatin layer or the holographic gel or both. AChE activity in solution was measured by a standard unmodified polyHEMA hologram at two assay buffer concentrations. The solution activity at 1 mM assay buffer (as used in all other experiments) appeared to conform to Michaelis-Menten behavior, and this indicates a negligible effect of buffering by the polyHEMA gel on the immobilized enzyme kinetics. An analogous sigmoid kinetic pattern was noted when the assay buffer concentration was increased by 10-fold, suggesting that the additional buffering capacity that was earlier presumed to distort the Michaelis-Menten kinetic behavior must have been exerted by the gelatin component. Therefore, a certain concentration of substrate and level of pH change must be reached to induce a detectable holographic response. The interrelationship among the cited parameters was further complicated in a proton-liberating reaction like acetylcholine hydrolysis, and the perturbation of the inherent kinetic behavior would be even more drastic in an enzyme system with high intrinsic catalytic efficiency like AChE. Consequently, the inhibition parameters IC50 and Ki could also be affected; however, given proper control experiments and appropriate assumptions made where necessary, their apparent values (IC50(app) and Ki(app)) could still be attained as a manifestation of the intrinsic inhibition characteristics of the enzyme (Figure 3). The IC50(app) for edrophonium, galanthamine, neostigmine, tacrine, and eserine were estimated as 23.6(±2.9), 16.4(±6.6), 2.76(±0.25), 1.77(±1.35), and 0.24(±0.09) µM, in that respective order. Between the two competitive inhibitors (edrophonium and galanthamine), comparable IC50(app) values were attained as seen with the fluorescence assay (Table 1). A similar phenomenon was also observed among the mixed/ noncompetitive inhibitors (tacrine, eserine, and neostigmine). Although the inhibitors may have a similar mode of action in solution, they are different in many aspects such as molecular 7586

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Figure 4. A single-sensor analysis of inhibition of immobilized AChE by different edrophonium concentrations at 3.7 (O), 11 (b), 33 (0), and 100 µM (9). All inhibitors were preincubated with the sensor for 10 min prior to additions of acetylcholine (0.5-10 mM) at 25 °C. The inset is a secondary plot of KM(app)/Vmax(app) derived from the data as a function of edrophonium concentration with a linear fit (R 2 ) 0.989). The Ki value is determined from the negative value of the x-intercept. Error bars represent the standard deviation from the mean (n ) 3).

weight, structure, chemical properties, binding sites, accessibility to the immobilized enzyme, and interaction with the matrix; therefore, it is not strictly accurate to compare IC50’s of different inhibitor type.35,36 Nevertheless, preliminary data demonstrate the viability of using the sensor to screen compounds, especially those with structurally and chemically similar properties, for reliable comparison and ranking order of their relative potencies. Determination of Ki(app) with the sensor prototype was exemplified with edrophonium, using data from triplicate experiments with a single sensor (Figure 4). A secondary plot of KM(app)/Vmax(app) as a function of edrophonium concentration was used to estimate the Ki(app), which was equivalent to the x-intercept (11 µM) (inset of Figure 4).35 To validate the reliability of this approach, data from three determinations using separate sensors were also compared, with a similar Ki(app) being achieved. Like substrates, inhibitors may also be subjected to partitioning and/or diffusion constraints. Moreover, the partitioning of the inhibitor in the presence and absence of substrate partitioning and/or diffusion limitation should also be considered. In the absence of any matrix effect on the substrate, the inhibitor concentration in the microenvironment would be either higher or lower than that in the bulk phase, depending on the ionic nature of the inhibitor and polymer.43 In this case where edrophonium and acetylcholine have relatively similar charge and molecular structure, the Ki(app) would also be subjected to the same form of constraint, yielding a similar partition coefficient to that of the KM(app) as shown earlier (F ∼ 0.02).43 Nevertheless, the results further confirm the reliability of the sensor in estimating Ki(app), not only for a particular inhibitor but also for other structurally and chemically similar inhibitors provided a suitable correction factor (for example, using the partition coefficient) is applied if necessary. Holographic Array Sensors. Four circular and equally spaced individual polyHEMA holograms were constructed on a backing glass slide.37 The alignment of these spot holograms essentially mimicked an array sensor format to permit screening assays to be conducted in parallel. The apparent absence of the reflected light from some spot holograms was due to the viewing angle at which the photographic image was captured, since the diffraction

