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Research Article Cite This: ACS Appl. Mater. Interfaces 2018, 10, 5175−5184

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Selective Discrimination of Key Enzymes of Pathogenic and Nonpathogenic Bacteria on Autonomously Reporting ShapeEncoded Hydrogel Patterns Zhiyuan Jia, Issa Sukker, Mareike Müller,* and Holger Schönherr* Physical Chemistry I & Research Center of Micro and Nanochemistry and Engineering (Cμ), Department of Chemistry and Biology, University of Siegen, Adolf-Reichwein-Straße 2, 57076 Siegen, Germany S Supporting Information *

ABSTRACT: This work reports on a new approach to rapidly and selectively detect and discriminate enzymes of pathogenic from those of nonpathogenic bacteria using a patterned autonomously reporting hydrogel on a transparent support, in which the selectivity has been encoded by the pattern shape to enable facile detection by a color change at one single wavelength. In particular, enzyme-responsive chitosan hydrogel layers that report the presence of the enzymes β-glucuronidase (β-Gus) and β-galactosidase (β-Gal), produced by the nonvirulent Escherichia coli K12 and the food-borne biosafety level 3 pathogen enterohemorrhagic E. coli, respectively, via the blue color of an indigo dye were patterned by two complementary strategies. The comparison of the functionalization of patterned chitosan patches on a solid support with two chromogenic substrates on one hand and the area-selective conjugation of the substrates on the other hand showed that the two characteristic enzymes could indeed be rapidly and selectively discriminated. The limits of detection of the highly stable sensing layers for an observation time of 60 min using a spectrophotometer correspond to enzyme concentrations of β-Gus and β-Gal of ≤5 and ≤3 nM, respectively, and to ≤62 and ≤33 nM for bare eye detection in nonoptimized sensor patches. These results confirm the applicability of this approach, which is compatible with the simple measurement of optical density at one single wavelength only as well as with parallel, multiplexed detection, to differentiate the enzymes secreted by a highly pathogenic E. coli from a nonpathogenic E. coli on the basis of specifically secreted enzymes. Hence, a general approach for the rapid and selective detection of enzymes of different bacterial species for potential applications in food safety as well as point-of-care microbiological diagnostics is described. KEYWORDS: hydrogels, biosensors, selective bacteria detection, enterohemorrhagic E. coli (EHEC), micropatterns

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INTRODUCTION In recent years, a significantly increasing number of incidences of food-borne diseases related to bacterial contaminations in food constitutes a major public health problem all across the globe.1,2 Likewise, after decades of immediate treatment of bacterial infections with antibiotics, which saved many millions of lives, antibiotic resistances are increasing at an alarming rate.3−6 Hence, bacterial infections have again become a threat to patients worldwide, also because antibiotic-resistant bacteria spread rapidly.7 It can be concluded that part of the antibiotics crisis is related to inappropriately prescribed antibiotics that accelerate the rate of resistance build-up in bacteria.7,8 In this general context, the simple yet rapid and selective detection of pathogenic bacteria and bacterial infections is of prime importance for allowing targeted and rapid treatment.9 For instance, rapid and economically affordable diagnostics may help to differentiate pathogenic bacteria from nonpathogenic bacteria to ensure adequate precautions and treatment. For pathogen detection and identification, there are traditionally standard microbiological procedures based on a © 2018 American Chemical Society

selective culturing method combined with the quantification of colony forming units. In recent years, polymerase chain reaction-based procedures have complemented these methods for pathogen detection.10,11 A very recent addition to the screening technologies that are available and approved, e.g., by the FDA, are mass spectrometric techniques, such as matrixassisted laser desorption/ionization imaging mass spectrometry or three-dimensional (3D) imaging (3D) mass spectrometry.12 Although these established methods can provide conclusive and unambiguous results, they required highly trained personnel and are either time consuming, relatively expensive, or may not work properly in remote areas without appropriate electricity or climate control.13 Among the more recently developed alternative methods for bacterial infection sensing, functional nanocapsules (liposomes14,15 and polymersomes16,17) and nanoparticles18,19 as Received: October 6, 2017 Accepted: January 18, 2018 Published: January 18, 2018 5175

