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Colorimetric Paper Bioassay for the Detection of Phenolic Compounds Ramiz S. J. Alkasir, Maryna Ornatska, and Silvana Andreescu* Department of Chemistry and Bimolecular Science, Clarkson University, Potsdam, New York 13699-5810, United States S Supporting Information *

ABSTRACT: A new type of paper based bioassay for the colorimetric detection of phenolic compounds including phenol, bisphenol A, catechol and cresols is reported. The sensor is based on a layer-by-layer (LbL) assembly approach formed by alternatively depositing layers of chitosan and alginate polyelectrolytes onto filter paper and physically entrapping the tyrosinase enzyme in between these layers. The sensor response is quantified as a color change resulting from the specific binding of the enzymatically generated quinone to the multilayers of immobilized chitosan on the paper. The color change can be quantified with the naked eye but a digitalized picture can also be used to provide more sensitive comparison to a calibrated color scheme. The sensor was optimized with respect to the number of layers, pH, enzyme, chitosan and alginate amounts. The colorimetric response was concentration dependent, with a detection limit of 0.86 (±0.1) μg/L for each of the phenolic compounds tested. The response time required for the sensor to reach steady-state color varied between 6 and 17 min depending on the phenolic substrate. The sensor showed excellent storage stability at room temperature for several months (92% residual activity after 260 days storage) and demonstrated good functionality in real environmental samples. A procedure to mass-produce the bioactive sensors by inkjet printing the LbL layers of polyelectrolyte and enzyme on paper is demonstrated.

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accompanied by a visible color change. Examples include sensors for the detection of glucose,11,12 heavy metals,13,14 and neurotoxins.15 Colorimetric sensors for the detection of phenol that measure optical changes of quantum dots16 or organic dyes such as 3-methyl-2-benzothiazolinone hydrazone17 and 2,6dichloro-quinonechloroimin18 have been reported, but these have not been adapted to paper platforms and most still require the use of chromogenic reagents and spectroscopic instrumentation for the quantification of the signal. We describe herein a paper-based colorimetric sensor with immobilized tyrosinase for the naked-eye detection of phenolic compounds. The method exploits the color change of chitosan, immobilized on a paper platform, upon interaction with enzymatically generated quinones. Selective binding of reactive quinone derivatives of phenol onto the nucleophilic amino functionalities of chitosan and the formation of a distinct color change in the presence of phenolic compounds have been reported by Payne et al.19 This principle has been applied previously in the environmental remediation for the removal of phenol20 and BPA.21 Here, the color change is used to develop a paper-based assay for the detection of a variety of phenolic

henolic compounds including phenol and simple substituted phenol derivatives such as bisphenol A (BPA), catechol, and cresols are widely used industrial byproducts that can bioaccumulate in the environment and the ecological food chain through consumption or uptake. Their presence in air, water, and food matrices poses toxic risks to human health and the environment. Conventional methods for the detection of these chemicals include laboratory-based spectrophotometric1 and chromatographic methods2,3 as well as several types of immunochemical4 and enzyme-based electrochemical biosensors.5,6 Simple, easy-to-operate, and inexpensive methods for screening and detection of these compounds are needed to simplify and extend field monitoring and evaluate exposure risks of real matrices which contain these chemicals. This paper reports development and optimization of a reagentless, selfintegrated enzyme-based paper bioassay with colorimetric readout for the detection of phenolic compounds. Colorimetric paper-based sensors or colorimetric “test strips” with immobilized enzymes have been reported in recent years as simple and rapid detection assays for field analysis.7,8 In their vast majority, these devices utilize conventional colorimetric reagents (e.g., soluble redox dyes, quantum dots,9 gold nanoparticles,10 and redox active cerium oxide nanoparticles11). ). In these assays, detection is based on the monitoring of optical changes in the dye, the redox state, or the physicochemical properties (e.g., aggregation or dispersion) of nanoparticles, induced by the presence of an analyte and © 2012 American Chemical Society

Received: April 27, 2012 Accepted: October 31, 2012 Published: October 31, 2012 9729

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Figure 1. Operational principle of the LbL paper-based bioassay. The schematic illustrates as an example the detection of BPA. The method involves enzymatic conversion of BPA followed by chemical binding of the resulting quinone to chitosan, which induced a change in color from the colorlesswhite of chitosan to the blue-green of BPA-quinone imine. The lower right panel shows examples of sensor responses to 50 ppb BPA, phenol, and dopamine and comparison with a control experiment in the absence of phenol.

