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Small-Molecule Triggered Cascade Enzymatic Catalysis in HourGlass Shaped Nanochannel Reactor for Glucose Monitoring Lei Lin, Jing Yan, and Jinghong Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac501983a • Publication Date (Web): 30 Sep 2014 Downloaded from http://pubs.acs.org on October 5, 2014

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Small-Molecule Triggered Cascade Enzymatic Catalysis in Hour-Glass Shaped Nanochannel Reactor for Glucose Monitoring

Lei Lin, Jing Yan, Jinghong Li* Department of Chemistry, Beijing Key Laboratory for Analytical Methods and Instrumentation, Tsinghua University, Beijing 100084, China.

*to whom corresponding should be addressed. Phone: 86-10-62795290; Fax: 86-10-62771149 Email: [email protected] 1

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Abstract Construction of an electrical signal-sensitive nanoreactor in response to small molecule remains a challenge in the developing fields of biomimetic device. Solid nanochannels are considered as promising candidates for constructing smart systems, which is highly sensitive to biochemical stimulus. Here, we report an hour-glass shaped nanochannel reactor based on cascade enzymatic catalysis and cation-selective nanochannel system. The employed glucose-specific dual-enzyme combination, glucose oxidase (GOx) and horseradish peroxidase (HRP), ensures the glucose catalytic efficiency and selectivity. Presence of glucose immediately induced the bienzymatic sequential reaction. The yielding gluconic acid decreased the micro-environmental pH in the channel gradually. Different concentration of glucose produced different amount of acid and thus altered the negative charge density inside the nanochannel to different extent. Modification convenience and mechanical robustness also ensure the stability of the test platform. Owing to its unique cation-selective property and high sensitivity toward micro-environmental alteration, this nanodevice shows robust glucose-responsive properties through monitoring ionic current signatures.

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Introduction Living systems utilize diverse electrical signal controlling systems to regulate a variety of essential cellular events, such as cell communication, nerve conduction1. A remarkable feature of those systems is that they endow cells with dynamic and sensitive response abilities to various intrinsic or environmental stimuli, e.g. pH, temperature, amino acid, glucose, oxygen etc.. This superior biological property has inspired the development of biomimetic concept and the following applications of various smart systems for sensing, gating, drug delivery and biomedicine2-4. In recent years, solid nanochannels are considered as promising candidates for constructing biomimetic systems to imitate diverse physiological processes within their limited but well-structured nano-zones5-8. Owing to their unique structure and high sensitivity toward micro-environmental change, they can promptly and directly convert the external stimulus into electrical signals through the stimuli-triggered alternation on physical properties of the nanochannels9. Nanochannels have emerged as an increasingly popular tool for protein interaction monitoring, bimolecular conformational investigation, molecular transport controlling, and biosensors10-12. Comparing with protein nanochannels, which still have to rely on the fragile lipid bilayer, solid-state nanochannel appears to be much more promising in practical applications for its mechanical and chemical robustness. Particularly, artificial nanochannels fabricated in track-etched polymer membranes attract a great deal of interest because of their excellent characteristics, such as well-defined dimensions and geometries, ease of miniaturization and compatibility with electronic measurement technique. Fabrication simplicity and modification convenience further promote the application of the nanochannel systems in the field of biomimetic materials13-16. Hou et al. developed a smart nanochannel device with pH-gating ion transport properties through asymmetric chemical modification of single nanochannels17.Jiang et al. also described a novel biomimetic nanochannel system utilizing DNA conformational switching, which was controlled by potassium concentration18. Unfortunately, despite its notable advantages, how to utilize 3

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nanochannel appropriately for developing metabolizable molecule-responsive platforms still remains in its early stage. In this work, we presented a novel hour-glass shaped nanochannel reactor for examining small molecular stimuli on the basis of cascade enzymatic catalysis and cation-selective nanochannel system. By using charge as a key discriminating factor, the functionalized nanochannel system exhibits high sensitivity toward charge distribution alteration inside the channel. The famous dual-enzyme catalytic combination, glucose oxidase (GOx) and horseradish peroxidase (HRP), was introduced to convert the external stimulus into readable signals promptly. Presence of glucose directly induced the bienzymatic sequential reaction. Accumulation of the yielding product gluconic acid gradually decreased the micro-environmental pH as well as the negative charge density inside the nanochannel, further leading to an obvious current decline of the nanochannel. Within this framework, integration of cascade enzymatic catalysis into double-conical nanochannel could create robust glucose-responsive devices.