DOI: 10.1021/acsami.7b15147 ACS Appl. Mater. Interfaces 2018, 10, 5175−5184

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ACS Applied Materials & Interfaces well as sensing films,20−25 which detect or differentiate bacteria or bacterial infections at an early stage, have received considerable attention. For instance, the reporter liposomes developed by Jenkins and co-workers could signal infection and discriminate Staphylococcus aureus from Pseudomonas aeruginosa via a released fluorescent reporter dye after the selective break down of appropriately designed multicomponent liposomes by bacterial virulence factors.15 A similar approach for sensing high concentrations of bacterial enzymes that differentiate the presence of few bacteria and the onset of infection by enzyme-labile vesicles was realized with enzyme-labile amphiphilic block copolymers, as was reported before.16,17 Bacteria detection or differentiation between bacterial species could also be realized with nanoparticles18,19 or sensing films, as mentioned. Wu et al. reported on a highly sensitive and specific multiplexed method to detect three pathogenic bacteria like S. aureus, Vibrio parahemolyticus, and Salmonella typhimurium using multicolor up-conversion nanoparticles.26 For applications that expand the mentioned potential topically applied approaches toward potential use inside patients, a polymerbased pH-responsive sensor system, which could be applied as an early warning system of urinary catheter blockage, was reported by Milo et al.27 Here, a pH-responsive polymeric coating within the catheter releases a fluorescent dye upon degradation, which occurs in the presence of Proteus mirabilis infections, providing a visual response. Other methods focus on bacterial enzymes or toxins able to trigger a visible response of sensing devices. For instance, a colorimetric sensor, which is based on a bacteria-specific RNAcleaving DNAzyme probe as the molecular recognition element, has been reported by Tram et al.28 Here, the sensing signal has been obtained by an increase in the pH value of the test solution due to the hydrolysis of urea by urease. As an alternative, we have recently reported on autonomously reporting chitosan films that are equipped with different fluorogenic or chromogenic substrates, such as the fluorogenic substrate 4-methylumbelliferyl-β-D-glucuronide and the chromogenic substrates 5-bromo-4-chloro-3-indolyl-β-D-glucuronide and 4-nitrophenyl-β-D-glucuronide.21,22,25 These substrates were conjugated to chitosan hydrogel film coatings to detect the presence of the characteristic enzyme β-glucuronidase (β-Gus) of Escherichia coli (K12). Related chitosanbased platforms for the detection and discrimination of different bacterial enzymes to prove the presence of Pseudomonas aeruginosa vs Staphylococcus aureus on one hand and nonpathogenic E. coli (K12) vs pathogenic enterohemorrhagic E. coli (EHEC) O157:H7 were reported.23,24 In general, the color change of the autonomously reporting hydrogels, resulting from a selective enzymatic cleavage reaction of the corresponding colorimetric substrate, was rapidly detectable (depending on the enzyme and its concentration etc. typically in ≤1 h) with very low limits of detection (LOD) for the enzymes on this observation time scale (in several cases of fluorescence-based detection ≪1 nM).22−24 For practical applications, a simple detection scheme that bypasses the need for separated excitation illumination and reporter dyes that are insoluble in aqueous media appear to be desirable. The enzyme β-galactosidase (β-Gal) is secreted by the foodborne biosafety level 3 pathogenic enterohemorrhagic E. coli (EHEC), which is a serious human pathogen and can cause lifethreatening diseases.29 By contrast, β-Gus is produced by the most widely used cultivated lab strain (E. coli K12), which is nonpathogenic.30 Thus, β-Gal and β-Gus could serve as

markers to differentiate pathogenic and nonpathogenic E. coli strains. For the current detection of EHEC, the analysis of the socalled shiga toxins is the most important diagnostic criterion in EHEC laboratory diagnostics because EHEC cannot be phenotypically distinguished from apathogenic E. coli. The ProSpecT STEC assay and the Premier EHEC assay employ polyclonal or monoclonal anti-Shiga toxin 1 and 2 antibodies to capture the toxin and horseradish peroxidase labeled monoclonal mouse or polyclonal anti-Shiga toxin 1 and 2-labeled antibodies to detect the bound toxin, respectively.31 It is also possible to confirm the presence of E. coli (EHEC) by the polymerase chain reaction of E. coli O157:H7 specific genes or next generation sequencing to identify bacterial genomic elements (16S rRNA) via molecular-based methods.32 The drawback of these established methods is that they are time consuming and demand high personnel and material costs. Here, we expand on our previously established concept of autonomously sensing hydrogel layers20−22,24,25 to differentiate the pathogenic E. coli (EHEC) from the nonvirulent E. coli (K12) with a nonleachable signaling dye and a simple concept that is compatible with a technologically least demanding detection approach. The expansion refers further to the implementation of multiple sensor elements on a single diagnostic film or coating that can be analyzed by a conventional optical density measurement at one single wavelength. Both target enzymes liberate the same waterinsoluble indigo dye from the autonomous sensing layers, which therefore does not diffuse away from the site of the reaction. By spatially separating the individual enzyme-reactive spots and by encoding the functionality (i.e., the selectivity for a given enzyme) by the shape of the patterned spots, an instrumentally very simple yet selective photometric analysis is enabled as well as the possibility for selective bare eye detection. To this end, i.e., to be able to differentiate β-Gal (secreted by EHEC) from β-Gus (produced by E. coli K12) on a single test strip in one shot, two complementary methods to obtain such shape-encoded patterns were developed and the selectivity and sensitivity in terms of the limits of detection (LOD) of their response to the bacterial enzymes mentioned above and their stability were systematically elucidated.