Figure 5. (a) Developed spot holograms (i) without and (ii) under illumination with white light. (b) Effect of initial enzyme loading on immobilized activities of spot AChE sensors. The AChE concentrations used were 3.13 (]), 6.25 (2), 25 (0), 50 (9), 100 (O), and 200 U/mL (b). Upon addition of 10 mM acetylcholine, all measurements were made at 25 °C. Error bars represent the standard deviation from the mean (n ) 3). (c) Apparent immobilized activities of the sensors in terms of the total peak wavelength shifts (as measured at t ) 5 min). The inset shows a linear correlation when the initial rates of the peak wavelength shifts are plotted against the different enzyme concentrations used (R 2 ) 0.997).

wavelength of the hologram is dependent upon the angle at which the reference beam is incident on the holographic fringe planes (Figure 5a). It should be noted, however, that all the spot holograms appeared green (λmax ∼ 540 nm) when viewed at the same angle. Individual reaction chambers were created to encompass each spot hologram to prevent chemical cross-talk between the sensors. A customized platform was assembled to accommodate and interrogate the array format, with the optics configured such that the light was directed and collected from the underside of the hologram. This arrangement could circumvent another key limitation associated with the current label-free optical systems, which is the masking of binding measurements that occurs in complex samples (such as crude cell lysates, culture media, or serum samples); extensive calibration or sample preparation procedures are often necessary for accurate analysis of the binding events.15 The successful use of holographic sensors in complex samples has also been reported.29 The effect of fabrication procedures on the holographic film was also assessed. While scaling down the holograms did not significantly affect their inherent optical and functional properties, excellent reproducibility of the spot holograms was also demonstrated. Because of a higher effective surface area to volume ratio for reaction provided by these “miniaturized” sensors, the possibility of reducing the initial enzyme loading while retaining satisfactory response kinetics was investigated. This was achieved

by monitoring the apparent activities of the sensors comprising different enzyme concentrations immobilized (between 3.125-200 U/mL) (Figure 5b). The total wavelength shift (as measured at t ) 5 min) increased nonlinearly, yielding a gradual apparent signal saturation when the maximal response capacity of the spot sensor (∆λmax ∼140 nm) was reached at 200 U/mL AChE initial loading (Figure 5c). A linear correlation was also achieved between the initial rates of peak wavelength shift and the amount of enzyme loaded (inset of Figure 5c). When 50 U/mL AChE was immobilized in the initial sensor prototype, a maximal rate of ∼25 nm min-1 was attained; at the same concentration, the rate of wavelength shift recorded by the spot sensor was ∼35 nm min-1. Aside from extension of the holographic response kinetics, the spot sensor format also reduced the amount of enzyme required for immobilization to achieve a comparable holographic response. Real-Time Screening of Inhibitor. The immobilized enzyme on the spot sensor was also found to deviate from Michaelis-Menten behavior, exhibiting a sigmoid dependence of initial velocity of enzyme reaction on substrate concentration (KM(app) ) ∼6.6 mM). Similarly, the inhibition profiles of the inhibitors on the immobilized enzyme would also be affected; any assumptions made for the initial sensor prototype would likely be applicable to the array sensors, since both immobilized systems were consistent in their overall apparent kinetic behavior. Enzyme inhibition assays were carried out in parallel with one assay Analytical Chemistry, Vol. 81, No. 18, September 15, 2009

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Figure 6. (a) Holographic responses of spot sensors comprising a series of edrophonium concentrations at 0 (b), 1.2 (O), 3.7 (2), 11 (0), 33 (9), 100 (]), and 300 µM ([). (b) Photographic images illustrating real-time holographic response and visible color change of the spot sensors. The inhibitors were preincubated with the sensors for 10 min prior to addition of 10 mM acetylcholine at 25 °C. An end-point measurement of the peak wavelength in each spot sensor was also measured at t ) 5 min.

