Article pubs.acs.org/ac
Electrochemical Assay for a Total Cellulase Activity with Improved Sensitivity Deby Fapyane and Elena E. Ferapontova* Interdisciplinary Nanoscience Center, Faculty of Science and Technology, Aarhus University, Gustav Wieds Vej 14, 8000 Aarhus C, Denmark S Supporting Information *
ABSTRACT: Electrochemical methods allow fast and inexpensive analysis of enzymatic activities. Here, we report a simple and yet efficient electrochemical assay for the total activity of cellulase, a hydrolytic enzyme widely used in food and textiles industries, and for production of bioethanol. The assay exploits the difference in electrochemical signals from a soluble redox indicator, ferricyanide, on nitrocellulose films treated by cellulases. Ferricyanide electrochemistry is totally inhibited on graphite electrodes modified with an insulating nitrocellulose film and is evoked after the cellulase treatment. Ferricyanide voltammetric responses correlate with the increased permeability of the films and electrochemically active surface area of electrodes becoming accessible to the ferricyanide reaction after nitrocellulose digestion by cellulase. Trichoderma and Aspergillus niger cellulases activities were determined in a 5 min assay with a sensitivity of 10−8 U per assay, being 103−104-fold more sensitive than the standard commercially available optical assays. That makes the developed electrochemical approach the most prospective cost-effective alternative both for research and automated industrial applications. The most widely used cellulase assay is a filter paper assay (FPA) for measurement of a total cellulase activity. The FPA method is quite inexpensive, highly reproducible, and simple, being performed by casting an enzyme sample on a cellulose filter paper.7 The enzyme cleaves the paper and releases glucose, which is then analyzed by the dinitrosalicylic acid (DNS) reducing sugar assay.8 However, the FPA needs a minimum of 2 mg of sugar released from the 50 mg sample, making this method not suitable for cellulase exhibiting low glucosidase activity. Moreover, the FPA method has a relatively low sensitivity and requires high concentrations of the enzyme (from 1 to 102 U mL−1), which can be lessened by 60% by decreasing the reactor volume.9 Alternatively, McCleary et al.10 have proposed a more specific cellulase activity assay for the endoglucanase activity using benzylidene end-blocked 2-chloro-4-nitrophenyl-ß-cellotrioside as a substrate. Due to its blocked terminal part, preventing hydrolysis by other types of cellulases, this substrate is subjected only to endoglucanase digestion. The hydrolyzed substrate releases 2-chloro-4-nitrophenyl-ß-glycoside, which is further cleaved by supplementary ß-glucosidase to produce Dglucose and 2-chloro-4-nitrophenol, and the latter is quantified spectrophotometrically, with a sensitivity of 10−3 U per assay. Mangan et al.11 suggested an alternative synthetic substrate for endoglucanase hydrolysis, 4-methylumbelliferyl-ß-cellotrioside,
D
ue to the high specificity of catalysis and environmentally friendly conditions of functioning, enzymes are widely used in a variety of biotechnological processes, from drug discovery and development to food and material engineering, providing enormous industrial opportunities for implementation of economically sensible biocatalytic conversion schemes and large-scale productions.1 One of the highly demanded technological enzymes is cellulase, a hydrolytic enzyme mainly used for biomass conversion into biofuel, specifically, for the production of cellulosic ethanol.2 It also finds its applications in pharmaceutical and food industries, in brewery for removal of colloidal particles of glucan,3 and in the textiles industry for color brightening, cotton softening, and removing the particulates from the textile pores.4,5 Cellulase hydrolyzes cellulose in a complex several-step reaction, by hydrolytic cleavage of ß-1−4 glycosidic bonds of the polymer (Figure 1). Depending on the site of cleavage, cellulase is classified as endoglucanases (E.C 3.2.1.4), exoglucanases or cellobiohydrolases (E.C 3.2.1.91), and ßglucosidases (E.C 3.2.1.21).6 Endoglucanases attack random sites in the cellulose matrix, by this producing oligosaccharides, while exoglucanases can cleave only edge-located glycosidic bonds (Figure 1). Glucosidases further “cut” oligosaccharides into monosaccharides such as glucose. In different species, cellulases are represented either by a single enzyme type (e.g., endoglucanase in Aspergillus niger) or by a complex of enzymes operating synergistically (e.g., in Trichoderma sp.), and precise assaying of the cellulase activity is an important step in any cellulase application process. © 2017 American Chemical Society
Received: November 8, 2016 Accepted: February 28, 2017 Published: February 28, 2017 3959
DOI: 10.1021/acs.analchem.6b04391 Anal. Chem. 2017, 89, 3959−3965
Article
Analytical Chemistry
Figure 1. (A) Cellulase classification based on the location of the ß-1−4 glycosidic bond it cleaves: endoglucanase, exoglucanase, and ß-glucosidase. (B) Schematic representation of the electrochemical assay for the cellulase activity. A nitrocellulose insulating film is formed on the electrode surface, and ferricyanide electrochemistry is inhibited on such surface. Cellulase cleaves the glycosidic bonds in the film, and the electrode surface becomes exposed to the electrochemical reaction.