Figure 2. Schematic representation of the fabrication procedure of the colorimetric tyrosinase paper-based sensor showing sequential LbL deposition of alternate layers of chitosan, tyrosinase, and alginate onto the filter paper.

compounds (e.g., phenol, BPA, catechol and m- and psubstituted cresols). The sensor is constructed using a layer-by-layer (LbL) electrostatic assembly technique that allows deposition of uniform multilayer films on solid surfaces.22 To fabricate the sensor, alternative layers of chitosan, tyrosinase, and sodium alginate were deposted onto a filter paper disk that constitutes the sensing platform. The LbL strategy increased enzyme and chitosan loadings. This enhanced the biocatalytic conversion of the phenolic compounds and the binding efficiency of the resulting quinones to chitosan, thus providing assay sensitivity. Chitosan and alginate are biocompatible, have rich surface functionalities, and film forming ability23 making them excellent materials for use as enzyme immobilization matrices.23,24 In addition, the cellulosic structure and high porosity of the filter paper is amenable to surface modification. The functioning principle of the sensor is shown in Figure 1. Chitosan and alginate have been deposited onto paper to form uniform LbL layers that incorporate and stabilize the tyrosinase enzyme by

alternate electrostatic adsorption. Chitosan is needed for detection, while alginate is used as the oppositely charged polymer to electrostatically attach the enzyme. Optimization of the LbL assembly on paper, enzyme immobilization, and study of the analytical performance of this assay for the detection of phenolic compounds are described. Large scale manufacturing of the sensors using an inkjet printer to sequentially deposit the LbL layers of chitosan, alginate, and enzyme on paper while retaining bioactivity and functionality is demonstrated.

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EXPERIMENTAL SECTION R e a g e n t s a n d S t o c k S o l u t i o n s . T y r o s in a s e (E.C.1.14.18.1, from mushroom, 4276 units/mg), sodium triphosphate pentabasic, NaTPP (practical grade), chitosan from crab shells (practical grade), alginic acid (sodium salt), phenol (99% purity), L-ascorbic acid, L-tyrosine, L-phenylalanine, uric acid (min 99%), and bisphenol A (BPA) (99%+ purity) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium phosphate monobasic, sodium phosphate 9730

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Figure 3. UV−vis spectra of chitosan with (dotted line) and without (solid line) tyrosinase in the presence of 0.1 mM (A) phenol, (B) catechol, (C) m-cresol, (D) p-cresol, (E) BPA, and (F) dopamine.

dibasic anhydrous, and Fisher brand filter paper (P5; 09− 801C) with a diameter of 11 cm and a medium porosity were supplied from Fisher Scientific. Stock solutions of 1 × 10−2 M phenol, BPA, catechol, m-cresol, and p-cresol were prepared in methanol and stored at 4 °C for one week. Working solutions were prepared daily in 0.1 M phosphate buffer (PBS) at pH 6.5. Stock solutions of chitosan at 1.25 and 0.15% (w/v) and sodium alginate at 2.0 and 0.4% (w/v) were prepared by dissolving chitosan and sodium alginate in 0.1 M acetic acid and

distilled water, respectively. To obtain homogeneous solutions, the stock solutions of chitosan and alginate were stirred for 12 h and then filtrated under vacuum. These solutions were stored at 4 °C for 1 month. The stock solution of cross-linking agent NaTPP at 1.5% (w/v) was prepared in distilled water. All reagents were used without further purification, unless noted, and all solutions were prepared with distilled deionized water (Millipore, Direct-Q system) with a resistivity of 18.2 MΩ. 9731

