Cellulose-Based Biosensors for Esterase Detection - ACS Publications

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Cellulose-based Biosensors for Esterase Detection Fatemeh Derikvand, DeLu (Tyler) Yin, Ryan Barrett, and Harry Brumer Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04661 • Publication Date (Web): 19 Feb 2016 Downloaded from http://pubs.acs.org on February 22, 2016

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Cellulose-based Biosensors for Esterase Detection Fatemeh Derikvand, DeLu (Tyler) Yin, Ryan Barrett, and Harry Brumer* The Michael Smith Laboratories and Department of Chemistry, University of British Columbia, 2185 East Mall, Vancouver, BC, V6T 1Z4, Canada * Corresponding author: [email protected]; FAX +1 604 822 2114

Cellulose is also widely used in biological and biomedical applications as the principal component of dialysis membranes (regenerated cellulose), non-woven fabrics, and gauzes. Likewise, bacterial cellulose (BC) and other nanocellulose hydrogels have found unique biomedical applications as tis11 sue scaffolds and wound dressings. In wound dressings, in particular, there is significant interest in the development of bioactive surfaces, including those capable of detecting pathogens or endogeneous enzymes. In an elegant example, Edwards and co-workers have devised a bioactive cotton gauze to detect human neutrophil

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Cellulose-based paper has emerged as an attractive substrate for the development of economical, disposable analyti1-10 cal devices, especially for point-of-care (POC) applications. In addition to its light weight, low cost, and status as a readily biodegradable, renewable material, the hydrophilic, porous nature of paper offers particular advantages vis-à-vis other solid supports, e.g. glass and plastic, including a high 7 surface area and intrinsic capillary fluidics. Hence, paper has been effectively utilized as a matrix in dip-sticks, lateral flow assays (LFAs), and microfluidic paper analytical devices (µPADs) for the detection of a wide range of inorganic, organic, and biological analytes. Direct, visual readout is typically provided by intrinsic analyte color, chromogenic biochemical reactions, or analyte labelling with chromophores, fluorophores, or particles, thereby obviating the need for 5 large or costly analytical instruments. This is a particular 1,4,7 advantage in resource-limited locations.

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strate for the production of economical, disposable point-ofcare (POC) analytical devices. Development of novel methods of (bio)activation is central to broadening the application space of cellulosic materials. Ironically, such efforts are stymied by the inherent biocompatibility and recalcitrance of cellulose fibers. Here, we have elaborated a versatile, chemoenzymatic approach to activate cellulosic materials for CuAAC “click chemistry,” to develop new fluorogenic esterase sensors. Gentle, aqueous modification conditions facilitate broad applicability to cellulose papers, gauzes, and hydrogels. Tethering of the released fluorophore to the cellulose surface prevents signal degradation due to diffusion, and enables straightforward, sensitive visualization with a simple light source in resource-limited situations.

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elastase (HNE) in chronic wounds, the over-abundance of 13 which is implicated as a key factor preventing healing. This POC medical device comprises a chromogenic peptide substrate conjugated via the N-terminus to cotton cellulose fibers. Enzymatic cleavage of the C-terminal amide causes release of the yellow p-nitroanaline (p-NA) chromophore, thereby allowing visual detection. A recent incarnation of this technology utilizes fluorogenic 4-amido-7-methylcoumarin peptide conjugates and has been adapted to cotton 14 cellulose nanocrystals. Despite its simplicity, this approach has two general limitations associated with the release of the chromophore into the surrounding medium: Firstly, small aromatic molecules such as p-NA, phenols, etc., are often 15,16 toxic to cells and may contribute to inflammation. Secondly, the chromophore will be lost via diffusion into the surrounding environment, thereby attenuating a continual diagnostic increase of color on the cellulosic substrate. This is also an issue for many dip-stick enzyme tests, in which the generated free chromophore can be lost into the solution 17 tested. Thus, there is significant scope to develop novel bioactive cellulose-based enzyme sensors that overcome these limitations.

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ABSTRACT: Cellulose has emerged as an attractive sub-

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Scheme. 1. Cellulose-based enzyme sensor approaches. A. Surface tethering via the biomolecule. B. Surface tethering via the chromogen or fluorogen.

