Supramolecular Approach to Enzyme Sensing on Paper Discs Using

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A supramolecular approach to enzyme sensing on paper discs using lanthanide photoluminescence Tumpa Gorai, and Uday Maitra ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.6b00341 • Publication Date (Web): 21 Jun 2016 Downloaded from http://pubs.acs.org on June 25, 2016

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A supramolecular approach to enzyme sensing on paper discs using lanthanide photoluminescence Tumpa Gorai and Uday Maitra* Department of Organic Chemistry, Indian Institute of Science, Bangalore-560012, Karnataka, India KEYWORDS: Gel coated disc, Enzymes, Biosensor, Lanthanide, Luminescence . ABSTRACT: A paper based photoluminescent biosensor has been developed, with green Tb-luminescence as the output, for the rapid detection of β-glucosidase and lipase, both in purified form and in biological/natural fluids. A Tb Cholate hydrogel doped with specially designed, specific enzyme substrates ("pro"-sensitizers) was integrated on filter paper discs.The addition of small volumes (~5 µL) of enzyme solutions on these discs led to enhanced green emission (UV lamp, λex 365 nm), allowing qualitative detection of the enzymes – when needed the intensity enhancement can be quantified using an image processing software, or for multiple samples using a commercial plate reader. This simple technique allowed the detection of β-glucosidase in almond extract and lipase in blood serum. Easy identification of the inhibition of β-glucosidase was also demonstrated. The paper based sensor is inexpensive, user friendly and needs low volumes of biological sample for analysis. This strategy has the potential to be useful for possible clinical applications.

The development of user-friendly biosensors1,2 is of great importance in the field of biological and medical sciences and in the diagnosis of different diseases especially in resource limited areas. Among different classes of biosensors, paper based biosensors have drawn much interest in recent years because of their easy availability, low cost of fabrication, biocompatibility and biodegradability.3-5 The porous nature of paper allows efficient immobilization of a sensing material and easy diffusion of a target analyte. As a result, paper based colorimetric,6-17 electrochemical,18 chemiluminescence,19 electrochemiluminescence20,21 and fluorescent22-24 sensors have found uses in sensing ions,6,7 glucose,8,10 H2O2,9-11 DNA,18 biomarkers13,20,21 etc. Enzymes are one of the important target bioanalytes in the field of biosensors as they play crucial roles in the regulation of metabolic functions in living systems.25-28 The detection of enzyme activity through an efficient and simple design is therefore of utmost importance. The most commonly employed enzyme assays are solution based and the detection is done either through colorimetry29-33 or fluorimetry.34,35 Although the fluorescence based assays provide a higher intrinsic sensitivity, the autofluorescence from biological samples interferes with the measurements and this is a drawback for the detection of bioanalytes. In this context, a paper based sensing platform to detect enzyme activity through a delayed luminescence read-out would be more convenient. Trivalent lanthanides are excellent candidates in this respect as they possess sharp, almost line-like, long lived luminescence,36-39 and have been used for the design of enzyme sensors40-42 which required multi-step synthesis and did not have a general design principle. We herein report the fabrication of a new paper based sensor which detects enzymes rapidly through an enhancement in lanthanide luminescence and can be envisaged as a portable and disposable analytical device. This sensor only requires the design and synthesis of appropriate pro-sensitizers and the ‘sensing units’ are generat-

ed through self-assembly. We have previously reported a unique way of sensitizing lanthanides by bringing the trivalent Ln(III) ions and sensitizers in Cholate hydrogel matrices through the self-assembly of commercially available components.43,44 We also successfully demonstrated a photoluminescent enzyme assay by using suitably modified “prosensitizers” (unnatural enzyme substrates) which upon enzyme action released the sensitizer leading to a concomitant enhancement in Tb(III) emission in the hydrogel.45 We now present our work on the design and application of a paper based sensor created by coating a filter paper disc with Tb(III) based hydrogel. These Tb(III) based paper discs can detect enzymes with a green luminescence response, which can be photographed and quantified by an image processing software (we used ImageJ). For rapid analysis of a large number of samples a commercial plate reader can be used. We have also demonstrated the detection of these enzymes in natural/biological samples. The advantage with this system is that the entire sensing material is integrated on the paper surface in a single step by coating the Tb(III) based hydrogel doped with Table 1. Comparison of enzyme assays Assay