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Fabrication of the Colorimetric Tyrosinase Paper Sensor. Disk-shaped Fisher-brand filter paper was used as a solid support to fabricate the sensor. The filter paper was cut (after the LbL modification) into small spherical disks, each with a diameter of 0.6 cm, using a hole puncher. The paper was modified with alternating layers of chitosan, alginate, and enzyme using the multilayer sequence as shown in Figure 2. To begin the LbL sequence, the paper was first soaked in 1.5% NaTPP for 10 min and then air-dried for 30 min. Adsorption of NaTPP onto paper facilitated rapid ionic cross-linking and stabilization of chitosan onto the solid platform. Individual layers of enzyme and polyelectrolytes were then formed by sequential addition using a micropipet of the following amounts (in this sequence): chitosan (9 μL/layer, three layers, 1.25% stock solution), 200 units/μL tyrosinase (2.5 μL/layer, five layers), and alginate (6 μL/layer, two layers, 2% stock solution). The bioactive sensor was air-dried for 45 min before use (or stored in different conditions as indicated). Measurements of phenolic compounds were performed by adding a 25 μL sample onto the sensor surface. Quantification of color and sensor calibration was performed on scanned images using Adobe Photoshop, as described in Figure S1 of the Supporting Information. Data were collected on dried paper disks, typically ∼1 h after addition of the phenolic substrate, after formation of a stable color. A FUJIFILM Dimatix jettable fluid printer (DMP-2800) was used to print the LbL layers of chitosan, alginate, and enzyme for the printed sensor configuration. Solutions of 0.15% (w/v) chitosan and 0.4% (w/v) sodium alginate were printed. Real Samples Analysis. The functionality of the sensor was tested on tap water and river water samples. River water samples were collected from the local Raquete River from two different locations in Potsdam, NY: the riverside beside Fall Island and beside the Clarkson Moore House. The water samples were spiked to bring the concentration to 50 μg/L BPA. Twenty-five microliters of the spiked water was added onto the paper disk, and the resulting color intensity was measured and compared with a standard BPA solution of 50 μg/L.

monitoring of the color change of the immobilized chitosan on a filter paper after the binding of a quinone, enzymatically generated by tyrosinase in the presence of molecular oxygen. To construct an integrated, reagentless-sensing platform, both the chitosan and the enzyme were immobilized onto the filter paper. Chitosan has been used previously as a coating material and an additive for cellulosic materials25,26 and has been known to facilitate strong adsorption of biomolecules.27 Functionalization of paper with chitosan improved the strength of paper,26 morphology, moisture barrier,28 mechanical, and optical properties.29 Due to their surface charges and structural similarities, cellulose and chitosan interact strongly through electrostatic adsorption and hydrogen bonding which facilitate strong attachment of chitosan onto cellulosic paper.26,27 To stabilize chitosan and enhance the binding, the NaTPP cross-linking agent for chitosan was first preadsorbed onto the paper. The preadsorbed TPP− diffuses onto the paper, increasing the number of negative charges onto the cellulosic fibers and promoting rapid polymerization of chitosan through ionic cross-linking. Deposition of positively charged chitosan (pKa 6.5) induces formation of a positively charged aminoterminated layer for further electrostatic stabilization of tyrosinase, which has a low isoelectric point (4.7−5) (Figure 2). Initial experiments carried out with filter paper functionalized with a single layer of chitosan and enzyme (500 units/ layer) did not show a color change after the addition of phenol. Increasing the concentration or the amount of chitosan and the enzyme in the single layer was not effective at providing colorimetric quantifiable responses to phenol. To optimize the sensor, in subsequent experiments we have adopted an LbL assembly approach in which several layers of chitosan, enzyme, and alginate were deposited sequentially onto the paper as shown in Figure 2. This approach provided a biocompatible environment for the enzyme and enhanced protein adsorption through electrostatic binding, thus stabilizing the enzyme. The electrostatic properties of chitosan facilitated formation of alternative layers in conjunction with a complementary-charged polyelectrolyte, alginate. Alginate is a negatively charged anionic polyelectrolyte containing carboxyl functionalities that can easily form multilayer films with chitosan through LbL assembly.30 In the LbL design of the sensor, tyrosinase is entrapped in between layers of chitosan and alginate, leading to the formation of a multilayer sandwichtype platform with increased chitosan and enzyme loadings onto paper. There was no enzyme leaching, as quantified by enzyme activity tests in the washing solution of the paper disk after bioimmobilization. When phenol is added, the enzymatic reaction, as well as the chemical binding of the enzymatically generated quinone to chitosan, is amplified by the use of multiple layers. Since the method relies on the binding of the enzymatically generated product to the immobilized chitosan, the multilayer preconcentrates the analyte (e.g., the phenolic substrate) onto the sensing platform (for each layer of chitosan there is one layer of quinone-imine formed), thus enhancing the detection sensitivity. Indeed, the use of the LbL approach increased the intensity of the color and provided quantifiable detection for the phenolic compounds tested. Since both the enzyme and chitosan are fixed onto the sensing platform and the only reagent needed for analysis is the sample containing the analyte (e.g., the phenolic compound), this assay functions as a “reagentless” sensor. The detection capability of this method varies with the number of layers deposited onto the