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EXPERIMENTAL PART

Materials. Glass slides (ECN 631-1550, Menzel Gläs er, Braunschweig, Germany) were used as supporting substrates for patterned samples. Glass slides (20 × 20 mm2 cover slips, Menzel Gläser, Braunschweig, Germany), quartz slides (0.5 mm, 01016T-AB, SPI Supplies/Structure Probe, Inc.). Polydimethylsiloxane (PDMS) prepolymer and curing agent (Sylgard 184) were purchased from Dow Corning. Chitosan (medium molar mass, 190−310 kDa, 75−85% deacetylated), 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (XGal), succinic anhydride, phosphate-buffered saline (PBS, tablet), N(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC· HCl), N-hydroxy succinimide (NHS), β-glucuronidase purified from E. coli (β-Gus, 694.3 units/mg, E.C. 3.2.1.31; type IX-A), and βgalactosidase from E. coli (β-Gal, 174 units/mg, E.C. 3.2.1.23) were purchased from Sigma-Aldrich. 5-Bromo-4-chloro-3-indoly-β-D-glucuronide sodium salt (X-Gluc) (98% pure, Alfa Aesar), ethanol (absolute, Merck), dimethyl sulfoxide (DMSO) (99% pure, Merck), and acetic acid (glacial, J.T. Baker) were purchased from listed suppliers. Milli-Q water obtained from Millipore Direct Q 8 system (Millipore, Schwalbach, Germany) with a resistivity of 18 MΩ cm was used for preparation of all aqueous media. UV−Visible Spectroscopy. The UV−vis spectra were recorded by a Varian Cary 50 Bio spectrometer (Mulgrave, Victoria, Australia) 5176


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Stability Tests under Dry Conditions. Chitosan films were formed and modified with the enzyme substrates on quartz. The modified chitosan was placed in a petri dish and was stored either in a drawer at ambient conditions or in an oven at 40 °C for 72 h. UV−vis spectra were recorded with a Varian Cary 50 Bio spectrometer at the different positions of the modified chitosan in triplicate for each time point. Clean quartz was used to record a baseline, which was subtracted in the data analysis. Stability Tests under Wet (PBS) Conditions. Pieces of chitosan on glass (#4, Menzel Gläser, Braunschweig, Germany), which were modified with enzyme substrates, were inserted inside a quartz cell (0.1 cm path length) with 150 μL of PBS buffer solution. After 2 h of swelling, the initial spectra were recorded on a Varian Cary 50 Bio spectrometer in triplicate. Afterward, the quartz cell with the modified chitosan sample under PBS was stored in a drawer at ambient conditions or in an oven at 40 °C for 72 h. Subsequently, spectra were recorded in triplicate at room temperature. The clean glass inside of the quartz cell in 150 μL of PBS was used to record a baseline, which was subtracted in the data analysis. Determination of the Limit of Detection (LOD). Grafted chitosan films on glass were inserted inside the quartz cell (0.1 cm path length), followed by adding 150 μL of buffered enzyme solution with various enzyme concentrations. Spectra were recorded during the enzymatic reactions for 2 h, with a time interval of 2 min for the enzyme β-Gal (0.125 μmol/L). In addition, the kinetics of the enzymatic reactions was recorded at a fixed wavelength of 615 nm for all of the other applied enzyme concentrations, with a time interval of 0.5 s. The baseline was recorded with a glass slide in a quartz cell filled with 150 μL of PBS solution. All absorbance values were corrected to zero at t = 0. To determine the LOD of enzyme detection with the reporter hydrogels, the kinetics of the enzymatic reaction was recorded by sequential UV−vis measurements at λmax = 615 nm for the product 5,5′-dibromo-4,4′-dichloro-indigo. In the first 30 min of the enzymatic reaction, a linear increase in optical density was observed (Supporting Information, Figure S1). The initial apparent reaction rate was defined as the slope in the first 30 min in the kinetic plot via a linear least squares fit. It was found that the initial apparent reaction rate increased linearly with the initial enzyme concentration, which is in agreement with the Michaelis−Menten kinetics.33 According to the previous work,21 the LOD for the liberated indigo dye is 0.37 μM. Hence, value of the lowest detectable signal for the UV−vis spectrophotometer was calculated by the Beer−Lambert law.34 The lowest detectable signal corresponding to a concentration of 0.37 μM of the liberated indigo dye was reached after different reaction times after the addition of enzyme solutions with different enzyme concentrations. Therefore, depending on the observation time, the LODs for the detection of the enzymes are different. The LOD was estimated in accordance with the previously reported method.21 Preparation of Patterned Samples. Modification and Reactions of Patterned Samples. PDMS Mask Preparation. The PDMS mask was prepared according to the literature.25 The PDMS prepolymer and curing agent (Sylgard 184) were mixed in a 10:1 ratio (by weight) and poured into a polystyrene petri dish after 30 min of degassing. The curing process was performed in an oven at 70 °C for 1 h. Two pieces of PDMS mask with differently shaped holes were used in this work. Rectangular (3 × 8 mm2) and circular (diameter 6 mm) holes were drill pierced after the cured PDMS cooled down to ambient temperatures (thickness 2.80 ± 0.08 mm). The other PDMS mask (thickness 3.5 ± 0.1 mm) with circular (diameter 6 mm) and square (6 mm) holes was prepared in the same way. Stamping of the Substrate Pattern on a Continuous Chitosan Layer (Approach I). Chitosan solution (1.975 mL, 0.5% (w/v)) was deposited on a cleaned glass (2.6 × 7.6 cm2), dried in an oven under vacuum (90 °C, 8 mbar) for 6 h, and afterward neutralized by immersing in NaOH solution (0.1 mol/L, 60 mL) for 1 min under mild stirring, followed by a washing step with 60 mL Milli-Q water for 10 min under stirring. Finally, the samples were dried in a nitrogen stream. According to IRS result, the chitosan film thickness was 3.0 ± 0.6 μm. The PDMS mask, which contains rectangular and circular