Probe

Medium

Protease

Lanthanide complex

Solution

N-acetyltransferase41

Lanthanide complex

Solution

Protease

Lanthanide complex

Solution

Alkalinephosphatase & β-galactosidase12

Colorimetric reagents

Paper based

This work: β-glucosidase and Lipase

Luminescent Tbsensitizer self assembly

Paper based

40

42

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an appropriate enzyme substrate and the response is generated in a few minutes. To the best of our knowledge such paper based sensors to detect enzyme activity through lanthanide luminescence have not yet been reported in the literature (Table 1).

EXPERIMENTAL SECTION Materials and Methods Sodium Cholate, Tb(III)-acetate, 2,3-dihydoxynaphthalene (1), β-glucosidase (6 U/mg, form almonds, mol wt ~135000), Lipase (2.9 U/mg, from Candida rugosa, mol wt ~67000 ) were purchased from Sigma Aldrich. Mono-β-glucoside derivative of DHN (2), DHN-diacetate (3), (chart 1) were synthesised using previously reported procedures.45 Whatman filter paper (no 1, 3) and black chart paper were locally purchased. Distilled water was used for all the studies. An ultrasonic bath sonicator (1.5 L) was used for the preparation of the gels. A TLC cabinet viewer UV lamp (365 nm) wavelength was used for qualitatively viewing the Tb luminescence after adding enzyme. A Sony Cyber-Shot DSC-H70 16.1 MP Digital still Camera with 10x Wide-Angle Optical Zoom G Lens/ 3.0-inch LCD was used for capturing images under the UV lamp. A Varioscan plate reader was used for the luminescence measurements of the gel coated paper discs. ImageJ software was used for measuring green colour intensity. POM images were recorded on an in Olympus BX 51 polarizing optical microscope. SEM images were recorded in FEI ESEM Quanta 200 instrument. The emission intensities from these discs were recorded on a plate reader with TRF delay time of 200 µs, integration time of 1000 µs, λex 335 nm and λem 545 nm. The error bars reflect the standard deviations of three/ four sets of measurements (n).

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to the reaction mixture and stirred at room temperature for 2h. The reaction mixture was then filtered, diluted with CHCl3 and washed with H2O (20 mL). The organic layer was collected over Na2SO4 and after removal of the solvent under vacuum, the residue was purified by column chromatography on silica gel using chloroform to obtain 4 (480 mg, 71%) (scheme 1) as a white solid.1H NMR (400 MHz, CDCl3) δ 7.78 (dd, J = 3.2, 6.4 Hz, 2H), 7.65 (S, 2H), 7.47 (dd, J = 6.4, 3.2 Hz, 2H), 2.625 (dd, J = 7.6, 15.2 Hz, 4H), 1.298 (t, J = 7.6 Hz, 6H); 13C NMR (100 MHz, CDCl3) δ 171.92, 140.91, 131.41, 127.32, 126.14, 120.72, 27.41, 9.03; IR (KBr, cm-1) 1763, 1508, 1352, 1248, 1121, 902, 755; HRMS: calcd for C16H16O4Na [M+Na] 295.0948; observed C16H16O4Na [M+Na] 295.0946 Synthesis of Naphthaelne-2, 3-diyl dihexanoate (5): N,N'-Dicyclohexylcarbodiimide (DCC) (1.18 gm, 5.73 mmol) and 4-dimethylaminopyridine (DMAP) (45 mg, 0.37 mmol) were added to the n-hexanoic acid (0.63 mL, 4.99 mmol) solution in acetonitrile (3 mL) and stirred at 0ºC for 30 min and a white precipitate appeared. 2,3-dihydroxynaphthalene (DHN) (400 mg, 2.5 mmol) solution in acetonitrile (3 mL) was added to the reaction mixture and stirred at room temperature for 2h. The reaction mixture was then filtered, diluted with CHCl3 and washed with H2O (20 mL). The organic layer was collected over Na2SO4 and after the removal of the solvent under vacuum, the residue was purified by column chromatography on silica gel using chloroform to obtain 5 (546 mg, 61%) (scheme 1) as a white solid.1H NMR (400 MHz, CDCl3) δ 7.79 (dd, J = 3.2, 3.2 Hz, 2H), 7.64 (S, 2H), 7.46 (dd, J = 2.8, 5.8 Hz, 2H), 2.58 (t, j = 7.6 Hz, 4H), 1.75-1.82 (m, 4H), 1.36-1.41 (m, 8H), 0.94 (t, J = 6.8 Hz, 6H); 13C NMR (100 MHz, CDCl3) δ 171.27, 140.97,131.42, 127.32, 126.12, 120.75, 34.0, 31.19, 24.51, 22.22, 13.78; IR (KBr, cm-1) 1764, 1508, 1466, 1313, 1246, 1135, 908, 749; HRMS: calcd for C22H28O4Na [M+Na] 379.1885; observed C22H28O4Na [M+Na] 379.1885 Tb Cholate gel and coated disc preparation procedures