Experimental Section Reagents. Glucose oxidase, horseradish peroxidase, 2-[N-morpholino]ethanesulfonic acid (MES), glucose anhydrous, D-fructose, D-(+)-mannose and D-galactose were obtained from Beijing DingGuo Biotech. Co.. N-Hydroxysuccinimide (NHS) and N-ethyl-N-(3-dimethylaminopropyl)carbodiimide (EDC) were gotten from Alfa Aesar. Ltd. Sodium hypochlorite (NaOCl) and potassium iodide (KI) were obtained from Beijing Lanyi Chemical Company. Polyimide membranes (PI; Hoechst, 12 µm thick, with single ion track in the center) were bought from GSI, Darmstadt. Other regents of analytical grade were obtained from Beijing Chemical Company (China). Current Measurements. The PI film was mounted between two chambers of the testing cell which were both filled with test buffer (100 mM KCl, pH 7.0). Transmembrane potential across 4

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the film was applied by placing two Pt electrodes into each half of the cell (Scheme S1 in the Supporting Information). Current measurement was carried out via a Keithley 2636A picoammeter (Keithley Instruments, Inc., Cleveland, OH). The scanning voltage used in this work was varied from -0.5 to +0.5 V. All the ion current signals were recorded and collected via ACS Basic 1.2 Software (Keithley Instruments, Inc., Cleveland, OH). The average current values at different voltages were obtained through three repeated tests. Preparation of the Double-Conical Nanochannel. The single nanochannel with symmetric and double-conical shape used in this work was fabricated through the ion track-etching technique as reported in previous literatures19-20. Generally, in order to gain a double-conical shaped nanochannel, chemical etching needs to be performed from both sides of the PI film using sodium hypochlorite as etching solution. Scanning voltage (+1 V) was applied to monitor the etching process. Once the nanochannel was opened, which can be reflected by the apparently increasing ion current, stopping solution (1 M potassium iodide) was immediately added to both sides of the cell to neutralize the etchant and thus quickly slow down the etching rate. The resulting PI membrane with single nanochannel was carefully washed with deionized water and stored in deionized water overnight before use. Diameter calculation of the nanochannel-tip is described in Supporting Information. Bienzyme Functionalization of the Single Nanochannel. To obtain bifunctional membrane, the as-prepared PI film was put into 0.1 M MES buffer (pH 6.0) containing 50 mM N-Hydroxysuccinimide (NHS) and 100 mM N-ethyl-N-(3-dimethylaminopropyl)carbodiimide (EDC) for 30 min to activate the carboxyl groups on the channel walls. After rinsing with deionized water, the resulted PI membrane was then reacted with 1 mg/mL glucose oxidase (GOx) and 0.55 mg/mL horseradish peroxidase (HRP) in 0.1 M MES buffer (pH 6.0) for 5 h at 25 °C. Finally, the functionalized PI membrane was gently washed with deionized water and then stored in the test buffer. pH-Dependent Ion Current Test and Procedure for Enzymatic Reaction. 5

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To investigate the pH influence on the ion transport properties of the double conical nanochannel, 100 mM KCl buffered from pH 3.0 to 7.0 was used for current measurement. In a typical enzymatic experiment, bienzyme functionalized PI film was first mounted between two chambers of the testing cell. After the addition of test buffer (100 mM KCl, pH 7.0, oxygen saturated, containing different concentration of glucose), current measurement was immediately applied. The graft ratio of GOx to HRP was optimized from 0.25 to 2. Interfering species analysis was conducted by adding 1 mM fructose, mannose and galactose into test buffer (100 mM KCl, pH 7.0, oxygen saturated), respectively. Following procedures were similar to the above.

