Logic Catalytic Interconversion of G-Molecular Hydrogel - ACS

Subscriber access provided by READING UNIV

Article

Logic Catalytic Interconversion of G-Molecular Hydrogel Ruibo Zhong, Mingshu Xiao, Changfeng Zhu, Xizhong Shen, Qian Tang, Weijia Zhang, Lihua Wang, Shiping Song, Xiangmeng Qu, Hao Pei, Cheng Wang, and Li Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b17926 • Publication Date (Web): 16 Jan 2018 Downloaded from http://pubs.acs.org on January 16, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Logic Catalytic Interconversion of G-Molecular Hydrogel Ruibo Zhong†1,2,7, Mingshu Xiao†2, Changfeng Zhu1*, Xizhong Shen1,6, Qian Tang2, Weijia Zhang3, Lihua Wang4, Shiping Song4, Xiangmeng Qu2, Hao Pei2, Cheng Wang5 and Li Li2* 1

Department of Gastroenterology, Zhongshan Hospital, Fudan University, Shanghai, 200032,

P.R China. 2

Shanghai Key Laboratory of Green Chemistry and Chemical Processes, School of Chemistry

and Molecular Engineering, East China Normal University, 500 Dongchuan Road, Shanghai, 200241, P. R. China 3

Institutes of biomedical sciences and Zhongshan Hospital, Fudan University, Shanghai

200032, P.R. China. 4

Division of Physical Biology & Bioimaging Center, Shanghai Synchrotron Radiation

Facility, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, P. R. China 5

Department of Nuclear Medicine, Renji Hospital, Shanghai Jiao Tong University School of

Medicine, Shanghai 200127, P. R. China 6

Shanghai Institute of Liver Diseases, Zhongshan Hospital, Fudan University, Shanghai,

China 7

Department of Biomedical Engineering, School of Basic Medical Sciences, Guangzhou

Medical University, Guangzhou, P. R. China

1 Environment ACS Paragon Plus

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

KEYWORDS: Artificial enzyme hydrogel, catalytic interconversion, G-quadruplex, Pb2+ detection, logic gate. ABSTRACT: By incorporating hemin into G-quadruplex (G4) during cation-templated self-assembly between guanosine and KB(OH)4, we have constructed an artificial enzyme hydrogel (AEH)-based system for the highly sensitive and selective detection of Pb2+. The sensing strategy is based on a Pb2+-induced decrease in AEH activity. Due to the higher efficiency of Pb2+ for stabilizing G4 compared with K+, the Pb2+ ions substitute K+ and trigger hemin release from G4, thus giving rise to a conformational interconversion accompanied by the loss of enzyme activity. The Pb2+-induced catalytic interconversion endows the AEH-based system with high sensitivity and selectivity for detecting Pb2+. As a result, the AEH-based system shows an excellent response for Pb2+ in the range from 1 pM to 50 nM with a limit of detection of ~0.32 pM, which is much lower than that of the previously reported G4-DNAzyme. We also demonstrate that this AEH-based system exhibits high selectivity toward Pb2+ over other metal ions. Furthermore, two two-input INHIBIT logic gates have been constructed via switching of the catalytic interconversion induced by K+ and Pb2+ or K+ and pH. Given its versatility, this AEH-based system provides a novel platform for sensing and biomolecular computation.


Page 2 of 25

Page 3 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION Natural enzymes play significant roles in biological reactions in living systems due to their remarkable activities, specificity, and efficiency1. However, some intrinsic drawbacks (e.g., laborious preparation, ease of denaturation, nominal thermal stability, high price, and poor reusability)2-3 make them unattainable in large-scale production. To tackle these problems, extensive studies have been conducted on the construction of artificial enzymes4-7. Compared with natural enzymes, artificial enzymes possess a variety of merits in terms of easy preparation, high stability, low cost, long-term storage, resistance to harsh conditions, and tunable activity8-10. Moreover, a handful of successful paradigms have been synthesized11-15. In general, there are two approaches to prepare artificial enzymes2: (1) designing a proper platform whose basic constituents feature the same functionalities as the enzyme, and (2) encapsulation of enzymes or their active-site synthetic analogues in suitable matrices. Nevertheless, the catalytic activities of artificial enzymes are much weaker and the turnover number (TON) is much lower than those of natural enzymes16-17. For example, the absence of a defined binding site causes artificial enzymes to react more slowly than natural enzymes18. Therefore, it is highly desirable, but challenging, to construct artificial enzymes with high selectivity and catalytic activity. Soft materials (e.g., hydrogels), capable of functioning under aqueous conditions, have sparked growing interest thanks to their potential applications in controlled drug release19,20, bioassays21, and tissue engineering22. Most hydrogels are composed of synthetic polymers or biopolymers via chemical or physical crosslinks. With good stability, biocompatibility, and softness, hydrogels have emerged as promising materials in biological applications23-26. In addition, tremendous research has revealed that hydrogels are capable of functioning as enzyme-encapsulated matrices27-30, in which the stability and activity of enzymes can be maintained for a long time. However, these artificial enzymes are constructed on the basis of



natural enzymes, which are susceptible to the intrinsic drawbacks of natural enzymes, to a certain degree. According to previous work, combinations of guanosine-quadruplex (G4) DNA with hemin have been demonstrated to function as artificial enzymes mimicking the oxidative function of peroxidase31-36. Additionally, these artificial enzymes also exhibit excellent biocompatibility and catalytic activity. On the other hand, G4 hydrogels with good physical properties can be easily prepared via the bridge connection of borate between G437-39. Inspired by these findings, we hypothesized that the constructed artificial enzyme hydrogel (AEH), based on the self-assembly of G-quartet combined with hemin, could work as a catalyst with high activity similar to peroxidase. Moreover, based on the induction of catalytic interconversion by conformation interconversion, we designed this AEH as a highly sensitive and selective sensor for Pb2+ detection.