Chart 1. Structures of sensitizer (1), pro-sensitizers (2-5) and the inhibitor (6) used for β-glucosidase and lipase detection.

For the preparation of pro-sensitizer (2-5) doped Tb-Cholate gels, Tb-acetate (10 mM) solution and sodium Cholate (NaC, 30 mM) solution containing 75-200 µM of DHN (1) or prosensitizer (2-5) were prepared. Tb-acetate (400 µL) solution was mixed with NaC (400 µL) solution and sonicated for 1215 seconds to make a transparent gel. The as-prepared gel was sonicated for an additional 3-5 sec to reduce the viscosity. On each filter paper disc 40 µL gel was applied (in 20 µL portions) such that it spread uniformly. After 15 minutes the gel was absorbed completely by the filter paper which was then air dried.

Scheme 1: Synthesis of compound 4 & 5

Preparation of almond extract

Synthesis of Naphthalene-2, 3-diyl dipropionate (4): N,N'-Dicyclohexylcarbodiimide (DCC) (1.2 gm, 5.8 mmol) and 4-dimethylaminopyridine (DMAP) (40 mg, 0.32 mmol) were added to the n-propanoic acid (0.4 mL, 5.4 mmol) solution in acetonitrile (3 mL) and stirred at 0ºC for 30 min and a white precipitate appeared. 2,3-dihydroxynaphthalene (DHN) (400 mg, 2.5 mmol) solution in acetonitrile (3 mL) was added

Almond seeds (1.1 gm) were finely ground in a mortar and pestle. To the powder taken in a vial (15 mL), distilled water (7 mL) was added and the mixture was shaken manually at 15 min intervals while the sample was kept cold in a refrigerator. After 3 h the solid was removed by centrifugation, and the filtrate was used for the detection of β-glucosidase.

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RESULTS AND DISCUSSION Tb-Cholate gel was prepared by mixing equal volumes of Tbacetate and Na-Cholate solution in 1:3 molar ratios. On a rectangular (1 cm Х 1 cm) Whatman no 1 filter paper the gel (Tb3+ 5 mM, Cholate 15 mM, ca. 200 µL) was applied to cover the surface of the paper and dried for 2 h. The uncoated and the dried, coated paper were observed by polarising optical microscopy (POM, Figure 1a-i and 1a-ii) and scanning electron microscopy (SEM, Figure 1a-iii and 1a-iv and for more SEM images see Figure S1 in the Supporting Information). Both POM and SEM images showed the fibrous network of the cellulose fibres (Images 1a-i, 1a-iii), while the dried gel on the paper (Images 1a-ii, 1a-iv) showed up as flakes located on and around the fibres. This suggested the feasibility of the assay using this xerogel coated paper.