Results and Discussion Design of the Glucose-Responsive Nanochannel Reactor. Scheme 1 illustrates the developed strategy of the glucose-responsive nanochannel reactor. Polyimide membrane (PI, 12 µm thick, with single ion track) has emerged as prime candidate for preparing single ion track pore with high aspect ratio in free-standing film. Chemical etching process generated plenty of carboxyl (-COOH) groups on the nanochannel walls. GOx and HRP were then successfully grafted on the surface of nanochannel by means of efficient amide reaction between carboxyl and amino groups. Modified nanochannel exhibited an attractive cation-selective behavior under neutral condition, which was endowed by the negative charge distribution on the inner channel wall and the immobilized protein. Consequently, the amplitude of the ion current directly depended on the negative charge density on the inner surface of the nanochannel. Addition of glucose started the bienzymatic sequential reaction quickly: GOx catalyzed the oxidation of glucose into gluconic acid and H2O2; H2O2 was further decomposed into H2O and O2 by the biocatalysis of HRP. The yielding gluconic acid gradually decreased the micro-environmental pH in the channel. Different concentration of glucose produced different amount of acid and thus altered the negative charge density inside the nanochannel to different extent. This glucose-induced microscopic change in charge distribution can be displayed promptly 6

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and apparently through ion current test using nanochannel system due to its sensitivity toward micro-environmental alteration. Thus more glucose were catalyzed, lower current signals were achieved, offering a quantitative readout of glucose.

Bienzymatic Functionalization of the Single Nanochannel. The double-conical nanochannel embedded in the single ion track-etched PI membrane was fabricated through a well-developed track-etching technique. This technique has been widely employed for the manufacture of particular polymer products with single nanopore. Tip (the narrowest center) diameter of the hour-glass shaped nanochannel was controlled by a commonly used electrochemical method20 and was calculated to be about 20 nm wide (Figure S1 in the Supporting Information). Chemical etching treatment produced large amount of carboxyl groups (-COOH) on the nanochannel walls21,22, providing adequate active sites for further immobilization of GOx and HRP. What’s more, efficient amide reaction between carboxyl and amino groups also contributed to the protein ligation efficiency23,24. The bienzymatic functionalization process was characterized by monitoring the ion transport properties of the nanochannel. Previous reports pointed out that on the condition of using the same transmembrane voltage and electrolyte ion concentration, the ion current of PI nanopore was mainly determined by its effective pore size25-27. It is observed from Figure 1A that bare nanochannel shows a linear current-voltage (I-V) curve without rectification effect, which is ascribed to its symmetrical shape and surface charge distribution (curve a)28. As a comparison, a decrease of ∼50% in the ionic current response was obtained after the bienzymatic modification (curve b). This phenomenon arises from the fact that covalently grafted protein monolayer partially blocks the inner nanochannel and thus underlies a visible reduction of the effective pore size29. Additionally, the bienzyme-decorated nanochannel also exhibited a linear current-voltage curve under the same condition, confirming the symmetric structure inside the channel after modification. We further recorded ATR-FTIR spectra of the membrane to get insight on the surface modification30. It is observed from Figure S2 that bienzyme-conjugated PI film shows one peak at 3290 cm-1, assigning the 7

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N—H/O—H stretching frequency of enzyme. Two characteristic peaks appear at 1544 cm-1 and 1648 cm-1 corresponding to the amide II (N—H bending) and amide I (—C=O) bands of the protein, respectively. The ATR-FTIR characteristics indicate the successful functionalization of bienzyme onto the inner surface of the double-conical nanochannel, which is consistent with the results obtained from the current measurements.

Ion Current Test of the pH-Responsive Nanochannel. It is assumed that the effective pore size of the nanochannel was dependent on two factors in this system: physical block and charge density inside the channel. Figure S3 in the Supporting Information demonstrates current-voltage (I-V) characteristics of the bare nanochannel in 0.1 M KCl solution buffered from pH 7.0 to 3.0. It is appeared that the conductance of the nanochannel gradually reduced along with the decreasing pH value. As mentioned above, the inner wall of the bare channel is full of carboxyl groups after chemical etching treatment. Thus it is highly negatively charged under the neutral condition because the pKa of the etching-resulted carboxyl moiety is estimated to be lower than 3.0. In other words, the carboxyl groups generated on the channel wall endow the nanochannel with a remarkable cation-selective property31-33, which is playing a key role in our system. Considering the dependence of negative charge density on pH, the tip of the nanochannel permeated K+ much more rapidly under higher pH conditions18, bringing on a higher ion current value. On the contrary, when pH gradually dropped around pKa of carboxyl group, due to the charge neutralization effect, negative charge density inside the nanochannel also decreased. Accordingly, lower conductance was obtained. After functionalization, the bienzyme-decorated nanochannel still maintained the pH-responsive capability. As can be seen in Figure 1B, the current-voltage (I-V) properties showed a similar variation trend with a smaller range ability than that before modification under the same test condition. It may be attributed to two main reasons. First, partial carboxyl moieties have been converted into neutral amide bonds after grafting reaction. Second, the isoelectric points of the newly introduced enzyme GOx and HRP are 4.2 and 7.2, 8

