Ultrathin Hydrogel Films for Rapid Optical Biosensing - ACS Publications

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Ultrathin Hydrogel Films for Rapid Optical Biosensing Xi Zhang, Ying Guan, and Yongjun Zhang* State Key Laboratory of Medicinal Chemical Biology and Key Laboratory of Functional Polymer Materials, Institute of Polymer Chemistry, College of Chemistry, Nankai University, Tianjin 300071, China S Supporting Information *

ABSTRACT: Novel biosensors have been designed by reporting an analyte-induced (de)swelling of a stimuli-responsive hydrogel (usually in a form of thin film) with a suitable optical transducer. These simple, inexpensive hydrogel biosensors are highly desirable, however, their practical applications have been hindered, largely because of their slow response. Here we show that quick response hydrogel sensors can be designed from ultrathin hydrogel films. By the adoption of layer-by-layer assembly, a simple but versatile approach, glucose-sensitive hydrogel films with thickness on submicrometer or micrometer scale, which is 2 orders of magnitude thinner than films used in ordinary hydrogel sensors, can be facilely fabricated. The hydrogel films can not only respond to the variation in glucose concentration, but also report the event via the shift of Fabry− Perot fringes using the thin film itself as Fabry−Perot cavity. The response is linear and reversible. More importantly, the response is quite fast, making it possible to be used for continuous glucose monitoring.

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the gel.16 Unfortunately, it remains a big challenge to fabricate an ultrathin hydrogel film that is still compatible with the chosen optical transducer. Here we show that quick-response hydrogel biosensors can be designed from ultrathin hydrogel films. Layer-by-layer assembly,17,18 a simple but versatile approach, was adopted for film fabrication. Hydrogel films with thickness on submicrometer or micrometer scale, which is 2 orders of magnitude thinner than films used in ordinary hydrogel sensors, can be facilely fabricated. They swell to a larger degree in the presence of glucose, showing good glucose sensitivity. In addition, they present Fabry−Perot fringes on their reflection spectra,19−21 thus report glucose-induced swelling via the shift of these fringes.(Scheme 1) The response is linear and reversible. More importantly, the response is quite fast, making it possible for the new sensor to be used for continuous glucose monitoring.

INTRODUCTION Stimuli-responsive hydrogels capable of undergoing sharp and reversible volume change in response to an external stimulus have found applications in many fields especially in biomedical area.1,2 Various novel biosensors have been designed by reporting an analyte-induced (de)swelling of a stimuliresponsive hydrogel (usually in the form of a thin film) with a suitable optical transducer.3−10 A typical example is the PCCA (polymerized crystalline colloidal array) sensor designed by Asher et al.,3,6,7 which is composed of a stimuli-responsive hydrogel film and a highly ordered crystalline colloidal array embedded inside. Analyte-induced (de)swelling of the film is read out from the shift of the Bragg diffraction of the crystalline colloidal array. Another hydrogel sensor was fabricated by incorporation of a reflection hologram into a hydrogel film.4,8 Similarly, changes in the diffraction wavelength of the hologram indicate the presence of a specific analyte that induces the hydrogel swelling. These simple, inexpensive biosensors are highly desirable, however, their practical applications have been hindered, largely because of their slow response.5,11 When PCCA glucose sensor is used as an example, it takes over 90 min for the sensor to respond to the introduction of 1 mM β-D-glucose. (New compositions were developed later that respond in a few minutes.)11 The slow response is directly associated with the slow (de)swelling of the hydrogel film.5 It is well-known that fully (de)swelling of bulky gels usually take hours or even weeks.12,13 This problem has limited the application of hydrogels not only in sensing, but also in artificial organs, actuators and on−off switches.14 Although many methods have been proposed to speed the gel swelling,14 a straightforward way is to scale down the gel size,15 as it is well-known that the swelling rate is inversely proportional to the square of the characteristic dimension of © 2011 American Chemical Society