Quantification of the luminescence can be done inexpensively by the measurement of the intensity of the green color by readily available image processing software. On the other hand, for more detail understanding of the enzyme activity, or for rapid analysis of multiple samples, a commercial plate reader can be used.5 The (green) color intensity measured for the 9 discs showed < 5 % deviation, almost completely agreeing with the plate reader data (< 5% deviation). (Figure S4 and Table S1 in the Supporting Information). After optimizing the procedure for disc coating, enzyme detection was carried out. β-Glucosidase is an enzyme present in all living systems which helps in the metabolism of cellulose and the other forms of carbohydrates for nutrition.46 For β-glucosidase detection the coated disc was prepared from compound 2 (200 µM) doped Tb-Cholate gel. This coated disc was nonluminescent under 365 nm UV lamp but after the addition of aqueous (aq.) β-glucosidase, it became intensely green luminescent (Figure 1c). Next, the experiment was done in the following way: water (16 µL) was added to the first disc as a control and increasing amount of (corresponding to 4, 8, 16 and 32 µg of enzyme, respectively) β-glucosidase was added on the remaining discs. As a result, enhanced emission was observed with increasing time and enzyme concentration. The images of the luminescent discs (under 365 nm UV lamp) were captured after 20 and 60 min (Figure 2a) and The intensity enhancement was quantified by using a plate reader and also using ImageJ software. For doing experiments in a plate reader, gel coated discs were placed in the wells of a 96-well plate. After adding 5 µL portions of the enzyme solutions the emission intensities were measured, which showed an increase in emission intensity with increasing enzyme concentration (Figure 2b and Figure S14a in the Supporting Information).

Figure 1. (a) POM images of the filter paper (a-i) before and (a-ii) after gel coating (scale bar 200 µm); SEM images of the filter paper (a-iii) (scale bar 300 µm) before and (a-iv) (scale bar 100 µm) after gel coating. (b) Schematic representation of gel coating procedure on the paper disc. (c) Green emission under UV lamp (λex 365 nm) due to generation of free sensitizer after enzyme action on gel coated paper discs.

Paper coating attempted by other non-gel routes proved to be ineffective for enzyme detection (Figure S2 & S3 in the Supporting Information), thereby confirming the effectiveness of the gel-based coating. Using a 3 mm hole puncher, circular holes were made on a black chart paper 2.5 cm apart. Square pieces (1.5 Х 1.5 cm) of Whatman no 3 filter paper53 were attached by glue on one side so that from the opposite side only the disc shaped filter paper was visible (Figure 1b). To establish the uniform nature of the coating, a luminescent gel (made from DHN (1), Tb3+) was initially used for coating nine identical discs. The as-prepared DHN doped Tb-Cholate gel was sonicated for an additional 5-6 sec to reduce the viscosity. On each disc 40 µL gel was applied (in 20 µL portions) such that it spread uniformly. After 15 minutes the gel was found to be absorbed completely by the filter paper which was then air dried for 30 minutes. The coated discs were first checked under a 365 nm UV lamp which showed uniform green emission.

Figure 2. (a) Images captured under 365 nm UV lamp after addition of increasing amounts of β-glucosidase. (b) Plate reader data (λem 545 nm) after 140 min of addition of variable amounts of β-Glucosidase (n = 4). (c) Plate reader data (λem 545 nm) as a function of time (with 1.2 µg of β-glucosidase) (n = 3)

In another experiment, the intensity of the gel coated paper was measured with 1.2 µg of the enzyme as a function of time (Figure 2c, Figure S14b in the Supporting Information). The limit of detection (LOD) of β-glucosidase by this technique was calculated to be 12.7 µg/mL (~94 nM). LOD was calculated using the formula LOD = 3σ/ b (σ represent the standard

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deviation of the signal obtained from the blank sample (676.61) and b represent the slope (31.97) of the titration curve) The color intensity measurement was also performed for a similar experiment using ImageJ. For this purpose, on several 2-doped Tb Cholate gel coated discs β-Glucosidase solution was added in low (nM) concentrations. After 105 min detectable emission was observed from all the enzyme added discs (Figure 3a). A photograph was taken, and the plot of color intensity vs enzyme concentration showed increase in color with increasing enzyme concentration (Figure 3b).