per spot sensor using edrophonium as an example. Each concentration of edrophonium was added into each reaction chamber containing assay buffer, followed by a preincubation of 10 min prior to addition of acetylcholine. As anticipated, the diffracted light of the spot holograms with the three highest inhibitor concentrations (11-100 µM) remained red-shifted, whereas those of the other holograms containing inhibitor concentrations between 0-3.7 µM were blue-shifted (Figure 6). The IC50(app) for edrophonium measured by the spot sensor was 16.2 µM, which was comparable to that estimated from the initial prototype (IC50(app) ) 23.6 µM). Evidently, adaptation of the holographic sensors into different formats is possible without compromising their fundamental working properties. CONCLUSIONS Holographic sensors suitable for enzyme inhibition assays have been developed from AChE immobilized on the holographic pH sensor. pH-sensitive polyHEMA holograms have been fabricated with MAA to monitor the pH change as a result of acetic acid production by the hydrolysis of the substrate acetylcholine. AChE was entrapped in a separate gelatin layer deposited on the hologram surface, followed by chemical cross-linking with form7588

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aldehyde. In addition to the simple immobilization procedure, harsh chemical conditions during hologram recording could be avoided, and thus increase the probability of retaining higher immobilized activity. More importantly, a faster localized pH was attained when immobilizing the enzyme in this way, resulting in a greater holographic response than that produced by the pH change detected in bulk solution. This could be attributable to the direct deposition and proximity of the gelatin-AChE layer on the sensor surface, possibly driving a higher number of protons liberated from the hydrolytic reaction to diffuse from the enzyme microenvironment into the holographic phase. Besides improvement of sensor kinetics and response range, the operational and storage stability were also established with a view to sensor development within the context of a robust detection modality for multiple, continuous screening assays. Because of the non-Michaelis-Menten behavior and an apparent positive cooperativity seen with the immobilized enzyme, the resultant inhibition parameters were also affected. The deviation from the Michaelis-Menten kinetic pattern of the immobilized enzyme was most likely brought about by the inherent buffering capacity of the gelatin layer per se. Other matrixes without buffering properties could be utilized to substitute gelatin

and thereby still retain the bilayer structure of the sensor; however, additional studies are required to ensure that a comparable holographic response range and kinetics can be fulfilled by the alternative approach. Despite the higher IC50(app) values recorded by the sensor, the relative potency of one inhibitor with respect to the other(s) of the same mode of action was comparable to that seen with the fluorescence assay. Although determination of Ki(app) with the sensor prototype was only exemplified using edrophonium, the single-sensor data were consistent with those determined from separate sensors. As such, both IC50(app) and Ki(app) could still be estimated with the sensor to represent the intrinsic inhibition properties of the enzyme provided that a proper calibration is used when analyzing their respective data. This applies not only to a particular compound but also for structurally and chemically related chemical entities, which are included in most screening libraries generated by the high-throughput synthetic chemistry domain in drug discovery research. Initial studies of the holographic array sensors demonstrated that their functional integrity have not been impaired by the fabrication procedure. The overall apparent kinetic and inhibition characteristics of the enzyme immobilized on the “miniaturized” array format were comparable to those of the initial prototype. In addition to a marked increase in assay throughput, it is envisaged that the array format allows high flexibility in experimental design. Key development studies of the sensors can be fully validated prior to adaptation into the final intended format, hence

providing ample scope for sensor improvement and potentially reducing the overall production cost and time. Although continuous real-time screening of compounds could be feasible, the proof-of-concept system is restricted to biological targets where a pH change associated with their binding and/or activity is involved, some of which may not be pharmaceutically relevant. Current work is thus focused on integrating novel (bio)recognition chemistries within the holographic matrix to render the system amenable to a wide range of targets of pharmaceutical interest, such as proteases, kinases, and membranebound G-protein coupled receptors (GPCRs). Throughput-wise, it is anticipated that the density of the current system can be increased and engineered as an array of holographic spots (possibly in micrometer dimensions). Coupled with an appropriate optics configuration, a new platform could be devised for multiple sensors to be scanned in parallel for a direct visual readout, providing an immediate “Yes/No” binding and/or activity answer with holographic responses recorded in real-time at various concentration ranges. ACKNOWLEDGMENT We thank GlaxoSmithKline plc for a research studentship and Theresa Pell, Colin Davidson, Jeff Blyth, and Xiaoping Yang for useful discussions. Received for review April 27, 2009. Accepted July 28, 2009. AC9008989

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