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EXPERIMENTAL SECTION

Materials. 3-Aminophenylboronic acid hemisulfate (APBA), (3aminopropyl) triethoxysilane (APTES), and 1-(3-dimethyl aminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) were purchased from Alfa Aesar. 2-Aminomethyl-5-fluorophenylboronic acid (AMFPBA) was purchased from Acros. Poly(vinylalcohol) (PVA; DP = 1750 ± 50), acryloyl chloride, acrylic acid (AA), acrylamide (AAm), 2,2′azo-bis-isobutyronitrile (AIBN), and other reagents were purchased from local providers. 3-(Acrylamido)phenylboronic acid (AAPBA) and 2-acrylamidomethyl-5-fluorophenylboronic acid (AAMFPBA) were synthesized from acryloyl chloride and APBA or AMFPBA according to ref 22. Synthes is of Poly[ac rylamide-co-3-(acrylamido)phenylboronic acid] ([P(AAm-AAPBA)]) and Poly[acrylamideReceived: September 12, 2011 Revised: October 21, 2011 Published: December 4, 2011 92

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Scheme 1. (A) Shift of Fabry-Perot Fringes as a Result of the Analyte-Induced Swelling of the Hydrogel Film; (B) Covalent Bonding between P(AAm-AAPBA) and PVA and Breakage of the Bondage by Glucose

co-2-acrylamidomethyl-5-fluorophenylboronic acid] ([P(AAmAAMFPBA)]). P(AAm-AAPBA) was synthesized by free radical polymerization. Briefly, 0.50 g of AAm, 0.15 g of AAPBA and 4.0 mg of AIBN were dissolved in 40 mL of DMF. The mixture was purged with nitrogen for 30 min to remove dissolved oxygen, and then heated to 70 °C to initiate the free radical polymerization under nitrogen bubbling. The reaction was allowed to proceed for 12 h. The product was precipitated in acetone, filtered, washed three times with acetone, and dried under vacuum. The yield was about 60%. The content of the PBA groups in the copolymer was determined to be 10 mol % from the peak integration ratio between the benzene protons and the methylene protons of the main chain. The Mn and Mw were measured by GPC to be 26500 and 65000, respectively. Copoylmer P(AAm- AAMFPBA) was synthesized by copolymerization of AAm and AAMFPBA similarly. Film Fabrication. The hydrogel films were fabricated on silicon wafers or quartz slides. Before use, the substrates were cleaned in boiling piranha solution (3:7 v/v H2O2−H2SO4 mixture), rinsed thoroughly with deionized (DI) water, and dried. To introduce amino groups, they were immersed in a 1 wt % toluene solution of (3aminopropyl)triethoxysilane for 12 h, washed in toluene for 2 min, and dried at about 100 °C in the oven. A layer of poly(acrylic acid) was first assembled by immersing the substrates in a 0.1 wt % aqueous solution of poly(acrylic acid) (pH 3.0) for 10 min followed by washing with DI water. PBA groups were introduced by treating the substrates in an aqueous solution containing 7.5 mM APBA and 12.5 mM EDC for 4 h. The substrates were then immersed in 0.1 wt % solutions of PVA and P(AAm-AAPBA) (in 0.050 M pH8.5 phosphate buffer) alternately, each for 4 min, intermediated with water washing. Films with various thicknesses were fabricated by repeating the deposition cycles. Loosely bound materials were removed by soaking in 0.050 M pH 8.5 phosphate buffer containing 10 mM glucose for 150 h. Glucose Sensing. The experimental setup for glucose determination was shown in Scheme 2. In a typical experiment, the hydrogel film fabricated on silicon wafer was first immersed in phosphate buffer. The reflection spectra of the film were measured with a fiber optic spectrometer. After swelling equilibrium was reached, the solution was