nase from Trichoderma sp. (3 U mL−1) and the CELLG5 endoglucanase activity assay kit were purchased from Megazyme (Wicklow, Ireland). Electrode Modification. The surface of graphite (Gr) electrodes (3.05 mm in diameter with geometrical surface area 0.071 cm2, Werk Ringsdorff, Germany type RW001) was polished stepwise with a silicon carbide emery paper (SIC paper no. 1000 HV 30-800, Struers A/S, Ballerup, Denmark) and on the A4 paper (Papyrus AB, Mölndal, Sweden) for 2 min each, to yield a flat shiny surface. A 10 μL volume of the nitrocellulose solution (0.06−2%) was cast on the electrode surface and dried under the argon flow (a 15 min step). Electrochemical Characterization. Cyclic voltammetry (CV) was performed in a standard three-electrode electrochemical cell connected to a μAutolab potentiostat (Type III, Eco Chemie B.V., Utrecht, Netherlands) supported with GPES (version 4.9) and NOVA (type 1.8.17) software. A Ag/AgCl (KClsat) electrode and a Pt wire were the reference and auxiliary electrodes, correspondingly. All electrochemical measurements were carried at 20 ± 1 °C. The working solution was 0.1 M PBS, pH 5, containing 1 mM K3(FeCN)6. Faradaic and capacitive charges were evaluated from the CV data: the faradaic charges by integrating the ferricyanide reduction peak area, and the capacitive charges by integrating the current− potential dependence in the region where no faradic reaction occurred (a so-called capacitive area), within the −0.1 to +0.1 V potential window. Electrode Treatment with Cellulase. An amount of 10 μL of a 1 mg mL−1 cellulase solution in 0.1 M PBS, pH 5 (A. niger cellulase), pH 6 (T. reesei and Trichoderma sp. cellulases), and pH 7 (T. longibrachiatum cellulase) or 1 U mL−1 of endoglucanase from Trichoderma sp. dissolved in 0.1 M acetate buffer, pH 4.5, was placed onto the nitrocellulose-modified electrodes and left to react from 5 to 60 min. After that, the electrodes were rinsed with water, dried in the Ar flow, and studied electrochemically. Reproducibility of the results was verified with at least six equivalently prepared electrodes per assay.
specifically hydrolyzed further by glucosidases with a formation of chromogenic 4-methylumbelliferyl that is detected fluorometrically with a sensitivity of 10−4 U per assay. Both methods for endoglucanase activity are simple, robust, and automatable. However, they are relying on native or supplementary glucosidase activity, while the natural cellulase complexes tend to have low or no glucosidase activity at all.12 Thus, any new assay accounting for all possible types of cellulases is highly requested to efficiently monitor the total activity of cellulase. Electrochemical methods are routinely used for assaying of the enzymatic activity of redox active enzymes, and they often offer simpler and still efficient analytical protocols for analysis of oxidoreductases,13−15 whose bioelectrocatalytic activity can be directly correlated with their homogeneous activity in solution. With redox-inactive enzymes, electrochemistry was also shown to be a powerful analytical tool for assaying activities of lipases16 and proteases17 in assays performed either with redox-labeled substrates or via enzymatic cascades. Here, we report a simple and inexpensive electrochemical method for fast, sensitive, and reliable screening of the total cellulase activity that exploits the electrical insulating properties of nitrocellulose filmsa typical cellulase substrate. The insulating film formed by nitrocellulose on the electrode surface prevents any electrochemical reaction of a soluble redox indicator at the nitrocellulose-modified electrode. After the nitrocellulose film is digested by cellulase, the electrode surface becomes more accessible for the electrode reactions. That allows assessment of the enzymatic activity via redox indicator reactions on the cellulase-treated surfaces (Figure 1).