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respectively, which are both higher than that of the etching-resulted carboxyl group. This is also the reason why current-voltage (I-V) signals collected at pH 3.0 and 4.0 were quite close to each other. The results not only offer evidence for the successful enzyme immobilization on the inner channel surface, but also suggest that PI nanochannel is sensitive to pH-induced microscopic change in charge distribution.

Glucose Triggered Cascade Enzymatic Catalysis in Nanochannel Reactor. Based on the intriguing pH-responsive property of the nanochannel, two important enzymes, GOx and HRP were introduced into the channel system.The cascade enzymatic properties of the modified nanochannel reactor were investigated by measuring the ionic current across the channel in test buffer. In our strategy, we used signal decrease percent, which is defined as (I0-I)/I0, to evaluate the current change of nanochannel reactor in response to glucose stimuli, where I0 and I are the signal intensity measured before and after glucose addition at -0.5 V, respectively. Higher (I0-I)/I0 value not only means higher efficiency of the bienzymatic reaction, but also provides more excellent signal response towards glucose triggered cascade enzymatic catalysis. Figure 2A demonstrates that HRP-modified nanochannel showed no significant (line a) change in the ionic current when exposing to glucose-containing test buffer. After functionalized with both GOx and HRP, the addition of glucose leads to a nearly 60% decrease in the conductance of the nanochannel and finally reached a maximum within 20 min (line c),suggesting a tendency to complete the glucose-induced bienzymatic sequential reaction. It is well known that thermally stable GOx34-36,a flavin-containing glycoprotein, specifically catalyzes the oxidation of glucose into gluconic acid and hydrogen peroxide (H2O2) in the presence of oxygen as shown below:

The yielding gluconic acid gradually decreased the micro-environmental pH in the nanochannel reactor. Subsequent decline in the surface negative charge density further 9

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results in an obvious drop of the conductance. While, as compared to bienzyme-modified nanochannel reactor (line c), nanochannel functionalized with only GOx (line b in Figure 2A) showed much smaller change in the ionic current. It is reasoned that the catalytic efficiency of GOx is mainly influenced by two factors: insufficient oxygen supply for the reaction and H2O2 cumulation induced decay of the GOx activity37-39. Because O2 is continuously consumed during the enzymatic process, thus the gluconic acid output is greatly limited by the O2 level in the test buffer. However, by reason of the poor solubility in water, O2 dissolved amount is quite low compared with the glucose concentration. In addition, glucose oxidation product hydrogen peroxide will underlie the degradation of GOx activity with time. To minimize both of these two problems, HRP was employed into the nanochannel reactor by taking advantage of the following enzymatic reaction:

Successive consumption of H2O2 by HRP helps to remove the GOx inhibitor from the test system. Furthermore, HRP catalytic product oxygen also assists in meeting the oxygen requirement of the GOx enzymatic reaction to some extent. Figure 2A indicates that glucose triggered cascade enzymatic catalysis can be investigated by the as-proposed pH-responsive nanochannel reactor rapidly and accurately. As mentioned above, HRP plays a crucial role in the glucose-induced coupled enzymatic reaction. Combination of HRP ensures the oxidation activity of GOx and the productivity of gluconic acid. On the other hand, it is also noted that GOx still acts as the principal part of the sequential catalytic reaction. Consequently, graft proportion of GOx to HRP on the nanochannel walls turns out to be an important influencing factor of bienzymatic reaction efficiency. Figure 2B manifests that relatively low signal decrease percent appear whenever the graft ratio of GOx to HRP was much higher or lower. It may probably arise from the fact that less graft amount of HRP would lead to the accumulation of H2O2 inside the nanochannel and thus the decay of the GOx activity. Analogously, less modified GOx on the channel surface could directly reduce the gluconic acid yield as well as the pH decreasing magnitude. 10

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Accordingly, the optimal graft ratio of GOx to HRP was chosen to be 1:2.