Scheme 2. Experimental Setup for Glucose Determination

withdrawn, and new buffer containing various concentration of glucose was added. Glucose-induced film swelling was detected from the changes in reflection spectra of the film. Before use, the glucose solutions were all left overnight to ensure mutarotation was complete. The temperature of the sample cell was controlled with a refrigerated circulator. Characterization. Reflection spectra of the hydrogel films (using silicon wafer as substrate) were measured with AvaSpec-2048 Fiber Optic spectrometer. UV−vis absorption spectra of the hydrogel films (using quartz slide as substrate) were measured on a TU 1810PC UV−vis spectrophotometer (Purkinje General, China). 1H NMR spectra of P(AAm-AAPBA) were recorded on a Varian UNITY-plus 400 NMR spectrometer using D2O as solvent. GPC measurement was carried out on a Viscotek TDA302. 93

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Figure 3. (A) Reflection spectra of a 60-bilayer PVA/P(AAm-AAPBA) film in 0.05 M pH 8.5 phosphate buffer containing various concentration of glucose; T = 25 °C. (B) Calculated optical path length as a function of glucose concentration.

Figure 1. Reflection spectra of PVA/P(AAm-AAPBA) films with various bilayer numbers. The plots are shifted along the vertical axis for clarity. (B) Calculated OPL of PVA/P(AAm-AAPBA) films as a function of bilayer number.

between the two polymers as driving force (Scheme 1B), as we previously reported.25 The formation of phenylboronate ester bond in the LBL film has been confirmed previously by the appearance of the marker mode of boronate ester in IR spectra of the film.25 As shown in Figure 1A, the reflection spectra of the PVA/ P(AAm-AAPBA) LBL films display oscillations in the UV, visible, and near-IR range we examined. These peaks are known as Fabry−Perot fringes, which stem from the interferences between beams reflected at the air−film and film−substrate interfaces (Scheme 1A).19 The film thickness θ and the optical path length (OPL = ne·θ, where ne is the refractive index) can be calculated from the two adjacent wavelengths, λp and λp+1, for which the absorbance is maximal, using the following relationship: θ=

Figure 2. Reflection spectra of a 50-bilayer PVA/P(AAm-AAPBA) film measured in air (dried film) and in water (swollen film). The substrate is silicon wafer.

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1 2ne(1/λ p − 1/λ p + 1)

OPL = ne θ =

RESULTS AND DISCUSSION

or

1 2(1/λ p − 1/λ p + 1)

(1)

Figure 1B plots OPL of the film against the bilayer number. A good linear relationship was found, indicating the film grows linearly at least in the range we studied. The resultant films swell when soaked in an aqueous solution, that is, they are hydrogel in nature. As shown in Figure 2, the PVA/P(AAm-AAPBA) film still present Fabry− Perot fringes when immersed in water. Compared with the spectra measured in air with dried film, the amplitude of the oscillations is reduced significantly. As mentioned above, Fabry−Perot fringes stem from the interferences between beams reflected at the air−film and film−substrate interfaces

We used the so-called layer-by-layer (LBL) assembly in which a pair of polymers with complementary functionalities are alternately deposited onto a solid surface to fabricate ultrathin hydrogel films. Using this method, films with a thickness down to several nanometers can be facilely fabricated.23 By introducing a sensing moiety, films sensitive to certain analyte can be designed.24 Here we fabricated glucose-sensitive hydrogel films from poly(vinylalcohol) (PVA) and poly[acrylamide-co-3-(acrylamido)phenylboronic acid] (P(AAmAAPBA)) using the covalent phenylboronate ester bonding 94

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Scheme 3. Binding of Glucose with PBA Groups in the Filma

a

It may bind with free PBA groups as indicated by the solid arrows, or compete with PVA as indicated by the empty arrows.

Figure 4. Change in optical path length Δ(OPL) of a 60-bilayer PVA/ P(AAm-AAPBA) film with time upon stepwise increasing and decreasing glucose concentrations. Glucose concentrations (in mM) are indicated at the equilibrium OPL. Other conditions are the same with Figure 3.