Figure 3. (a) Image captured under 365 nm UV lamp 105 min after addition of β-glucosidase in nanomolar concentrations. (b) Green color intensity (∆I = Isample - IBlank) plotted against βglucosidase concentrations (n = 3).

A control experiment was done by directly applying aq. Tb-acetate solution doped with 2 on disc 1 (Figure 4a) and checking its photoluminescence after adding aq. βglucosidase. No emission was observed from disc 1, but disc 2 prepared with the Tb-Cholate gel doped with 2 showed bright green emission after adding aq. β-glucosidase.

Figure 4. (a) Image under 365 nm UV lamp for 2 containing Tbacetate solution added disc (disc 1) and 2 doped Tb-Cholate gel coated disc (disc 2) after the addition of β-glucosidase (16 µg). (b) Plate reader data (λem 545 nm) as a function of time for 2 containing Tb-acetate solution added disc (disc 1) and 2 doped Tb-Cholate gel coated disc (disc 2) after the addition of βglucosidase (n = 3).

Measurement on a plate reader confirm the visual observations (Figure 4b) thereby proving that only the gel-derived material has the ability to exhibit luminescence enhancement. 54

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To check the presence of β-glucosidase in natural samples, almond was chosen as a source of glycosidase enzymes.47 When water, heat treated (90 °C/ 15 min) almond extract (it was expected that heat treatment denatured β-glucosidase present in the almond extract) and fresh almond extract were added on discs 1, 2, 3, respectively (Figure 5a), emission enhancement was observed only on disc 3 (due to the presence of native β-glucosidase). Color intensity (software) and plate reader measurements (Figure 5b, Supporting Figure S5 and Figure S6) were done to confirm these observations. Many enzyme inhibitors are of clinical importance,48 and thus a rapid screening of an enzyme inhibitor is always desirable. To check the efficiency of the coated disc for inhibitor screening, a bicyclic aza sugar 6, a known inhibitor for β-glucosidase with an IC50 of 86 µM, was chosen.49 For the inhibition studies, aq. β-glucosidase was pre-incubated with varying concentrations of this inhibitor. The test discs were prepared as before and treated with inhibitor-incubated enzyme solutions which showed reduced enzyme activity compared to the control (Figure 5c). Plate reader measurements (Figure 5d) confirmed the expected results.

Figure 5. (a) Images captured under 365 nm UV lamp after 30 min of addition of 10 µL portions of Water (disc 1), Heat treated almond extract (disc 2), Almond extract (disc 3). (b) Green color intensity (∆I = Isample - IBlank) plot of the gel coated discs after 60 min of addition of 10 µL portions heat treated almond extract (disc 2), Almond extract (disc 3) (n = 3). (c) Images captured under 365 nm UV lamp for gel coated discs after adding βglucosidase pre-incubated with inhibitor 6. (d) Plate reader data (λem 545 nm) as a function of time after addition of inhibitor incubated β-glucosidase on gel coated discs.

Lipase is another enzyme of interest as it is routinely assayed for the diagnosis of several cardiac and liver related diseases.50 Initial studies for lipase detection with DHN-diacetate (3) showed that it was not very stable in the coated paper disc (Figure S7 in the Supporting Information). Therefore DHN dipropionate (4) and DHN-dihexanoate (5) were tested (Figure S8 in the Supporting Information), and DHN-dipropionate (4) was found to be optimum with respect to stability and rate of reaction (5 reacted too slowly). The higher stability for longer chain esters on paper surface follows the usual rates of hydrolysis as a function of the steric bulk of the alkyl chain. Therefore, 4 doped Tb-Cholate gel coated discs were made and as before, images were captured for the emissive discs (after adding varying amounts of lipase) at specific time intervals (Figure 6a). Time delayed luminescence on a plate reader was also measured (Figure 6b, Figure 6c, and Figure S15 in the Supporting Information). By this technique, the lowest amount (LOD) of lipase, we could detect was 1.6 µg/mL (σ =153.5, d