with the wavelength range from 200 or 300 to 800 nm and a scan rate of 300 nm/min. The baseline was recorded with a quartz (0.9 × 1.2 cm2) or glass slide (0.75 × 2.00 cm2) in a quartz cell with 100 μL PBS solution. All spectra reported have been recorded with a baseline correction. A quartz cell with 0.1 cm path length was used for transmission measurements of coated samples on quartz or glass slides. Interferometry Reflectance Spectroscopy (IRS). The reflectance spectra of the chitosan film with a 5 nm Au layer on top were obtained from an Ocean Optics spectrometer and a charge-coupled device detector (USB 2000+) equipped with a tungsten halogen light source (LS-1). The data were collected using the SpectraSuite program, and the effective optical thickness was obtained by performing a fast Fourier transformation of the recorded interferometric reflectance spectra using Igor program (Wavemetrics Inc. Igor program). The refractive index of chitosan film is 1.51.20,22 Modification of Chitosan with Enzyme Substrates. General. Chitosan films were prepared according to previously published work.25 Instead of silicon, quartz slides (0.9 × 1.2 cm2) and glass slides (0.75 × 2.00 cm2) were used as supporting substrates. The quartz and glass slides were cleaned in a UV-Ozone cleaner (ProCleaner TM system, supplied by Bioforce Nanosciences) for 30 min. Aqueous chitosan solutions (0.5% (w/v), 0.7% (w/v)) were prepared with 1 wt % acetic acid. Any impurities and particles (d ≥ 2.5 μm) were removed by filtration (Whatman no. 5 qualitative filter paper). Chitosan solution (108 μL 0.5% (w/v) and 150 μL 0.7% (w/v)) were deposited on quartz and glass slides, respectively. Afterward, the samples were dried in an oven under vacuum (90 °C, 8 mbar) for 6 h and neutralized by immersing into NaOH solution (0.1 mol/L) for 1 min under mild stirring. Then, the films were rinsed with copious amounts of Milli-Q water. Finally, the samples were dried in a nitrogen stream. The thicknesses of the chitosan films on quartz (108 μL 0.5% (w/v)) and on glass (150 μL 0.7% (w/v)) were 3.0 ± 0.6 and 4 ± 1 μm, respectively, as determined by interferometry reflectance spectroscopy (IRS). Grafting of X-Gluc to Chitosan Hydrogels. The modification was performed according to a previously published work:25 Briefly, 5.0 or 4.5 mmol/L X-Gluc solution was prepared in phosphate-buffered saline (PBS, pH 7.4) in ambient atmosphere, followed by addition of N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) (6.7 mol/mol of X-Gluc) and N-hydroxy succinimide (NHS) (6.7 mol/mol of X-Gluc). Then, the solution was stirred for 1 h. Each chitosan hydrogel film on quartz or glass was immersed in 3 mL of this 5.0 or 4.5 mmol/L X-Gluc modification solution for 6 h under shaking (rate: 60 Hz) at ambient condition, followed by exhaustive washing in 20 mL Milli-Q water under shaking for 2 h (60 Hz, Milli-Q water replacement intervals of 30 min) and subsequent drying in a nitrogen stream. Preparation of N-Succinyl Chitosan (NSC). NSC was prepared according to the literature.23 For this, chitosan film was immersed in 3 mL of 0.5 mol/L solution of succinic anhydride in dimethyl sulfoxide (DMSO) under rotary shaking (35 rpm) at 60 °C for 24 h. Afterward, each modified sample was washed with 3 mL of DMSO for 30 min under shaking (60 Hz) at ambient condition. Finally, the samples were rinsed with Milli-Q water and dried in a nitrogen stream. Grafting of X-Gal to Chitosan Hydrogels. X-Gal solution (2.5 mmol/L) was prepared in a mixed solution of DMSO and PBS, with a volume ratio of 1:4. Mixed solutions of EDC (30 mmol/L) and NHS (30 mmol/L) solutions were prepared in PBS (pH 7.4). Each NSC sample was first immersed in 3 mL of EDC/NHS solution in PBS for 60 min under shaking (60 Hz) at ambient conditions. Then, the activated NSC film was immediately immersed in 3 mL of a 2.5 mmol/ L X-Gal solution for 6 h under shaking (60 Hz) at ambient conditions, followed by a rinsing step with Milli-Q water and drying in nitrogen stream. Enzymatic Reactions in the Hydrogels. To the grafted chitosan samples, which were inserted inside the quartz cell (0.1 cm path length), 100 μL of buffered enzyme solution with various enzyme concentrations were added. Spectra were recorded during the enzymatic reaction at 200 or 300 to 800 nm for 2 h, with a time interval of 3 or 5 min. 5177