(Scheme 1A). The amplitude of the oscillations depends on how effectively light is reflected at the two interfaces, which is further dependent on the refractive index difference.26 When the film is transferred from air into water, the refractive index of the medium increases from 1.0 (air) to 1.33 (water), while the refractive index of the film is expected to decrease as a result of the swelling of the film in water (see below). Therefore, the difference in refractive index at the water−film interface decreases significantly, resulting in a dramatic decrease in the amount of light reflected. Although there will be a slight increase in the amount of light reflected at the film−substrate interface because of the reduced refractive index of the film, the combined result is a significant reduced amplitude of the Fabry−Perot fringes. Fortunately, these fringes are still very clear, making it a suitable tool for optical transducing. A second change in the reflection spectra is that more fringes appear when the film is soaked in water (Figure 2). According to eq 1, OPLs of the dried and wet film were calculated to be ∼1112 and 2949 nm, respectively, clearly indicating the swelling of the film in water. Note there will be a slight decrease in the refractive index of the film as a result of the film swelling, therefore, the film thickness increase is more than 165% upon immersion in water. In other words, the swelling degree of the film is more than 165%. Next we study if glucose can alter the swelling degree of the hydrogel film and if the glucose-induced swelling be reported by Fabry−Perot fringes. As we reported previously, the PVA/ P(AAm-AAPBA) film disassembles slowly in aqueous solutions, because the phenylboronate ester bonding is reversible.25 The presence of glucose accelerates the film disassembly as it competes with PVA for binding with P(AAm-AAPBA).

Figure 5. (A) Glucose-induced changes in optical path length of a 40bilayer PVA/P(AAm-AAPBA) film measured in 0.05 M phosphate buffer with various pHs at 25 °C. (B) Glucose-induced changes in the optical path length of a 60-bilayer PVA/P(AAm-AAPBA) film measured in 0.05 M pH 8.5 phosphate buffer at 25 and 37 °C, respectively. (C) Glucose-induced changes in optical path length of a 30- (□), 60- (○), and 90-bilayer (△) PVA/P(AAm-AAPBA) film measured in 0.05 M, pH 8.5, phosphate buffer at 25 °C, respectively. 95

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(Scheme 3). It may bind with the free PBA groups and convert them from a neutral, hydrophobic form to a negatively charged, hydrophilic form.6,28,29 The increased charge density of the P(AAm-AAPBA) polymer is favorable for film swelling. More importantly, glucose may compete with PVA for the PBA binding sites (Scheme 1B). As a result, the cross-link density of the film decreases.7,11 In both ways, the binding of glucose with PBA groups in the film results in a larger swelling of the film. For an ideal sensor, linear response is highly desirable because it leads to a simple calibration and, more importantly, a constant sensitivity and precision over the entire linear range. As shown in Figure 3B, a good linear relationship between OPL of the film and glucose concentration was found. In addition, the glucose-induced film swelling is totally reversible. As shown in Figure 4, OPL of the film increases with increasing glucose concentration while it decreases with decreasing glucose concentration. The reversibility of the sensor will make it a good candidate for continuous blood glucose monitoring in diabetes. The effects of pH and temperature on glucose detection were shown in Figure 5A and B, respectively. As shown in Figure 5A, the sensitivity of the film increases with increasing pH in the pH range we studied. The highest sensitivity toward glucose was observed at pH9, while at physiological pH a rather low glucose-sensitivity was observed. Similar pH-dependent glucose-sensitivity has been reported previously for other PBAbased glucose-sensitive materials.28,29 As shown in Scheme 1B, PBA used here is a weak acid with a pKa of 8.2.30 Both the undissociated, planar trigonal structure and the dissociated, tetrahedral structure can react reversibly with glucose; however, only the tetrahedral structure can form a stable complex.31 As a result, the film only exhibits good glucose-sensitivity at relatively high pHs. By using a PBA with a lower pKa (5fluorophenylboronic acid),8 good glucose-sensitivity can be achieved at physiological pH (Figure 3S). In contrast to the significant effect of pH, the effect of temperature is much smaller. The response of a 60-bilayer film to glucose was measured at 25 and 37 °C, respectively (Figure 5B). Only a slight decrease in glucose sensitivity was observed when the temperature rises from 25 to 37 °C. The result is reasonable considering that both polymers do not show thermosensitivity over the temperature range we studied. The slight decrease in glucose sensitivity at 37 °C may be explained by a slightly reduced solubility of the polymers at a higher temperature. Figure 5C shows the effect of film thickness on glucose detection. Upon the addition of the same concentration of glucose, films with a larger bilayer number and, therefore, a larger thickness display larger changes in optical path length. Therefore, it will be an effective way to improve the sensitivity of the sensor by using thicker films. As the hydrogel films used here have a thickness on the submicrometer and micrometer scale, which are 2 orders of magnitude thinner than the films used in other hydrogel sensors (e.g., the film thickness is 125 μm in Asher’s PCCA sensor11), the new sensors are expected to have a much faster response. Figure 6A shows the response of a 30-bilayer film to the addition of 2 mM glucose. The characteristic time to obtain 1/e of the total change in optical length, τ, was obtained from the single exponential fit of the data. At 25 °C, τ is determined to be only 0.47 min, while an even shorter τ of 0.27 min was obtained at 37 °C. To the best of our knowledge, this is the fastest response for hydrogel-based glucose sensors.