Glucose-Responsive Capacity and Specificity of the Nanochannel Reactor. To investigate the glucose-responsive capacity of the nano-reactor, different amounts of glucose were adding to the test buffer (Supporting Information, Figure S4A) on the basis of the optimal experimental conditions. As can be seen in Figure 3A, the transmembrane ionic currents for bare channel exhibited no obvious changes upon treatment with glucose. However, when these measurements were repeated with the bienzyme-modified nanochannel, an obvious difference in the results was observed. Ionic currents decreased gradually with the increasing glucose concentrations varied from 15 to 100 µM. The dependence of signal decrease on the glucose concentration is described in Figures S4B. The linear relationship can be described as signal decrease (%) = 6.76+0.50 C with the correlation coefficient of R2 = 0.986, where C is the glucose concentration and signal decrease is defined as (I0-I)/I0 (I0 and I are the signal intensity measured before and 20 min after glucose addition at -0.5 V). Responsive specificity is also one important aspect of the nanochannel reactor. The selectivity of the bienzyme-functionalized nanochannel depends on the modified enzyme. It is well known that GOx possesses a very high specificity toward glucose comparing with any other sugars40. Experiments with interferences including 1 mM fructose, mannose and galactose were conducted to test the selectivity of the bienzymatic nanochannel platform. Figure 3B exhibits that as high as 1 mM control samples were investigated, however, no detectable current variation signal was observed under the same test condition. It is consistent with the high specificity of GOx for glucose41-42. The results indicate that the as-proposed bienzymatic nanochannel reactor can be applied for constructing robust glucose-responsive devices.

Reusability of the Nanochannel Reactor. Another unique feature of our nanochannel reactor is the switching effect. To 11

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verify this, the sensing device was challenged separately measuring the ion currents when it was exposed to three cycles of 100 µM glucose and buffer, respectively. It is manifest in Figure 4 that ionic current decreased whenever the presence of glucose at each cycle, confirming rapid response of bienzyme-functionalized nanochannel reactor to external stimulus. Additionally, the results also suggest that designed nanochannel platform could recover from stimuli-triggered physical alternation promptly without losing the bioactivity of the immobilized proteins during the repetitive experiments. Good switching property and short renewing time of the bienzymatic nanoreactor in circular stimulation indicated its great potential in biomimetic device development.

Conclusion In summary, a small-molecule triggered nanodevice based on bienzyme functionalized nanochannel system and cascade enzymatic catalysis has been developed. Combination of GOx and HRP ensures the catalytic efficiency and specificity. Different concentration of glucose produced different amount of gluconic acid and thus altered the micro-environmental pH inside the nanochannel to different extent. This glucose-induced microscopic change in charge distribution can be monitored conveniently and sensitively through ion current signals using the cation-selective nanochannel system. We believe that the small molecule-responsive nanochannel reactor holds a great potential in biological process researches, drug discovery and diagnostic applications.

Acknowledgement This work was financially supported by National Basic Research Program of China (No. 2011CB935704), the National Natural Science Foundation of China (No. 21235004, No. 21128005), the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement No. 260600 ("GlycoHIT"). 12