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Figure 1. Schematic of the fabrication of autonomously reporting hydrogel patterns with shape-encoded selectivity. Top (approach I): (1) chitosan solution was deposited on the cleaned glass to form a chitosan film. (2) N-Succinyl chitosan was synthesized on the film only in a rectangular patterned area with the help of a PDMS mask, followed by the modification process (3) with the chromogenic substrates X-Gal and X-Gluc in the rectangular and circular holes, respectively. The bacterial enzymes β-Gus and β-Gal were applied to the patterned and nonpatterned areas (4) of the chitosan film, respectively, where the enzymes in different patterned areas could be differentiated by a color change to blue with a certain shape compared with the nonfunctionalized chitosan layer (5). Bottom (approach II): (1) the PDMS mask that exhibits square and circular holes was attached on a cleaned glass. (2) The chitosan hydrogel patterns with different shapes were formed by depositing chitosan solution into the holes of the PDMS mask, where also the modification took place. Finally, the mixed enzyme solution was applied on each functionalized chitosan pattern (4) and the blue color in the differently shaped areas was detected. (5) Upon exposure to the enzymes, the same insoluble blue indigo dye was formed by the enzymatic cleavage of the β-linked indole derivatives in the substrates with differently shaped patterns after dimerization within oxygen. XGluc is digested by β-glucuronidase (β-Gus), which is secreted by E. coli (K12), whereas β-galactosidase (β-Gal), which is secreted by the enterohemorrhagic E. coli (EHEC), cleaves the target bond in the X-Gal-modified hydrogel areas. The shape of the colored areas can be used to differentiate β-Gal (EHEC) from β-Gus (E. coli K12). holes, was attached on the chitosan film. N-Succinyl chitosan (NSC) was formed only in the rectangular pattern area by the addition of succinic anhydride solution (100 μL, 0.5 mol/L, in DMSO) in the rectangular hole with rotary shaking (35 rpm) at 60 °C for 24 h, followed by rinsing with 100 μL of DMSO and then Milli-Q water five times. Hundred microliters of EDC/NHS (in PBS) solution was added into the rectangular hole. Then, the sample was shaken for 60 min (60 Hz) at ambient conditions. X-Gal solution (100 μL 2.5 mmol/L) was added into the rectangular hole after removing the rest of the solution (EDC/NHS (in PBS) solution). Then, the X-Gluc modification solution (100 μL, 5.0 mmol/L) was added into the circular holes. The modification procedure proceed with shaking (60 Hz) at ambient conditions for 6 h. Afterward, any unbound substrate was rinsed out with Milli-Q water, followed by drying the sample in a nitrogen stream without the PDMS mask. Enzymatic Reaction in the Shape-Encoded Biosensor According to Approach I. Forty microliters of the enzyme solution (20 μL 4 μmol/L β-Gus and 20 μL 1 μmol/L β-Gal mixture, in PBS solution, pH 7.4) was dropped on each patterned and nonpatterned area of the chitosan films. The color changes on a single-strip sensor were recorded by an iSight camera (iphone 5s) under white light illumination in front of a white background to enhance the contrast of the image. Substrate Modification on Embossed Chitosan Patterns (Approach II). A cleaned glass (2.6 × 7.6 cm2) was covered with the PDMS mask, which possessed three circular (diameter 6 mm)- and three square-shaped (6 mm) holes on two parallel lines. Chitosan solution (100 μL 0.7% (w/v)) was deposited in each hole and dried in an oven for 3 h at 70 °C. The chitosan thickness in circular and square