■
EXPERIMENTAL SECTION Materials. Nitrocellulose (4% solution in ethanol/diethyl ether supplied as a collodion solution, >98% purity), cellulase from A. niger (1 U mg−1), Trichoderma reesei (6 U mg−1), Trichoderma longibrachiatum (≥1 U mg−1), and Trichoderma sp. (≥5000 U mg−1), ethanol, K3(FeCN)6, NaN3, and all components of buffer solutions were purchased from SigmaAldrich (Broendby, Denmark). Milli-Q water was used all over the work (18Ω, Millipore, Bedford, MA, U.S.A.). Endogluca3960
DOI: 10.1021/acs.analchem.6b04391 Anal. Chem. 2017, 89, 3959−3965
Article
Analytical Chemistry
Graphite Surface Pretreatment. Reproducibility of the nitrocellulose film production was found to directly correlate with the surface roughness and, thus, with the way the electrodes were polished. In contrast to previous publications, in which the emery paper polished, high-surface area Gr surfaces were shown to be advantageous for protein and peptide adsorption and electrochemistry,18−21 in the current work, the micrometer-scale roughness of the Gr surface detrimentally affected the reproducibility of the nitrocellulose film formation and its insulating properties. No uniform films could be produced on highly rough Gr surfaces, and the electrode surface was finally polished with an A4 paper to the mirrorlike luster state. Those A4-polished surfaces were flat enough to be uniformly insulated even with quite thin polymer films (Figure S1A), which allowed us a further development of the assay. Optimization of Nitrocellulose Concentration. The effect of the thickness of the nitrocellulose films on the on the electrical insulation and response of the polymer electrodes to the cellulase action (namely, to cellulase digestion) was studied with polymer films produced with 0.06−2% nitrocellulose solutions (Figure S1B, Supporting Information). The polymer film thickness varied between 6 and 30 μm (for 0.25−1% nitrocellulose films on basal plane HOPG, Figure S3, Supporting Information, while for concentrations less than 0.25% no visible layer could be detected by SEM). Among those nitrocellulose films, the most reproducibly produced ones where prepared using 0.5% nitrocellulose solutions (15 ± 3 μm insulating films). They showed the best balance between electrical insulating properties and sensitivity to the cellulase action (Figure 2). Those films were highly susceptible to
Scanning Electron Microscopy (SEM) Imaging. An amount of 10 μL of the nitrocellulose solution was drop-cast either on the polished Gr electrode or on basal plane highly oriented pyrolytic graphite (HOPG, for cross-sectional view) surface and dried under the Ar flow. Images of the nitrocellulose films before and after exposure to cellulases were taken with a Nova NanoSEM (FEI, Oregon, U.S.A.) equipped with a true lens detector TLD. Determination of the Cellulase Activity by the CELLG5 Assay (Endoglucanase-Specific Assay). The assay was performed according to the manufacturer’s protocol. Briefly, the CELLG5 reagent was prepared by mixing 100 μL of thermostable ß-glucosidase (600 U mL−1) with 3 mL of a 4,6O-(3-ketobutylidene)-4-ß-D-cellopentaoside solution in 10% DMSO/H2O and 0.02% w/v NaN3. The assay was performed at rt in a 3 mL cuvette by mixing 50 μL of the CELLG5 reagent and 50 μL of 0.1−1 mg mL−1 enzyme solution; the reaction was allowed to proceed for 5−15 min and stopped by adding 1.5 mL of a stop buffer, Tris buffer (2% w/v), pH 9.0. Enzyme solutions were prepared in buffer solutions corresponding to the pH optimum of used cellulases (A. niger at pH 5, T. longibrachiatum at pH 6, T. reesei at pH 7, and Trichoderma sp. at pH 6, Supporting Information, Figure S2) and according to the manufacturer’s protocol (endoglucanase from Trichoderma sp. at pH 4.5). The endoglucanase activity was determined by following the 4-nitrophenol absorbance at 400 nm in Tris buffer solution, pH 9 (the absorbance coefficient ε400 of 18.1 mM cm−1) with a UV−vis spectrophotometer (Agilent Technologies Cary 60 UV−vis). The unit activity of a endoglucanase per milligram of protein was determined according to eq 1: U (mg −1) =
A400 − Ablank vol total 1 1 t volenzyme ε400 w
(1)
where A400 is 4-nitrophenol absorption at 400 nm, Ablank is absorption of the assay mixture at 400 nm without enzyme added, t is the incubation time in min, voltotal is the total volume of the reacting mixture (1.6 mL), volenzyme is the volume of the enzyme aliquot added (0.05 mL), and w is the amount of the protein added per milliliter (in milligrams). The endoglucanase activity of cellulases was 0.07, 0.17, 1.2, and 1.3 U mg−1 for cellulases from A. niger, Trichoderma sp., T. reesei, and T. longibrachiatum, respectively.