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RESULTS AND DISCUSSION Spectroscopic Investigation of the Biocatalytic Conversion of Phenolic Compounds in the Presence of Chitosan. While formation of a colored compound of chitosan with the enzymatically generated quinone has been reported for phenol, the kinetics of the process and evaluation of this method for use in analytical assays for the determination of phenolic compounds have not been studied. In initial work, we conducted UV−vis measurements in solution to determine spectral properties of quinone imines for six phenolics (BPA, phenol, catechol, dopamine, m-cresol, and p-cresol) upon reaction with chitosan. Addition of each of the six phenols to a solution of 0.125% (w/v) chitosan containing tyrosinase (1500 units/mL) produced distinct spectral changes in the UV−vis spectrum (Figure 3). The color of the solution (red-brownish for phenol and catechol and blue-green for BPA) and the kinetics of the reaction varied with the type and the chemical structure of the phenolic compounds tested. Experimental details on the UV−vis measurements are included in Figure S2 of the Supporting Information. 3.2. Fabrication of the Colorimetric LbL TyrosinaseBased Paper Sensor. The operational principle of the sensor is shown schematically in Figure 1. The method is based on the 9732

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Figure 4. Optimization of experimental parameters of the colorimetric sensor: (A) Effect of the number of layers (the x-axis represents the number of tyrosinase layers), (B) effect of the chitosan amount, (C) effect of the alginate amount, and (D) effect of the pH. The graphs show triplicate measurements and standard deviation of color intensity after addition of 100 μg/L phenol (color was quantified using Adobe Photoshop of scanned images of dried paper discs after color stabilization).

with 6 μL of alginate per each layer. Increasing the amount of alginate or chitosan decreased the intensity of the color (Figure 4C), likely due to diffusion limitations. Effect of pH. Several factors affect the pH sensitivity of the system including the dependence of enzyme activity on pH, pKa, and the degree of ionization of the two complementary polyelectrolytes, alginate and chitosan. The multilayer structure, porosity, and diffusion characteristics, as well as the composition of the functional groups within and outside the layers, are all controlled by the pH.31 The optimum color intensity of the LbL tyrosinase sensor in response to phenol additions was obtained at pH 6.4 which corresponds to the optimum pH of the free enzyme (Figure 4D). The response decreased slightly for a pH higher than 7. Interestingly, when the sensor was tested at a very low pH of around 2, the sensor surface still changed color (the intensity was lower than that observed at pH 6.4). This indicates that the immobilized tyrosinase is still active and able to catalyze phenolic substrates even in harsh pH conditions. Enhanced activity of immobilized tyrosinase at an acidic pH of ∼3 was reported previously for a cross-linked enzyme aggregate with ammonium sulfate and glutaraldehyde, which was explained by a change in amino acid