To address this challenge, we envisioned a reversedsubstrate approach, in which the chromogenic moiety, rather than the biomolecular component, is tethered to the insoluble cellulosic substrate (Scheme 1). This approach simultaneously addresses the interrelated problems of signal attenu-

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ation and potential toxicity due to chromophore diffusion after substrate cleavage. At the same time, the liberated biomolecule (e.g., fatty acid or peptide) can be cleared by subsequent metabolism in vivo. We present here an initial proof-of-concept, in which general, fluorogenic esterase biosensors have been produced from a range of cellulosic substrates relevant to in vitro and in vivo diagnostics. Esterases, including lipases, represent a broad enzyme class of widespread importance in biology and biotechnology. A fundamental issue in the development of cellulose-based biosensors is the method of attachment of the bioactive moi18,19 ety to the poorly reactive cellulose fiber surface. Direct covalent modification of the cellulose hydroxyl groups has been effectively applied to paper sheets and cotton fabrics, but has some limitations with respect to reaction conditions, degree of functionalization, and maintenance of fiber matrix 20-23 In particular, non-aqueous solvents are generstructure. ally incompatible with maintaining nanocellulose hydrogel structures, such as BC. Non-covalent approaches for bioactive paper functionalization include printing techniques 24-31 and entrapment by surface-adsorbed polymers. In this spirit, we recently developed a chemo-enzymatic method to produce general, activated cellulose surfaces, to which a diversity of functional groups may be subsequently appended via the facile copper(I)-catalyzed azide-alkyne cycloaddition 32 (CuAAC), a type of “click chemistry” (Scheme 2). This approach relies on the intrinsically strong binding interaction of the plant cell wall matrix polysaccharide xylogucan (XyG) 33 with cellulose. Notably, binding of native and modified XyGs to diverse types of cellulose occurs spontaneously from aqueous solution, thus facilitating non-disruptive modification of matrices such as paper sheets and nanocellulose hy23,32,34-39 This interaction is essentially irreversible, drogels. 33 except at extremely high pH (e.g. in 1 M NaOH). To expand this versatile technology to produce sensitive cellulose-based biosensors for esterases, we prepared diacetyl, dibutryl, and diheptanoyl derivatives of 5(6)carboxyfluorescein-tetraethyleneglycol (TEG)-azide (FTA), using an scalable, cost-efficient, two-step synthesis starting from the parent fluorophore and the hydrophilic, biocompatible linker amino-TEG-azide (11-Azido-3,6,9-trioxaundecan-1amine), both of which are commercially available. Diacylation blocks the fluorescence of the fluorescein moiety, and this approach has previously been used to produce fluoro40,41 genic esterase substrates. Despite high yields for all three derivatives (see Supporting Information), the diheptanoyl derivative was poorly soluble in water. Thus, only the diacetyl and dibutryl compounds were studied further. To validate the FTA diesters as substrates, we first tested activity in solution the model esterase, porcine liver esterase 40,42 (PLE). The standard esterase substrate p-nitrophenyl acetate (p-NPA) was used as a benchmark. Noting a limited solubility of the FTA diesters under the p-NPA assay conditions (5 mM p-NPA in 20 mM tris buffer, pH 8, including 4 % -1 -1 acetonitrile; PLE specific activity 27 µmol min mg ), we examined the effect of increasing the amount of the organic co-solvent on PLE activity. In the presence of 10% acetonitrile, the specific activity of PLE toward p-NPA was reduced -1 -1 ca. 30 % to 19 µmol min mg PLE (see Supporting Information). Subsequently, initial-rate kinetic analysis in 10% acetonitrile indicated that the solubility limit of both FTA diesters was reached well before enzyme saturation (see

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Supporting Information). FTA diacetate had an apparent solubility limit of ca. 90 µM and a specific activity at this -1 -1 concentration of 0.45 µmol min mg PLE. FTA dibutyrate had an apparent solubility limit of ca. 35 µM and a specific -1 -1 activity at this concentration of 0.16 µmol min mg PLE (see Supporting Information). OH OH O HO OH