■
RESULTS AND DISCUSSION The cellulase activity assay is based on the cellulase digestion of nitrocellulose films formed on the Gr electrode surface. Those films should be thick enough to be insulating and prevent any redox reaction on the modified electrode surface not treated by cellulases and thin enough to be digested, in a reasonable time, by cellulases to the extent that enables electrochemical detection and calibration of the redox signal from the redox indicator such as ferricyanide (Figure 1). Such assay to a large extent relies on the properties of nitrocellulose films formed on the Gr electrode surface, both on their insulating properties and their “digestibility” by cellulases that affects their electrical conductivity. Main efforts were thus focused on reproducible preparation of nitrocellulose film electrodes susceptible to the cellulase action. For that, different thicknesses of films (produced by applying different amounts of nitrocellulose to the Gr electrode surface) and different times of exposure of those films to the cellulase were investigated.
Figure 2. Dependencies of the ferricyanide CV peak area (black dots) before and (white dots) after 5 min of exposure to 1 mg mL−1 cellulase from A. niger on the concentration of the nitrocellulose solution (% v/ v) used for film preparation. Inset: representative CVs of the 0.5% nitrocellulose-modified Gr electrode in 1 mM K3(FeCN)6/0.1 M PBS, pH 5, scan rate 0.1 V s−1, before (solid line) and after (dotted line) 5 min of treatment with 1 mg mL−1 A. niger cellulase.
digestion by cellulases, which exhibited both via the higher intensities of the ferricyanide faradic signals and the changes in the capacitive currents after their exposure to cellulases (Figure 2, inset). Most important, before cellulase treatment, no faradaic signal from ferricyanide was observed, i.e., those films exhibited perfect insulting properties, and no background correction of the detected signals for the faradic reaction was necessary. For nitrocellulose concentrations below 0.5%, the formed films were nonuniform and insufficiently insulating, which allowed electrochemistry of ferricyanide be observed 3961
DOI: 10.1021/acs.analchem.6b04391 Anal. Chem. 2017, 89, 3959−3965
Article
Analytical Chemistry
Figure 3. Representative SEM images of 0.5% nitrocellulose films formed on the Gr surface before and after treatment with 1 mg mL−1 solution of A. niger cellulase. Cellulase treatment times are denoted in figures. The grainlike structures is marked by red circles. The SEM image scale is 10 μm. Insets: representative CVs recorded with the same electrodes in 1 mM K3(FeCN)6/0.1 M PBS, pH 5. Potential scan rate 0.1 V s−1.
Figure 4. (A) Optical assay: the dependence of absorbance at 400 nm on the cellulase−substrate reaction time. (B and C) Electrochemical assay: the dependence of (B) faradaic and (C) capacitive charges on the time of the film exposure to the cellulase action: (black dots) endoglucanase from Trichoderma sp. and cellulases from (red dots) A. niger, (green dots) Trichoderma sp., (yellow dots) T. reesei, and (blue dots) T. longibrachiatum. For each data point the results from six electrodes were used; standard deviation ranging between 5% and 15%.