paper surface, the enzyme, chitosan and alginate amounts, and the pH. Optimization of these parameters with respect to the analytical performance characteristics is discussed in the following sections. Phenol was used as a model phenolic substrate in these studies. Addition of phenol to the sensing paper induces a color change from colorless to brownish. Effect of Number of Layers. The color intensity to the addition of phenol increases with increasing the number of layers of chitosan/enzyme/alginate as shown in Figure 4A. The maximum response was obtained with five layers of enzyme (200 units/μL) deposited sequentially in between chitosan (1.25%) and alginate (2%). Further increase in the LbL sequence did not result in an enhanced response. This could be due to the formation of a densely packed structure that prevents diffusion of the substrate to the bioactive site of the immobilized enzyme. Effect of Chitosan and Alginate Amounts. Figure 4 (panels B and C) show the effect of increasing the chitosan and alginate amounts on the sensor response. An optimum response was obtained with 9 μL of chitosan per layer which provided a sufficient coverage and amino functionalities to form a LbL structure with alginate and to which enzymatically formed oquinone would bind. Maximum color intensity was obtained 9733

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Figure 5. (A) Colorimetric responses of the sensor to the addition of phenolic compounds in the concentration range of 1−500 μg/L for phenol, BPA, dopamine, catechol, and m- and p-cresol. The first column shows a control sample in the absence of an enzyme after the addition of 100 μg/L phenolic substrate. (B) Color formation over time of the LbL tyrosinase paper for addition of 100 μg/L (A) phenol and (B) BPA and the corresponding dynamic range.

side chain ionization in the microenvironment surrounding the active site of the enzyme.32 Analytical Characteristics of Colorimetric LbL Tyrosinase Paper Sensor. The optimized sensors have been tested for the detection of several phenolic compounds, including phenol, BPA, dopamine, catechol, m-cresol, and p-cresol. Figure 5A shows the colorimetric responses to concentrations of these compounds ranging from 1 to 500 μg/L. After addition of the phenolic substrates, the color of the sensor changed significantly. Different colors have been formed depending on the phenolic compound tested: reddish-brown for phenol, bluegreen for BPA, dark-brown for dopamine, orange for catechol and m- and p-cresols. The reaction kinetic was studied by monitoring the color formation every 30 s for 60 min (Figure 5B). The color started to appear within the first 60 s and stabilized after ∼6 min for phenol and 17 min for BPA. The digital images of color formation over time for phenol and BPA are shown in Figure S3 of the Supporting Information. The rate

of color formation corresponds to 18.13 color intensity units (CIU)/min for phenol and 5.08 CIU/min for BPA, respectively. The time needed for the sensors to reach a stable color intensity, or the response time of the sensor, varied with the phenolic compound tested and typically ranged between 6 and 17 min, as follows: 6 min for phenol, 17 min for BPA and dopamine, 10 min for catechol and m- and p-cresol. The intensity of the color increases with increased substrate concentration. Figure 6 shows the corresponding linear calibration curves and linear ranges, quantified from the color intensity of scanned images of the paper discs in dry state, after reaching a steady-state value. Calibration plots show bimodal responses toward phenol, BPA, dopamine, catechol, m-cresol, and p-cresol with two distinct linear ranges of 1−25 μg/L and 50−500 μg/L. High concentrations gave a lower sensitivity than lower concentrations. The bimodal distribution of the linear range is a consequence of the two sequential processes involved (e.g., enzymatic catalysis) followed by the chemical 9734

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Figure 6. The response calibration curves and linear plots (inset) of the colorimetric tyrosinase paper sensors for detection of phenol, BPA, dopamine, and p-cresol in 0.1 M PBS, pH 6.5 (average responses and standard deviation for n = 3 paper sensor disks).