holes was 14 ± 2 and 11 ± 2 μm, respectively, as determined by IRS. Neutralization proceeded in the hole with NaOH solution (100 μL, 0.1 mol/L) for 1 min, and then the sample was rinsed with Milli-Q water and dried in a nitrogen stream. The remaining modification procedure was the same as that in approach I. (Circular hole: [X-Gluc] = 4.5 mmol/L; square hole: [X-Gal] = 2.5 mmol/L.) Enzymatic Reaction in the Shape-Encoded Biosensor According to Approach II. β-Gal (0.6 μmol/L 40 μL on each pattern) and β-Gus (2.0 μmol/L, 40 μL on each pattern) enzyme solutions were dropped on the patterned area of the first and second rows, respectively. Buffered enzyme solution (mixture of 20 μL of 2.0 μmol/L β-Gus and 20 μL of 0.6 μmol/L β-Gal, in PBS solution, pH 7.4, 40 μL on each pattern) was dropped on the third row. The color changes on the patterned areas were recorded by an iSight camera (iphone 6) under white light illumination in front of a white background.

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RESULTS AND DISCUSSION The two complementary approaches to encode selectivity in enzyme-responsive chitosan hydrogel layers for the rapid detection and discrimination of specific enzymes of the nonvirulent E. coli K12 (E. coli K12) and pathogenic E. coli (E. coli EHEC) are shown schematically in Figure 1. Either the chromogenic substrates were coupled to specific areas of a neat continuous chitosan film using a PDMS master with different shapes (X-Gluc was applied to the circular areas and X-Gal to the rectangular areas, approach I) or the chitosan hydrogel was deposited and subsequently functionalized using a similar PDMS mask to result in shaped and chromogenic substrate5178


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Figure 2. Details of the modification of chitosan by grafting the chromogenic substrates X-Gluc and X-Gal, respectively, as well as of the selective enzymatic cleavage of the reporter dye by β-Gus from nonvirulent E. coli and β-Gal from pathogenic E. coli (EHEC).

Figure 3. UV−vis absorption spectra of the β-glucuronidase catalyzed reaction in X-Gluc-g-chitosan hydrogel. Inset: absorbance at λmax = 615 nm for the dimerized indigo derivative in enzyme solution versus time as well as the blank experiment in PBS solution; (a) enzymatic reaction in a piece of X-Gluc-g-chitosan hydrogel on quartz (approach I). The thickness of the chitosan layer was around 3 μm; [X-Gluc]mod = 5.0 mmol/L; [β-Gus] = 2.0 μmol/L. Measurements are repeated every 3 min for 2 h, in ambient conditions. (b) enzymatic reaction in a piece of X-Gluc-g-chitosan hydrogel on glass (approach II). The thickness of the chitosan layer was around 4 μm; [X-Gluc]mod = 4.5 mmol/L; [β-Gus] = 2.0 μmol/L; measurements are repeated every 5 min for 2 h, in ambient conditions.