Figure 6. (A) Response kinetics of a 30-bilayer PVA/P(AAm-AAPBA) film upon addition of 2 mM glucose. The data were measured in 0.050 M, pH 8.5, phosphate buffer at 25 (○) and 37 °C (□), respectively. The solid lines show the best single exponential fits to the data. (B) Characteristic times for 30-, 60-, and 90-bilayer PVA/P(AAm-AAPBA) films in response to the addition of 2 mM glucose. The data were measured in 0.050 M, pH 8.5, phosphate buffer.

Therefore, the stability of the films should be improved before they are used for glucose sensing. Two efforts were taken to obtain stable films. The first one is to increase the molar ratio of AAPBA in P(AAm-AAPBA) polymer from ∼5% in the previous study to ∼10% in the present study. With increasing AAPBA ratio in P(AAm-AAPBA), more covalent bonds will form between the two polymers, thus, results in a more stable film.27 A second effort is to remove the loosely bound chains by immersing the film in glucose solution. As shown in Figure 1S, when immersed in glucose solution, film material loss was observed. It is fast at the beginning, but slows down gradually with time. Little film material loss was found after a 150 h immersion, indicating the remaining film is stable. In the following work, all films were treated in this way before any further test. As shown in Figure 2S, the thickness of a treated film remains unchanged during a 2 h test, confirming high stability of the film. The glucose-responsivity of the resultant PVA/P(AAmAAPBA) films was then tested. As shown in Figure 3A, Fabry−Perot fringes of the film shift as glucose concentration increases. From the reflection spectra, OPL of the film was calculated and plotted against glucose concentration (Figure 3B). As expected, OPL increases with increasing glucose concentration. It is well-known that glucose, as a 1,2-diol, can bind with the PBA group to form boronate ester.6,28,29 In the present case, glucose may react with PBA groups in the film in two ways 96