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native xyloglucan a&b

OH NH O HO OH

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propargylated xyloglucan c

OH NH O HO OH

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cellulose substrate d

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Scheme 2. Chemo-enzymatic preparation of cellulose-based esterase sensors. a) GalOx/Catalase/HRP, R.T., H2O; b) PPA, NaBH3CN, AcOH, R.T., H2O/MeOH (2:1); c) Cellulose substrate, aqueous; d) diacyl-FTA, CuSO4.5H2O, sodium ascorbate, THPTA, H2O : acetonitrile (7 : 3); e) PLE, tris buffer, pH 8. General structure of xylogucan is depicted with Consortium for Functional Glycomics standard symbol nomenclature: blue circle = β-Glc(1,4), red star = α-Xyl(1,6), yellow circle = β-Gal(1,2).

Turning our attention to enzyme detection on cellulose surfaces, we prepared clickable Whatman no.1 filter paper

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discs as model substrates by using our established method (Scheme 2). Thus, propargylated XyG was adsorbed from aqueous solution to a level of 40 mg/gram cellulose (0.27 32 mg/disc; 1 cm diameter); see ref. and Supporting Information. We previously used a mixed solvent system of 7:3 (v/v) water/t-BuOH to expedite the CuAAC reaction on pa32 per surfaces. Here, we observed that mixed solvents composed of water and t-BuOH or DMSO resulted in an unacceptable level of spontaneous ester hydrolysis. Instead, the use of a 7:3 mixture of water and acetonitrile reduced the rate of FTA diester hydrolysis during CuAAC, and thus background fluorescence of the resulting discs, to negligible levels (Fig. 1). Subtractive analysis (see Supporting Information) indicated that the amount of the FTA-dibutyrate covalently attached to the surface was 200 nmol/disc (1 cm diameter discs, average mass 6.7 mg). Excess, non-conjugated FTA diester was readily removed by washing with gentle shaking.

Fig. 1. Enzyme concentration- and time-dependent hydrolysis of FTA-dibutyrate on paper surfaces. (A) raw image data obtained by fluorescence scanning of Whatman No. 1 filter paper discs on which FTA-dibutyrate was anchored by xyloglucan using CuAAC. Disc 1, blank Whatman No. 1 filter paper loaded with 20mM tris buffer, pH 8; Discs 2-6, Whatman No. 1 filter papers with attached FTAdibutyrate loaded with 20 µL of 0, 1.2, 5.9, 24 and 120 µM PLE in 20 mM tris buffer, pH 8; Disc 7, FTA-dibutyrate disc treated with 0.1 mM NaOH(aq) to fully hydrolyze the diester. Incubation times at room temperature (20 °C) are indicated. (B) Relative quantitation of ester hydrolysis by integration total of fluorescence intensity of the entire disc surface. Lines are B-spline curves intended as ocular guides only.

Quantitative imaging using a highly sensitive fluorescence scanner was subsequently used to demonstrate the effectiveness of the resulting paper-based esterase sensors. We ob-

served an unacceptably high spontaneous hydrolysis rate for FTA diacetate-modified paper discs. The generally poor hydrolytic stability fluorescein diacetate esters in solution is 40 well-known, and attachment to the paper surface did not significantly mitigate this instability. Thus, we turned our attention to the analysis of FTA dibutryate-modified discs (Fig. 1). A blank sample (unmodified Whatman No. 1, Disc 1), and a negative control containing FTA dibutyrate to which no PLE was added (Disc 2), indicated that negligible buffercatalyzed background hydrolysis occurred over time. A disc (Disc 7) that had been treated with 0.1 mM NaOH(aq) served as a reference for complete ester hydrolysis. Discs 3-6 (Fig. 1) were loaded with increasing concentrations of PLE, and image analysis clearly revealed an enzyme concentrationdependent and time-dependent response, which reached saturation for all samples. In consideration of real-world applications in resource limited situations, we demonstrated the ability to detect cellulose surface-bound, free FTA using either a benchtop illuminator or an LED flashlight of the type used routinely in forensic analyses (Fig. 2). The intrinsic excitation and emission maxima, and bright fluorescence, of the fluorescein scaffold make it particularly suited for epi-illumination and observation using readily available, low-cost light sources and filters. Thus, we were able to confidently visualize the hydrolysis of as low as 2.5 percent of the total FTA dibutyrate, equivalent to 1.75 nmol FTA, on a single disc using a low-cost laboratory electrophoresis gel illuminator (Figs. 2A & 2B). Using the specific activity value of PLE for FTA dibutyrate in solution -1 -1 (0.16 µmol min mg PLE), a limit-of-detection of 0.36 µg (4.3 pmol) PLE in a 30 min incubation can be estimated, with the additional caveat that diacylfluorescein hydrolysis kinet42 ics are non-linear (Fig 1B, Disc 2; ref. ). Alternatively, this fluorescence could be readily visualized using an LED flashlight and orange-lensed glasses in a darkened room (Fig. 2C).