after 45 min and which is most pronounced after 60 min of exposure to cellulase (Figure 3, SEM images after 45 and 60 min of cellulase exposure, marked by red circles). The formation and development of “hole” structures in the polymer film was electrochemically supported both by the enhanced redox activity of ferricyanide on more enzymatically digested films and increased capacitive currents, stemming from the transformation of a compact, water-insoluble nitrocellulose film into the rougher, more hydrophilic one, with water molecules entrapped in and some parts of Gr the surface starting to be exposed to the solution (Figure 3, insets). (N.B.: exposure of the polymer film electrodes to the blank buffer solution had no effect on the film morphology and its insulating properties.) Interestingly, the ferricyanide CV on cellulasetreated polymer films showed the potential peak separation close to the theoretical value of 57 mV (at 298 K and the number of electrons involved n = 1),24 60 mV after 5 min of treatment with cellulase. Upon further digestion of the film by cellulase, the intensity of the ferricyanide redox CV signals increased (Figure 3), and that increase could be directly correlated with the enzymatic activity of cellulase used. Electrochemical Analysis of the Cellulase Activity. The extent of the nitrocellulose film digestion by cellulase as a
before any cellulase treatment, while with higher nitrocellulose concentrations the produced films were too thick to be timely digested by cellulases (Figure 2). In all further experiments, polymer films formed with 0.5% nitrocellulose solutions (referred to as “0.5% films”) were interrogated. Surface and Electrochemical Characterization of Nitrocellulose Films. SEM studies of morphological transformations of nitrocellulose films on Gr upon the cellulase treatment demonstrated that, with the increasing time of exposure to cellulase action, fractures and cavities (or “holes”) appeared and enlarged on the polymer film surface (Figure 3 and Figure S3B, Supporting Information with a tilt angle of 40°). As can be seen, the most pronounced fractures can be observed already after 15 min of the cellulase action. Earlier SEM studies of the cellulose degradation by cellulase22 suggest that cellulose exists in a twisted, ribbonlike structure, and cellulase attaches to its surface preferably at kink sites and then cuts those ribbons into shorter microfibrils, followed by their thinning into rod or even grainlike particles, as also evidenced by atomic force microscopy (AFM) studies23 and our SEM results. The SEM images show the structural degradation of the nitrocellulose films and their partial transformation into grainlike particle structures, which formation can be followed 3962
DOI: 10.1021/acs.analchem.6b04391 Anal. Chem. 2017, 89, 3959−3965
Article
Analytical Chemistry Table 1. Rate of Nitrocellulose Digestion by Cellulase Evaluated by Optical and Electrochemical Assays dQ faradaic/dt μC/min cellulase endoglucanase Trichoderma sp. A. niger Trichoderma sp. T. longibrachiatum T. reesei
Vmax, Abs/t 0.124 0.08 0.03 0.007 0.007
± ± ± ± ±
0.013 0.001 0.0011 0.001 0.001
V(t0−5 min) 0.27 0.03 0.023 0.015 0.015
± ± ± ± ±
dQ capacitive/dt μC/min
Vmax
0.3 0.004 0.002 0.002 0.002
0.75 0.56 0.22 0.05 0.05
± ± ± ± ±
0.001 0.001 0.000 0.000 0.000
V(t0−5 min) 2.25 1.40 0.55 0.18 0.19
± ± ± ± ±
Vmax
0.60 0.10 0.10 0.001 0.003
3.53 2.38 1.03 0.248 0.30
± ± ± ± ±
0.30 0.14 0.08 0.03 0.02
Figure 5. Optical assay: (A) dependence of the absorbance at 400 nm on the logarithmic activity units for cellulases from different sources (endoglucanase from Trichoderma, cellulase from A. niger and Trichoderma sp.). Electrochemical assay: dependence of the normalized Qfaradaic (B) and Qcapacitive (C) extracted from CVs recorded with nitrocellulose film electrodes after 5 min of cellulase treatment on the logarithmic activity units of cellulase (endoglucanase from Trichoderma, cellulase from A. niger, and Trichoderma sp.). The pH optimum of individual cellulases at which the activity was assayed: pH 4.5 (Trichoderma endoglucanase), pH 5 (A. niger), and pH 6 (Trichoderma sp.). Inset: the dependence of the Q on the logarithmic concentration of cellulase fitted the linear regression (R2 = 0.97 and 0.99 for Qfaradaic and Qcapacitive, respectively).