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Figure 4. UV−vis absorption spectra of the β-galactosidase-catalyzed reaction in EHEC-reporting X-Gal-g-chitosan hydrogel. Inset: absorbance at λmax = 615 nm for the dimerized indigo derivative in enzyme solution versus time as well as a blank experiment in PBS solution. (a) Enzymatic reaction in a piece of X-Gal-g-chitosan hydrogel on quartz (approach I). The thickness of the chitosan layer was around 3 μm; [X-Gal]mod = 2.5 mmol/L; [β-Gal] = 0.5 μmol/L; measurements are repeated every 5 min for 2 h, at ambient conditions. (b) Enzymatic reaction in a piece of X-Gal-gchitosan hydrogel on glass (approach II). The thickness of the chitosan layer was around 4 μm; [X-Gal]mod = 2.5 mmol/L; [β-Gus] = 0.6 μmol/L; Measurements are repeated every 5 min for 2 h, at ambient conditions.

rate during the first 30 min was (2.50 ± 0.08) × 10−3 min−1. Compared with the sample in Figure 3a, the modified hydrogel in Figure 3b was prepared with thicker chitosan films (around 4 μm), which was formed by a higher concentrated chitosan solution (150 μL, 0.7% (w/v)) and lower concentration of the X-Gluc chromogenic substrate (4.5 mmol/L). This is likely the reason for the fact that the sample in Figure 3b exhibits apparently a slightly lower initial apparent rate in comparison to that of the sample, which was used in Figure 3a. In addition, the initial apparent rate also depends on the oxidation of the initially formed 5,5′-dibromo-4,4′-dichloro-indigo. The oxidation reaction may proceed faster on the surface than inside the hydrogel, which might explain why the thinner hydrogel in Figure 3a exhibited a slightly higher initial rate than that in Figure 3b. To prove in both cases that the released indigo dye formed due to enzymatic cleavage of the C−O bond in modified chitosan by β-Gus, a blank experiment was carried out in PBS. Chitosan modified with X-Gluc was inserted inside a quartz cuvette with PBS solution. The spectra were recorded by a UV−vis spectrometer after each 10 or 60 min in thin or thicker film, respectively. Afterward, the absorbance at 615 nm was plotted vs time (Figure 3, inset). These blank experiments confirmed that no changes in the absorbance at λmax = 615 nm occurred, when the β-Gus sensing hydrogel was exposed to PBS solution only. Further, the initial results indicated that the target bond in X-Gluc-g-chitosan is stable for at least 2 h in PBS solution under ambient conditions. More data on the stability of the chromogenic substrates is provided below. Enzymatic Reaction in EHEC Sensing Hydrogels. The β-Gal (EHEC) reporting hydrogels function analogously to the β-Gus sensing hydrogels. The β-linked indole derivative of the X-Gal chromogenic substrate is cleaved by β-Gal and forms the same water-insoluble indigo dye that stays inside the hydrogel. For more detailed analyses of the β-Gal sensing films prepared with a thin chitosan layer (∼3 μm thickness, related to approach I) on quartz (0.9 × 1.2 cm2) and thick chitosan layer (∼4 μm thickness, related to approach II) on glass (0.75 × 2.00 cm2), the enzymatic reaction of the β-Gal sensing hydrogel was performed in aqueous buffered solution (PBS, pH 7.4). The apparent kinetics of the reaction was recorded in time lapse

equipped chitosan patches on the supporting glass. Both methods thus afford shape-encoded areas modified with the two different substrates (X-Gluc was located in a circular area and X-Gal in the square area, approach II). Because the functionalization protocols are practically identical and because the initial reaction rates have already been shown to be independent of the hydrogel layer thickness,20 a very similar response to the enzymes of interest was expected. Hydrogel Synthesis and Enzymatic Reactions. Chitosan hydrogels were modified with the chromogenic X-Gluc substrate by EDC/NHS chemistry via amide bond formation between the carboxyl group of enzymatic substrates and the amine groups of chitosan. The β-Gal reporting hydrogel was obtained by treatment of chitosan hydrogels on quartz or glass substrates with (i) succinic anhydride in a ring-opening modification to form N-succinyl chitosan (NSC), followed by (ii) EDC-based covalent coupling of the chromogenic substrate X-Gal. The subsequent enzymatic reaction of the modified hydrogels was performed in neat buffered β-Gus and β-Gal solutions. The liberated indole derivative dimerizes in the presence of oxygen from the blue water-insoluble dye 5,5′dibromo-4,4′-dichloro-indigo after both corresponding enzymatic reactions (Figure 2). Enzymatic Reaction in Nonvirulent E. coli Sensing Hydrogels. The enzymatic reaction of the β-Gus (E. coli K12) sensing hydrogels prepared according to both approaches was performed in aqueous buffered solution (PBS, pH 7.4). The apparent kinetics of the reaction was recorded in time lapse measurements by UV−vis spectroscopy at ambient conditions. The increasing absorbance in the wavelength range of 550−700 nm is associated to the dimerized indigo derivative that was formed by the reaction shown in Figure 2. The absorbance at λmax = 615 nm showed a monotonous increase as a function of time for 2 μmol/L β-Gus (Figure 3a, inset) for the sample prepared according to approach I. The initial apparent rate during the first 30 min was (3.29 ± 0.09) × 10−3 min−1. A similar result was obtained for a sample, which was modified with the chromogenic X-Gluc substrate on glass corresponding to approach II (Figure 3b). The absorbance at λmax = 615 nm showed a monotonous increase as a function of time in 2 μmol/L β-Gus (Figure 3b, inset). The initial apparent 5180