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(9) Nakayama, D.; Takeoka, Y.; Watanabe, M.; Kataoka, K. Simple and precise preparation of a porous gel for a colorimetric glucose sensor by a templating technique. Angew. Chem., Int. Ed. 2003, 42, 4197−4200. (10) Lee, Y. J.; Pruzinsky, S. A.; Braun, P. V. Langmuir 2004, 20, 3096−3106. (11) Ben-Moshe, M.; Alexeev, V. L.; Asher, S. A. Anal. Chem. 2006, 78, 5149−5157. (12) Matsumoto, A.; Kurata, T.; Shiino, D.; Kataoka, K. Macromolecules 2004, 37, 1502−1510. (13) Kaneko, Y.; Sakai, K.; Kikuchi, A.; Yoshida, R.; Sakurai, Y.; Okano, T. Macromolecules 1995, 28, 7717−7723. (14) Zhang, X.; Xu, X.; Cheng, S.; Zhuo, R. Soft Matter 2008, 4, 385−391. (15) Xing, S.; Guan, Y.; Zhang, Y. Macromolecules 2011, 44, 4479− 4486. (16) Tanaka, T.; Fillmore, D. J. J. Chem. Phys. 1979, 70, 1214−1218. (17) Decher, G. Science 1997, 277, 1232−1237. (18) Tang, Z. Y.; Wang, Y.; Podsiadlo, P.; Kotov, N. A. Adv. Mater. 2006, 18, 3203−3224. (19) Guan, Y.; Yang, S. G.; Zhang, Y. J.; Xu, J.; Han, C. C.; Kotov, N. A. J. Phys. Chem. B 2006, 110, 13484−13490. (20) Lin, W.; Guan, Y.; Zhang, Y. J.; Xu, J.; Zhu, X. X. Soft Matter 2009, 5, 860−867. (21) Zhang, W.; Zhang, A.; Guan, Y.; Zhang, Y.; Zhu, X. X. J. Mater. Chem. 2011, 21, 548−555. (22) Shiomori, K.; Ivanov, A. E.; Galaev, I. Y.; Kawano, Y.; Mattiasson, B. Macromol. Chem. Phys. 2004, 205, 27−34. (23) Zhang, X.; Chen, H.; Zhang, H. Y. Chem. Commun. 2007, 1395−1405. (24) Ding, Z. B.; Guan, Y.; Zhang, Y. J.; Zhu, X. X. Polymer 2009, 50, 4205−4211. (25) Ding, Z. B.; Guan, Y.; Zhang, Y.; Zhu, X. X. Soft Matter 2009, 5, 2302−2309. (26) Yang, S. G.; Tan, S. X.; Zhang, Y. J.; Xu, J.; Zhang, X. L. Thin Solid Films 2008, 516, 4018−4024. (27) Jay, J. I.; Langheinrich, K.; Hanson, M. C.; Mahalingam, A.; Kiser, P. F. Soft Matter 2011, 7, 5826−5835. (28) Kataoka, K.; Miyazaki, H.; Bunya, M.; Okano, T.; Sakurai, Y. J. Am. Chem. Soc. 1998, 120, 12694−12695. (29) Zhang, Y. J.; Guan, Y.; Zhou, S. Q. Biomacromolecules 2006, 7, 3196−3201. (30) Matsumoto, A.; Yoshida, R.; Kataoka, K. Biomacromolecules 2004, 5, 1038−1045. (31) Nishiyabu, R.; Kubo, Y.; James, T. D.; Fossey, J. S. Chem. Commun. 2011, 47, 1106−1123.

We also studied the effect of film thickness on the response speed of the sensor. As shown in Figure 6B, thicker films respond slower, confirming again the key role of film thickness in the determination of response speed. Note the sensitivity is higher for thicker films, as we mentioned above. Therefore, when choosing film thickness, one should balance its opposite effects on response speed and sensitivity. In all cases, the sensor responds faster at 37 °C than at 25 °C, which may be attributed to faster diffusion of the polymer network at a higher temperature.

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CONCLUSIONS We fabricated a quick response optical glucose-sensor based on ultrathin, glucose-sensitive hydrogel film. These films, with thickness on the submicrometer and micrometer scale, were fabricated by layer-by-layer assembly. Both dried and swollen films present Fabry−Perot fringes on their reflection spectra. They swell to a larger degree in the presence of glucose. The glucose-induced swelling can be read out optically from the shift of Fabry−Perot fringes. Thanks to their thin thickness, the new sensor responses quite fast. This quick response sensor may have potential in real-time, continuous glucose monitoring. A weakness of the present design is its relatively low signal. As we have shown, the signal is strong enough for glucose sensing, however, from the viewpoint of application, a stronger signal will be better. We are now making efforts to improve the signal of the sensor by optimizing the film composition.

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ASSOCIATED CONTENT S Supporting Information * Disassembly of untreated film, film stability, and improved glucose-sensitivity at physiological pH. 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].

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ACKNOWLEDGMENTS We appreciate the financial support for this work from the National Natural Science Foundation of China (Grant Nos. 20974049, 20974050, and 2117407) and the Ministry of Sc i e n c e a n d T e c h n o l o g y o f C h i n a ( G r a n t N o . 2007DFA50760).

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