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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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(19) Kalman, E. B.; Vlassiouk, I.; Siwy, Z. S. Adv. Mater. 2008, 20, 293-297. (20) Xie, Y. B.; Wang, X. W.; Xue, J. M.; Jin, K.; Chen, L.; Wang, Y. G. Appl. Phys. Lett. 2008, 93, 163116. (21) Guo, W.; Cao, L. X.; Xia, J. C.; Nie, F. Q.; Ma, W.; Xue, J. M.; Song, Y. L.; Zhu, D. B.; Wang, Y. G.; Jiang, L. Adv. Funct. Mater. 2010, 20, 1339-1344. (22) Guo, W.; Xia, H. W.; Cao, L. X.; Xia, F.; Wang, S. T.; Zhang, G. Z.; Song, Y. L.; Wang, Y. G.; Jiang, L.; Zhu, D. B. Adv. Funct. Mater. 2010, 20, 3561-3567. (23) Jiang, Y. N.; Liu, N. N.; Guo, W.; Xia, F.; Jiang, L. J. Am. Chem. Soc. 2012, 134, 15395-15401. (24) Ali, M.; Tahir, M. N.; Siwy, Z.; Neumann, R.; Tremel, W.; Ensinger, W. Anal. Chem. 2011, 83, 1673-1680. (25) Siwy, Z. S. Adv. Funct. Mater. 2006, 16, 735-746. (26) Harrell, C. C.; Siwy, Z. S.; Martin, C. R. Small 2006, 2, 194-198. (27) Xia, F.; Guo, W.; Mao, Y.; Hou, X.; Xue, J.; Xia, H.; Wang, L.; Song, Y.; Ji, H.; Ouyang, Q.; Wang, Y.; Jiang, L. J. Am. Chem. Soc. 2008, 130, 8345-8350. (28) Ali, M.; Yameen, B.; Cervera, J.; Ramírez, P.; Neumann, R.; Ensinger, W.; Knoll, W.; Azzaroni, O. J. Am. Chem. Soc. 2010, 132, 8338-8348. (29) Nilsson, J.; Lee, J. R. I.; Ratto, T. V.; Letant, S. E. Adv. Mater. 2006, 18, 427-431. (30) Radhakumary, C.; Sreenivasan, K. Anal. Chem. 2011, 83, 2829-2833. (31) Cervera, J.; Schiedt, B.; Neumann, R.; Mafe, S.; Ramirez, P. J. Chem. Phys. 2006, 124, 104706. (32) Cervera, J.; Schiedt, B.; Ramirez, P. Europhys. Lett. 2005, 71, 35-41. (33) Siwy, Z.; Fulinski, A. Phys Rev Lett 2002, 89, 198103. (34) Jung, D. Y.; Magda, J. J.; Han, I. S. Macromolecules 2000, 33, 3332-3336. (35) Wen, Z.; Ci, S.; Li, J. J. Phys. Chem. C 2009, 113, 13482-13487. (36) Zhang, Q. A.; Qiao, Y.; Hao, F.; Zhang, L.; Wu, S. Y.; Li, Y.; Li, J. H.; Song, X. M. Chem. Eur. J. 2010, 16, 8133-8139. (37) Zhang, K.; Wu, X. Y. J. Controlled Release 2002, 80, 169-178. (38) Lucisano, J. Y.; Gough, D. A. Anal. Chem. 1988, 60, 1272-1281. 15

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Legends

Scheme 1 Schematic representation of the hour-glass shaped bienzymatic nanochannel reactor. Chemically etched double-conical nanochannel was first modified with GOx and HRP through the amide reaction between carboxyl groups on the nanochannel wall and amino groups of the protein. Presence of glucose in the test buffer directly induced the bienzymatic sequential reaction: GOx catalyzed glucose into gluconic acid and H2O2; H2O2 was further decomposed into H2O and O2 by the biocatalysis of HRP. Accumulation of the yielding gluconic acid gradually decreased the microenvironmental pH as well as the negative charge density inside the nanochannel, further leading to an obvious current decline of the cation-selective nanochannel. Figure 1. (A) Current-voltage (I-V) properties of the double-conical nanochannel embedded in chemically etched PI membrane before (a) and after (b) modification of GOx and HRP in the test buffer. (B) Current-voltage (I-V) characteristics of the bienzyme functionalized nanochannel in 0.1 M KCl buffered from pH 7.0 to 3.0.

Figure 2. (A) The time-dependent current changes of nanochannel modified with HRP (a), GOx (b), HRP and GOx (c) after the addition of 100 µM glucose. The current chagne is defined as signal decrease percent, i.e. (I0-I)/I0, where I0 and I are the signal intensity measured before and after glucose addition at -0.5 V, respectively. (B) Optimization of the graft proportion of GOx to HRP on the nanochannel walls. All the assays were carried out in the test buffer at room temperature.

Figure 3. (A) Dose response of bare (a) and bienzyme-functionalized nanochannel (b). (B) Selectivity analysis for glucose response. The analyte concentrations were as follows: 0.1 mM glucose, 1 mM fructose, 1 mM mannose and 1 mM galactose, respectively. 17

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Figure 4. Reversible variation of the ionic current measured at +0.5 V with (▲) and without (■) 100 µM glucose. The assays were all conducted in the test buffer at room temperature.

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Scheme 1

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Figure 1 1.5

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0.0 pH 7.0 pH 6.0

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Figure 2 60

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40 30 20 10 0 0.0

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Figure 3

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Figure 4 0 µM Glucose 100 µM Glucose

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0.5

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