function of time was detected via the changes of both the capacitive and faradaic currents of the redox indicator transformation on the modified polymer film surface. In general, the increase of the diffusion-limited faradaic peak currents, ip, correlates with the increase in the electroactive surface area of the electrode, A, once other parameters are kept constant (the Randless−Sevcik’s equation).24 Thus, the ip values immediately reflect the extent of the digestion of the polymer film by cellulase. However, the total charge, Q, spent in the electrochemical reaction of ferricyanide (Qfaradaic) allows a more precise and sensitive analysis of the process, since it takes into account the overall peak area (current integrated over time). In the assay development we further used the Q values plotted versus either the time or amount of the assayed enzyme. The changes of the Qfaradaic were also correlated with the changes of the capacitive charge, Qcapacitive, stemming from the changes in the electric double layer (EDL) capacitance due to the structural changes of the nitrocellulose films exposed to cellulases. Since cellulases from different sources have different pH optimums (varying between pH 4.5 and 7 for assay in solution),25−27 their on-surface activity was assessed at their specific pH optimums (Figure S2, Supporting Information) and then electrochemically interrogated. Cellulase activity was assayed both electrochemically and by the standard optical assay (Figure 4). In the optical assay, two regions can be distinguished in the absorbance−time plots (Figure 4A). It is the region the enzyme actively converts the substrate to the product, reflected by the steeply increased slope of the curve, where it transforms to the plateau once the total saturation of the enzyme binding sites with the substrate is achieved, and there is no further increase in the product formation. In the electrochemical assay, an additional ca. 10 min lag period region, preceding the steep-slope region, was observed (Figure 4, parts B and C), where the Q−time dependences showed a less steep slope. It is assumed that the lag period
reflects some rate-limiting step of cellulose depolymerization of the nitrocellulose solid substrate.28,29 Depolymerization of cellulose mainly depends on the endoglucanase activity of cellulase as the enzyme attacks random sites and edges on the accessible polymer surface, cleaving preferably the ß-glycosidic bonds and releasing shorter (up to six monomers) polymer chains.29 Nevertheless, despite different modes of the cellulase−substrate interactions exhibited in the optical and electrochemical assays, both methods show the same tendency in cellulase activity variations for different species. Endoglucanase from Trichoderma sp. evidently exhibits the highest activity among all studied cellulases (A. niger, Trichoderma sp., T. reesei, and T. longibrachiatum), and for other cellulases the relative change in their activity determined by two different methods follows the same pattern (Figure 4). By scrutinizing the electrochemical assay data, it becomes clear that the Q−time plots may also assist in discriminating the activity of different type cellulases (Figure 4, parts B and C). More active endoglucanases from Trichoderma sp. and A. niger share a very similar curvature, reaching the plateau region within 60 min. It also can be seen that cellulase from Trichoderma sp. has comparable activity with A. niger’s cellulase in the optical assay, but much less in electrochemical assay, suggesting that the overall Trichoderma sp.’s endoglucanase activity is lower, and the nitrocellulose film depolymerization proceeds slower, with electrochemical signals still increasing after the 60 min of reaction time. Other studied T. reesei and T. longibrachiatum’s cellulases evidently have even lower activity detected both in optical and electrochemical assays, consistent with previous studies.10 The rate of the enzymatic transformation, V, was calculated from the derivatives of the absorbance versus time and the Q versus time dependencies, reflecting the rate of the product generation/nitrocellulose film digestion by cellulases (Table 1 and Figure S4, Supporting Information). In electrochemical 3963
DOI: 10.1021/acs.analchem.6b04391 Anal. Chem. 2017, 89, 3959−3965
Article
Analytical Chemistry assay, the rate of the nitrocellulose film destruction by the enzyme was the actual measure of the enzyme activity. In the optical assay, when the small-molecular substrate (4,6O-(3-ketobutylidene)-4-ß-D-cellopentaoside) is in solution and easily accessible for cellulase cleavage, the reaction proceeds rapidly and the maximum reaction rate, Vmax, is achieved in 2.5 min, then the reaction rate drops down. In the electrochemical assay with a solid polymer substrate, the reaction proceeds in a different way, with an essential lag period as discussed earlier, and the apparent V constantly increases and reaches its maximum after 10 min for all type of cellulases, evaluated both from the Qfaradaic and Qcapacitive time dependencies (Figure S4). Interestingly, all parameters reflecting to the cellulase activity toward the solid nitrocellulose substrate and calculated from the Qcapacitive appeared to be approximately 4−5-fold higher than that evaluated from the Qfaradaic (Figure 4, parts B and C), which can be also