binding of the reaction product to chitosan. Several factors contribute to this behavior, including a slower rate of substrate diffusion through the LbL layers, enzyme inactivation, or blocking of the chitosan binding site at high-substrate concentrations. The use of a logarithmic calibration plot provides wider linearity of the method, in the following concentration ranges for the phenolics tested (not shown): 1− 400 μg/L for phenol, 1−200 μg/L for BPA, 1−300 μg/L for dopamine, 1−300 μg/L for catechol, 1−500 μg/L for m-cresol, and 1−200 μg/L for p-cresol. The minimal sensor response detectable with the naked eye is 5 μg/L for each phenolic substrate tested; this value represents the sensor response that provided a color change noticeable by the naked eye as compared to that of the blank, in the absence of phenolics. When the color was quantified with a scanner and Adobe Photoshop, the detection limit was 0.86 (±0.102) μg/L (determined using the 3σs/S criteria, where S is the slope of the linear calibration curve, and σs is the standard deviation of the color intensity of the blank). To study interferences, the color intensity after the addition of potential interfering compounds like ascorbic acid, uric acid, and phenylalanine was quantified and compared with the sensor response to the addition of two common tyrosinase substrates: phenol and tyrosine. There was no response to either one of the interferents tested, while the sensor showed well-defined color for both phenol and tyrosine (Figure S4A of the Supporting Information). The color intensity was not affected by the presence of competitive interferences (Figure

S4B of the Supporting Information shows sensor response to phenol in the presence of ascorbic acid). Sensor Stability and Reproducibility. The reproducibility of the sensor was evaluated for three identical paper sensors, prepared independently, following the same experimental protocol. The average color intensity after the addition of 100 μg/L phenol was 139.6 (±3.7) for n = 3 sensors with a calculated RSD value of 2.7% and 108.0 (±3.4) with a RSD of 3.2% for 100 μg/L BPA (Figure S5 of the Supporting Information). The sensors show excellent storage stability over a period of 260 days. The stability was evaluated by measuring the colorimetric response of sensors stored in the dried state at three different conditions: at room temperature (approximately 25 °C), in a refrigerator (2−8 °C), and in a freezer (−20 °C) (Figure S6 of the Supporting Information). A remarkable stability of up to 92% of the initial response was retained after 260 days of storage at room temperature. Sensors stored in the refrigerator or freezer showed 99% residual activity after 54 days and 97% after 260 days of storage. These results clearly indicate that the LbL assembly provides an excellent environment for preserving enzyme activity for long periods of time at room temperature. This is a consequence of the biocompatibility of the chitosan and alginate, in which the enzyme is entrapped, and the strong electrostatic intermolecular interactions through the LbL layering, which might prevent conformational changes thus reducing the risk of enzyme deactivation. The enhanced storage stability at room temperature is far superior to that of other systems with immobilized tyrosinase reported in the literature (most of which have been 9735

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studied at 4 °C).33,34 For example, the stability of tyrosinase immobilized in a binary alginate/poly(acrylamide-co-acrylic acid) hydrogel was of 100 days at 4 °C, while the free enzyme lost 50% of the initial activity after ∼20 days at the same temperature.33 The storage stability of our system is higher than that of glutaraldehyde cross-linked tyrosinase aggregates, which retained 83% of the initial activity after 3 months at room temperature.32 Application to Real Water Sample Analysis. The LbL paper sensor was used to determine the presence of phenolics in tap water and two river water samples collected from a local river from two different locations. The sensor showed no color change when the water samples were added. This indicates that phenolic compounds are not present in these samples. To demonstrate the potential application of this technology in analysis of environmental samples, the sensor was tested on spiked water samples after an addition of 50 μg/L BPA. Figure S7 of the Supporting Information shows the colorimetric responses of the sensor to the addition of spiked tap or river water. Using the colorimetric signal, the recovery ranged between 94 and 98% for the spiked samples, which indicates good functionality of the sensor in real matrices. The results of this study demonstrate that this sensor has potential for real world applications. Fabrication of the LbL Sensor by Inkjet Printing. Possibilities of large scale manufacturing of the LbL sensor on paper have been explored by automatically printing layers of chitosan, alginate, and enzyme using an inkjet printer. The inkjet-printing technology provides mass production capabilities with high reproducibly and low cost. Sensing spots with a diameter of 0.6 cm were digitally generated in Adobe Photoshop CS 8.0 and printed on regular A4 paper with 95 μm thickness. Ink formulations and the number of layers were optimized to allow reproducible printing and avoid blockage of the print head nozzle of the inkjet cartridge. Printing formulations of polyelectrolyte solutions were optimized by adjusting viscosity of chitosan and alginate to 0.15% and 0.4%, which corresponds to 10.22 and 11.72 cPs respectively. Inks were jetted with a 20 μm diameter nozzle cartridge with a typical drop volume of ∼10 pL. To achieve comparable amounts of reagents as in the manual LbL configuration, 10 layers were jetted for each manually deposited layer. A strong dark-blue color developed upon the addition of BPA to the printed LbL spot on the paper, similar to that observed with the manually fabricated device. The development of color confirms successful printing of the LbL assembly with retention of the catalytic activity of the printed enzyme and demonstrates the functionality of the inkjet-printed configuration. The calibration curve of the printed sensor for the detection of BPA is shown in Figure S8 of the Supporting Information. The sensitivity of the printed sensor (2.03 CI/μg L−1) was lower than the one that was manually deposited (2.86 CI/ μg L−1) (a summary comparative table is provided in S8 of the Supporting Information). Further enhancement of sensitivity can be made by increasing the number and sequence of layers. These preliminary results demonstrate the robustness of the method and suitability for large-scale manufacturing.