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= 59.4) (24 nM), which is lower than some of the literature reported values.51,52

Figure 6. (a) Images captured under 365 nm UV lamp after addition of increasing amounts of lipase. (b) Plate reader data (λem 545 nm) after 120 min of addition of variable amounts of Lipase (n = 3). (c) Plate reader data (λem 545 nm) as a function of time (with 156 ng lipase) (n = 3).

As with glucosidase, lipase detection can also be done. 2 h after enzyme addition to 4 doped Tb Cholate gel coated discs (Figure 7a). Green color intensity from the images captured under UV lamp was plotted against lipase concentration which showed a linear increase initially, later it attain a plateau at higher enzyme concentration (Figure 7b).55

in blood serum. As shown in Figure 8a, the disc with blood serum added on it, showed enhancement of luminescence within 15 min, while with water and denatured blood serum (80 °C/15 min) no enhancement was seen.

Figure 8. (a) Images captured under UV lamp (i) before and (ii) 15 minutes after addition of 10 µL H2O (disc 1),10 µL denatured blood serum (disc 2), 10 µL blood serum(disc 3). (b) Plate reader data (λem 545 nm) as a function of time, after adding 5 µL H2O (black line) 5 µL denatured blood serum (red line) and 5 µL blood serum (green line) on gel coated disc. (c) Color intensity (∆I = Isample - IBlank) plot for 4 doped gel coated disc 30 min after adding 5 µL denatured blood serum and 5 µL blood serum (n = 3).

The observation under UV lamp was quantified by use of plate reader instrument and also by the color intensity measurement (Figure 8b, Figure 8c and Figure S9 in the Supporting Information). The material cost for the disc fabrication was calculated to be less then INR 1/- (Supporting Information Table S4).

CONCLUSIONS In summary, a new paper based luminescent biosensor has been designed for β-glucosidase and lipase. Intensity enhancement can be quantified using an image processing software, or by a commercial plate-reader. The material cost for gel coated disc preparation was less then INR 1/-, so it is a low cost luminescent assay system. This simple paper-based sensor is useful for detecting the presence of enzymes in real samples, and for rapid screening of an inhibitor. We are currently expanding the scope of this enzyme assay, and also use other lanthanides for a possible 2-color detection system. We hope that this straightforward approach to enzyme sensing would be useful to many others. Figure 7. (a) (a) Image captured under 365 nm UV lamp 120 min after addition of Lipase in nanomolar concentrations. (b) Green color intensity (∆I = Isample - IBlank) plotted against Lipase concentrations (n = 3).

To confirm that the generation of the sensitizer is indeed responsible for the luminescence enhancement, HPLC analysis of the extract from the discs was done. The data showed the formation of the sensitizer only after adding the enzyme solution on the gel coated paper (Supporting Information note 10 and Figure S12, S13 in the Supporting Information). The following experiment illustrates the application of this paper based assay for detecting (non-specfic) lipases/esterases

ASSOCIATED CONTENT Supporting Information Available: The following files are available free of charge. Figure S 1: Additional SEM images of the gel coated paper, Figure S 2-3: Comparison of coated discs prepared by different techniques, Figure S 4 and Table S1: Data for checking reproducibility of gel coating, Figure S5 & S6: Photograph for almond β-glucosidase detection in three sets and corresponding plate reader data, Figure S7, Figure S8 and Table S2: Stability study of three different DHN-diesters on paper disc,

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Figure S 9: Photograph for blood serum lipase detection in three sets, Figure S 10, Figure S11 & Table S3: Sensor disc stability study, Figure S 12 & S13: HPLC study for the enzyme assays, Figure S 14 & S15: Luminescence spectra for β-glucosidase and Lipase detection, Figure S16: Checking activity of the β-glucosidase doped Tb Cholate gel coated disc, Table S4: Material cost for coated disc preparation and spectroscopic data for DHN-dipropionate and DHN-dihexanoate.