followed from the V and Vmax values (Table 1). Thus, it is clear that the Qcapacitive changes represent more sensitive tools for monitoring the total cellulase activity, since they enable detecting the cellulase-induced structural changes in the nitrocellulose films that precede the electrochemical reaction itself and anyway produce changes the EDL capacitance. Along with that, it seems that the faradaic activity can be followed only after the “holes” are formed in the polymer film and the Gr surface becomes accessible for the electrochemical reaction. Limit of Detection (LOD) and the Sensitivity of Analysis. In the optical assay, no distinct changes of absorbance were observed until the cellulase concentration reached 10−3 U mL−1 (5 × 10−5 U per assay, Figure 5A). In the electrochemical assay, the increase of the Qfaradaic started from the 10−6 U mL−1 cellulase concentration (Figure 5B), while a noticeable increase in the Qcapacitive values began even at lower cellulase concentrations, 10−7 U mL−1 (Figure 5C). No noticeable changes of the Q were observed at lower cellulase concentrations for all type of cellulases (Figure S5C). Though the prolonged cellulase treatment of the films improves the signals from ferricyanide (Figure 4), the 5 min assay time (Figure 3) was chosen for further assessment of the assay performance, in order to maximally shorten the assay time. The sensitivity of analysis was examined from the slope of the linear part of the calibration curves.30 (Figure 5, insets, data for Trichoderma sp.). In the electrochemical assay, the sensitivity was 42.3 ± 8.5 and 28.2 ± 1.4 μC cm−2 per log(U mL−1) for the assay relying on the Qcapacitive and the Qfaradaic, respectively. Analysis of the relative signal changes in the optical (Abs/ Absmax) and electrochemical (Q/Qmax,) assays shows that the electrochemical signal changes almost 80% (the optical signal changes are less than 10%) within the 10−7−10−3 U mL−1 cellulase concentration range, demonstrating an essentially higher sensitivity of cellulase detection (Figure S6). The optical and electrochemical methods can be linearly correlated only within a narrow 0.1−1 U mL−1 cellulase concentration range (Figure S6, parts A and B, inset), due to the significant difference in the detection limits of those two assays. Thus, the electrochemical assay exploiting both the Qfaradaic and the Qcapacitive changes produced by the cellulase action on the nitrocellulose film electrodes provides much higher sensitivity and lower detection limit of the cellulase activity analysis. The lowest concentration of cellulase detected is equivalent to the 10−6 U mL−1 of cellulase activity (10−8 U per assay) and 10−7 U mL−1 cellulase activity (10−9 U per assay), evaluated from the Qfaradaic and the Q capacitive signals,
respectively. The working concentration range in the electrochemical assay was also improved compared to the optical assay, depending on the type of cellulase used, ranging between 10−7 and 1 U mL−1, by this eliminating the necessity of cellulase preconcentration and/or concentration adjustment requested, e.g., in the FPA method.12
■
PERSPECTIVES AND OUTLOOK In conclusion, we have demonstrated that the electrochemical assay allows fast and robust analysis of the total enzymatic activity of different types of cellulases in a cost-effective and simple manner (the estimated price of the electrochemical assay performed, e.g., on the carbon paper electrode, is $1.2 per assay, including the standard endoglucanase from Trichoderma, while for the optical assay with DNS it is at least $4.5, both based on the Sigma-Aldrich catalog prices). The whole assay takes ca. 30 min, including the electrode preparation and modification steps and the assay itself. The rate of the nitrocellulose films decomposition by cellulases can be derived both from the faradic and capacitive signals, with the capacitive signal enabling an order of magnitude lower detection limit compared to the faradaic one, 10−7 versus 10−6 U mL−1 of cellulase concentrations. This method can be applied for a wide range of cellulase enzymes and enzyme concentrations, and fast screening of total cellulase activity from different sources can be done in relatively short assay time (5 min), without any glucosidase addition as required by the standard method or any currently available commercial kit. Furthermore, this method can be developed further to be applied for automated and multiplex assays by incorporating carbon-based printed electrodes within the microarray format.
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b04391. Additional SEM images of Gr and nitrocellulosemodified HOPG, additional CV data characterizing differently modified electrodes and their digestion by different cellulases at different pH, and additional data on kinetic analysis data of cellulase activity in solution and at electrodes (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Elena E. Ferapontova: 0000-0003-1177-3204 Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS This work is supported by the NUMEN project funded by the Danish Council for Independent Research (DFF-FTP-400500482B).
■
REFERENCES
(1) Kirk, O.; Borchert, T. V.; Fuglsang, C. C. Curr. Opin. Biotechnol. 2002, 13, 345−351. (2) Rubin, E. M. Nature 2008, 454, 841−845. (3) Campbell, G.; Bedford, M. Can. Vet. J. 1992, 72, 449−466.