mechanism with all the reagents needed for detection immobilized on the paper. The method is based on the combined use of enzymatic oxidation of the phenolic compounds by tyrosinase and the subsequent covalent binding of the reaction product to chitosan which also serves as an immobilization matrix for the enzyme on the sensing paper. This binding induces a distinct color change in the presence of phenolic compounds: blue-green for BPA, red for phenol, and orange-brown for cresols. The color change is concentration dependent and is visible to the naked eye, providing a semiquantitative (naked eye observation) or quantitative (e.g., using more advanced color quantification tools such as an IPhone camera, an x-Rite Capsure device, or a scanner/Adobe Photoshop) colorimetric way to detect targeted phenolic compounds without additional instrumentation. Furthermore, the method does not require the use of chromogenic dyes or external reagents. The sensor provided good sensitivity and detection limits, in the ppb range, for six well-known phenolic compounds including phenol, BPA, catechol, dopamine, and cresols. Another unique aspect reported in this investigation is the fabrication of biofunctional paper with an immobilized enzyme, using a LbL assembly approach consisting of deposition of several sequential layers of complementarily charged chitosan and alginate biopolymers. The LbL approach increased the efficiency of the biocatalytic conversion of the phenolic compounds and augmented the number of chitosan binding sites to which the enzymatically generated quinone would attach, thus enhancing the sensitivity of the assay. The method provided excellent conditions for enzyme immobilization, with good retention of enzymatic activity and excellent stability. The sensor was characterized by excellent reproducibility, pH dependency, and long-term stability of more than 260 days at room temperature. The study also demonstrates the possibility to mass-produce the LbL configuration by inkjet printing providing opportunities for large-scale manufacturing. The overall results and stability data obtained in this work suggest that the method can be potentially used as a generic approach for immobilizing biomolecules on paper and cellulose-based platforms, thereby enhancing bioactivity and increasing longterm stability. The method can thus facilitate development of novel bioactive paper-based platforms for a variety of biosensing applications.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Erica Sharpe, Rıfat Emrah Ö zel, and George Apau for their help with setting up the initial experimental procedure. We also thank Professor A. Rossner for helpful discussions. We are grateful to Professor D. Goia for the help with the DMP-2800 inkjet printer. This material is based on work supported by the National Science Foundation under

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CONCLUSION This study demonstrates a new paper-based colorimetric bioassay format for the detection of phenolic compounds that is portable, easy-to-use, inexpensive, and suitable for field analysis. The sensing platform has a build-in detection 9736

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Grant 0954919. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.

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dx.doi.org/10.1021/ac301110d | Anal. Chem. 2012, 84, 9729−9737