AUTHOR INFORMATION

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Corresponding Author *E-mail: [email protected]

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Notes The authors declare no competing financial interests.

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ACKNOWLEDGMENT This research was supported by the Department of Science and Technology (grant no. SR/S1/OC-68/2011 and SR/NM/NS88/2010). We thank Prof. Y.D. Vankar for a sample of inhibitor 6. We thank Hetal Vaishnav for helping with some experiments. The AFMM centre, IISc is acknowledged for SEM facility. The Council of Scientific and Industrial Research (CSIR) is thanked for the award of a research fellowship to TG.

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ABBREVIATIONS DHN, 2,3-dihydroxynaphthalene. (20)

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(45) Bhowmik, S.; Maitra, U. A novel ‘‘pro-sensitizer’’ based sensing of enzymes using Tb(III) luminescence in a hydrogel matrix. Chem. Commun. 2012, 48, 4624–4626. (46) Jeng, W. -Y.; Wang, N. -C.; Lin, M. –H.; Lin, C. -T.; Liaw, Y. -C.; Chang, W. -J.; Liu, C. -I.; Liang, P. -H.; Wang, A. H.-J. Structural and functional analysis of three b-glucosidases from bacterium Clostridium cellulovorans, fungus Trichoderma reesei and termite Neotermes koshunensis. J. Struct. Biol. 2011, 173, 46–56. (47) He, S.; Withers, S. G. Assignment of sweet almond βglucosidase as a family 1 glycosidase and identification of its active site nucleophile. J. Biol. Chem. 1997, 272, 24864-24867. (48) Asano, N. Glycosidase inhibitors: update and perspectives on practical use. Glycobiology 2003, 13, 93R-104R. (49) Mallick, A.; Pal, A. P. J.; Vankar, Y. D. Synthesis of L-3epi-isofagomine, its homo-, n-butyl and bicyclic analogues from D-glucose as glycosidase inhibitors. Tetrahedron Lett. 2013, 54, 6549-6552. (50) Littlewood, J. M.; Wolfe, S. P.; Conway, S. P. Diagnosis and Treatment of Intestinal Malabsorption in Cystic Fibrosis. Pediatric Pulmonology 2006, 41, 35–49. (51) Zhang, W.; Tang, Y.; Liu, J.; Jiang, L.; Huang, W.; Huo, F. -W.; Tian, D. Colorimetric Assay for HeterogeneousCatalyzed Lipase Activity: Enzyme-Regulated Gold Nanoparticle Aggregation. J. Agric. Food Chem. 2015, 63, 39−42. (52) Tang, Y.; Zhang, W.; Liu, J.; Zhang, L.; Huang, W.; Huo, F.; Tian, D. A plasmonic nanosensor for lipase activity based on enzyme-controlled gold nanoparticles growth in situ. Nanoscale 2015, 7, 6039–6044. (53) Gel coating on Whatman 3 filter paper was more uniform than on Whatman 1, and thus Whatman 1 was used only for recording POM images (Whatman 3 filter paper has greater thickness than Whatman 1 so light can't pass through it) and for all the other experiments Whatman 3 filter paper was used. Minimum 40 µL gel was needed for a uniform coating of a 3.5 mm disc. (54) Hydrophobic pockets formed during gel formation partially dehydrate Tb(III). The gel matrix also keeps close proximity between Tb(III) and the sensitizer released after enzyme action. In solutions concentrations are not adequate for this complexation to occur. (55) For the plate reader measurements, the intensities at 545 nm from the discs were measured as a function of enzyme concentration. In the image processing software the green color intensity from the discs were measured as a function of enzyme concentration. The dynamic range of intensity variation is different for these two methods. Both the methods showed linearity at lower enzyme concentrations. The nonlinearity in color intensity plot at higher enzyme concentrations is possibly due to saturation.

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