3964
DOI: 10.1021/acs.analchem.6b04391 Anal. Chem. 2017, 89, 3959−3965
Article
Analytical Chemistry (4) Niehaus, F.; Bertoldo, C.; Kähler, M.; Antranikian, G. Appl. Microbiol. Biotechnol. 1999, 51, 711−729. (5) Cherry, J. R.; Fidantsef, A. L. Curr. Opin. Biotechnol. 2003, 14, 438−443. (6) Knowles, J.; Lehtovaara, P.; Teeri, T. Trends Biotechnol. 1987, 5, 255−261. (7) Wood, T. M.; Bhat, K. M. Methods Enzymol. 1988, 160, 87. (8) Miller, G. L. Anal. Chem. 1959, 31, 426−428. (9) Xiao, Z.; Storms, R.; Tsang, A. Biotechnol. Bioeng. 2004, 88, 832− 837. (10) McCleary, B. V.; Mangan, D.; Daly, R.; Fort, S.; Ivory, R.; McCormack, N. Carbohydr. Res. 2014, 385, 9−17. (11) Mangan, D.; McCleary, B. V.; Liadova, A.; Ivory, R.; McCormack, N. Carbohydr. Res. 2014, 395, 47−51. (12) Coward-Kelly, G.; Aiello-Mazzari, C.; Kim, S.; Granda, C.; Holtzapple, M. Biotechnol. Bioeng. 2003, 82, 745−749. (13) Abdellaoui, S.; Bekhouche, M.; Noiriel, A.; Henkens, R.; Bonaventura, C.; Blum, L. J.; Doumèche, B. Chem. Commun. 2013, 49, 5781−5783. (14) Kartashov, A. V.; Serafini, G.; Dong, M.; Shipovskov, S.; Gazaryan, I.; Besenbacher, F.; Ferapontova, E. E. Phys. Chem. Chem. Phys. 2010, 12, 10098−10107. (15) Ferapontova, E. E.; Ruzgas, T.; Gorton, L. Anal. Chem. 2003, 75, 4841−4850. (16) Valincius, G.; Ignatjev, I.; Niaura, G.; Kazemekaite, M.; Talaikyte, Z.; Razumas, V.; Svendsen, A. Anal. Chem. 2005, 77, 2632−2636. (17) Ionescu, R. E.; Cosnier, S.; Marks, R. S. Anal. Chem. 2006, 78, 6327−6331. (18) Lopes, P.; Ferapontova, E. E. Monatsh. Chem. 2015, 146, 781− 786. (19) Shipovskov, S.; Bonamore, A.; Boffi, A.; Ferapontova, E. Chem. Commun. 2015, 51, 16096−16098. (20) Fapyane, D.; Poulsen, C. H.; Ferapontova, E. E. Electrochem. Commun. 2016, 65, 1−4. (21) Castaing, V.; Á lvarez-Martos, I.; Ferapontova, E. E. Electrochim. Acta 2016, 197, 263−272. (22) White, A. R.; Brown, R. M. Proc. Natl. Acad. Sci. U. S. A. 1981, 78, 1047−1051. (23) Liu, H.; Fu, S.; Zhu, J.; Li, H.; Zhan, H. Enzyme Microb. Technol. 2009, 45, 274−281. (24) Bard, A. J.; Faulkner, L. R. Electrochemical Methods: Fundamentals and Applications; Wiley: New York, 1980; Vol. 2. (25) Okada, G. Agric. Biol. Chem. 1985, 49, 1257−1265. (26) Akiba, S.; Kimura, Y.; Yamamoto, K.; Kumagai, H. J. Ferment. Bioeng. 1995, 79, 125−130. (27) Whitaker, D. Advances in Enzymatic Hydrolysis of Cellulose and Related Materials; Reese, E. T., Ed.; Pergamon Press: New York, 1963; pp 51−70. (28) Zhang, Y. H. P.; Lynd, L. R. Biotechnol. Bioeng. 2004, 88, 797− 824. (29) Percival Zhang, Y. H.; Himmel, M. E.; Mielenz, J. R. Biotechnol. Adv. 2006, 24, 452−481. (30) Pardue, H. L. Clin. Chem. 1997, 43, 1831−1837.
3965
DOI: 10.1021/acs.analchem.6b04391 Anal. Chem. 2017, 89, 3959−3965