Fig. 2. Visual detection of esterase activity on bioactive cellulose surfaces. (A) Observation of a gradient of free FTA on Whatman No.1 filter paper surfaces using a laboratory epi-illuminator with orange filter. The amount of FTA on the Discs from left to right: 3.5, 8.7, 17, 52, 87, 130, 170, 260, 350 nmol. (B) Close-up view of the discs observed in panel A. (C) Visualization of fluorescence using a UV LED flashlight and orange-lensed glasses (n.b. the room is fully illuminated to allow photography of the surrounding environment; actual visualization would be performed in a darkened room). (D)

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Visualization of esterase activity on cotton gauze wound dressing containing clicked FTA dibutyrate. (E) Visualization of esterase activity on bacterial cellulose pellicles. Panels D and E were obtained using the epi-illuminator shown in panel; scale bars = 1 cm. A. PLE was added to the right gauze or BC pellicle, respectively, only; the background fluorescence of the left gauze and BC pellicle was similar to the unmodified substrates (not shown).

To demonstrate the broad applicability of this approach to cellulose bio-activation beyond traditional paper supports, we prepared esterase sensors from cotton gauze purchased at a local pharmacy, and BC produced by culturing Acetobacter xylinum. Thus FTA-dibutyrate was clicked onto both substrates, which had been previously activated by adsorption of multi-propargylated XyG in water. As shown in Fig. 2D, this gentle method maintains both the loose-weave structure of the gauze. Similarly, the hydrogel structure of the BC is completely maintained, which is otherwise not be possible 37 using traditional, organic solvent-based methods. For both substrates, the activity of added PLE was readily revealed by epi-illumination and observation through an orange filter (Figs 2D & 2E). In conclusion, we have extended our versatile chemoenzymatic method of cellulose modification to the development of a general fluorogenic esterase biosensor. The use of multivalent propargylated XyG as a molecular anchor readily enables the method to be extended to a diverse range of cellulosic substrates, including paper, gauze, and nanocellulose hydrogels. Here, initial surface activation in water, followed by CuAAC in water-acetonitrile, maintains the unique matrix structures of these materials, which is of particular importance to their application in specific diagnostic or biomedical settings. Although in the present proof-of-concept, we opted to use direct fluorescein esters for synthetic simplicity, further reduction in background hydrolysis can be achieved using more sophisticated fluorophore caging strate40 gies. A further advantage over previous approaches to develop cellulose-based, hydrolase enzyme sensors is that the fluorophore remains covalently attached to the substrate, preventing diffusion and potential deleterious effects in vivo. The particular versatility of the click-chemistry conjugation and the potential to employ other fluorophore scaffolds implies that this technology may be readily extended to produce a range of enzyme-specific, cellulose-based biosensors for POC applications.

ASSOCIATED CONTENT Supporting Information

This material is available free of charge via the Internet at http://pubs.acs.org Experimental procedures; NMRs; Kinetic measurements; Standard curves; (PDF)

AUTHOR INFORMATION Corresponding Author

*Email: [email protected]. Phone: +1 604 827 3738

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENTS This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) Innovative

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Green Wood Fibre Products Network, an NSERC Discovery Grant, the Canada Foundation for Innovation, the British Columbia Knowledge Development Fund, and UBC faculty funding. We thank Dr. Guillaume Dejean for assistance with culturing Acetobacter xylinum. We thank Dr. Shaheen Shojania for helpful discussions and critical reading of the manuscript.

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