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Figure 5. Plots of the LOD for (a) β-Gus and (b) β-Gal detection determined by spectrophotometry versus the reaction time using X-Gluc- and XGal-grafted hydrogels, respectively.

Figure 6. Schematic (a) and photographs (b) of the enzymatic reaction result from autonomously reporting hydrogel patterns with shape-encoded selectivity. Photographs were taken after an enzymatic reaction time period of 1 h by an iSight camera (iphone 5s, iphone 6) under white light illumination with a white background. Left (approach I): chitosan at the rectangular and circular pattern was modified with X-Gal and X-Gluc substrates with the help of PDMS mask, respectively. A mixed enzyme solution ([β-Gus] = 4.0 μmol/L and [β-Gal] = 1.0 μmol/L with a volume ratio of 1:1) was applied on the patterned and nonpatterned area. The thickness of the chitosan layer was approximately 3 μm. [X-Gal]mod = 2.5 mmol/L; [X-Gluc]mod = 5.0 mmol/L. Right (approach II): the square and circular chitosan patterns were modified with X-Gal and X-Gluc substrates, respectively. Either β-Gal (0.6 μmol/L) or β-Gus (2.0 μmol/L) solution was dropped on the first and second pattern pair, respectively. A mixed enzyme solution ([β-Gal] = 0.6 μmol/L and [β-Gus] = 2.0 μmol/L with a volume ratio of 1:1) was applied on the third patterned chitosan pair. The thickness of the chitosan in the square and circular patterned areas was around 11 and 14 μm, respectively; [X-Gal]mod = 2.5 mmol/L; [XGluc]mod = 4.5 mmol/L.

For the thicker chitosan film corresponding to approach II, the apparent initial rate of the enzymatic reaction within the first 10 min in 0.6 μmol/L β-Gal solution is (2.9 ± 0.2) × 10−3 min−1 (Figure 4b, inset). The reason for the faster apparent initial rate observed for the thicker chitosan film is the higher concentration of enzyme solution that was applied during the enzymatic reaction. Apart from that, the initial apparent

measurements by UV−vis spectroscopy at ambient conditions (Figure 4). Both plots of the absorbance at λmax = 615 nm show a monotonous absorption increase at 615 nm as a function of time (Figure 4, insets). The apparent initial rate of the enzymatic reaction within the first 30 min of the thin chitosan film corresponding to approach I exposed to a 0.5 μmol/L βGal solution is (1.50 ± 0.06) × 10−3 min−1 (Figure 4a, inset). 5181


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present in lower concentration after mixing compared with the enzyme solutions with only one enzyme. Hence, the dimerized water-insoluble indigo dye was formed in both chromogenic substrates’ patterned areas, when the corresponding enzyme was present. In independent spectroscopic measurements, it was confirmed that even in solution, the inappropriate enzymes could not break the target bond in the enzyme substrate to form the indigo dye. Although UV−vis absorption spectra of enzyme substrate solutions exposed to the correct enzyme showed a rapid response, no absorbance increase at 615 nm was detectable for the “wrong” enzyme in both cases. (Supporting Information, Figure S4). Hence, the blue-colored differently shaped areas can indeed be used to indicate the presence of the different bacterial enzymes and further to detect and differentiate the two different target strains. The LODs for the patterns were determined in a manner similar to that for the homogenous films and were found to be comparable using spectrophotometry. However, for bare eye detection, the LOD of the dye itself was assessed by visual inspection. An absorbance of 0.00560 ± 0.00002, which equals a dye concentration of 0.05 mM, represents the onset of blue color discernible by the bare eye (Supporting Information, Figure S5). From the data, the high reproducibility can be judged qualitatively from the very similar color. More quantitative insight is afforded by readings taken with a standard microplate reader (Supporting Information, Figure S6). A relative error of

Selective Discrimination of Key Enzymes of ... - ACS Publications

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