Figure 1. Schematic illustration of catalytic interconversion of AEH for Pb2+ detection. As the schematic illustration shows in Figure 1, a guanosine-borate monoester was initially formed from the reaction between guanosine (G) and 0.5 equivalent of B(OH)3. The cation-templated assembly of the G4-quartet motif was induced upon addition of K+ ions. Thereafter, this G4-quartet motif acts as the building block to construct the hydrogel. The


Page 4 of 25

Page 5 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


simultaneous incorporation of hemin in the stacking of G4-quartets gave rise to a hydrogel with an enzyme-like property. Consequently, this AEH showed enhanced catalytic activity, in which H2O2/TMB oxidation was chosen as a model reaction. It should be noted that Pb2+ has a higher efficiency for stabilizing G4 compared with K+ due to its shorter M-O and O-O bonds40-42. As a result, these compact structures deriving from Pb2+ substitution of K+ caused simultaneous loss of hemin upon addition of Pb2+ in the system43, which resulted in conformational interconversion. It is proposed that such conformational transition is accompanied with a remarkable change in the catalytic activity of the AEH. That is to say, conformational interconversion induces catalytic interconversion. Hence, we have constructed an AEH-based sensor for sensitive and selective detection of Pb2+ by exploiting the property of this catalytic interconversion.

EXPERIMENTAL SECTION Chemicals. Hemin (bovine) was purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China). The metal salts (Pb(Ac)2, CdCl2, CrCl3, FeCl3, MgCl2, CaCl2, Zn(Ac)2, and CuCl2), TMB, and 30% H2O2 were provided by Aladdin Co., Ltd. (Shanghai, China). All reagents were used without further purification. The stock solution of hemin (10 mM) was prepared in DMSO, stored in the dark at -20 °C, and diluted to the required concentration with aqueous buffer.

Instrumentation. The absorption spectra of TMB oxidized by H2O2 were recorded with a Cary 60 scan UV/Vis spectrophotometer (Agilent) at room temperature. Circular dichroism spectroscopic measurements of 10 vt% microgel were carried out by JASCO J-815.

Preparation of the AEH. A typical procedure to prepare the AEH is as follows. The desired amount of guanosine was weighed into a vial before the appropriate amount of B(OH)3 solution (and water, if necessary) was added. Then, the mixture was sonicated for



approximately 30 s, followed by addition of the appropriate amount of KOH solution. The suspension was heated to 95 °C in a water bath until G was dissolved and the solution was clear. Subsequently, hemin was added with a final concentration of 100 mM after the solution was cooled to ~60 °C. For the preparation of microgels, a microfluidic water-in-oil emulsion approach was employed to segment a pre-gel aqueous phase into uniform building blocks44, in which one flow contains G and borate and the other flow contains K+ ions (Figure 2a). The mixture was microencapsulated using a droplet microfluidic device with HFE-7500 fluorocarbon oil containing 1.5% (wt/wt) fluorinated surfactant as the oil phase (continuous phase). The aqueous and oil phases were subsequently injected into the microfluidic device via a syringe. To guarantee the formation of the AEH, the microgel was stabilized for 2 h at room temperature before use. The microgels were prepared with a ratio of 2:1 of G:KB(OH)4 unless otherwise stated.

Kinetic experiments for the oxidation of TMB. The kinetic experiments for the oxidation of TMB were conducted using a 400 µL H2O2/TMB substrate solution mixed with 100 µL of AEH. In addition, the absorbance at 652 nm was monitored with a spectrophotometer to obtain the rate of oxidation of TMB.

The stability of the AEH in different pH buffers. The pH dependence of H2O2/TMB catalysis was tested using a 10 mM HCl-HEPES-Tris buffer spanning range of pH 2-10; the absorbance at 652 nm was measured by a spectrophotometer.

Detection of Pb2+ ions. For Pb2+ detection, an HEPES buffer (pH=7.4) was used as a unified buffer in this work. Typically, a variety of different 10 µL Pb2+ solutions (1 pM to 1 µM) were mixed with 10 µL of AEH for 10 min, and an H2O2/TMB substrate solution was quickly added to initiate the oxidation reaction. Following this, the absorption spectra were obtained at room temperature. The number of AEH-based microgel particles was calculated


Page 6 of 25

Page 7 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


as approximately 3×105 based on the average size of gel particles (~40 µm) and the sample volume typically used for Pb2+ detection (10 µL). For the selectivity study, other metal ions instead of Pb2+ were mixed with the AEH and the H2O2/TMB substrate, and the absorption spectra were subsequently obtained. UV-Vis spectroscopic study for devising a logic gate. Four experimental setups were used to explore the possibility of devising a logic gate: (a) vial containing water (200 µL), (b) vial containing hemin-incorporated K+ gel in water (200 µL), (c) vial containing K+ gel incubated in Pb(NO3)2 solution (1×10-4 M, 200 µL) and hemin, and (d) vial containing hemin-incorporated Pb2+ gel in water (200 µL). These sets were then treated with H2O2 (176 mM, 40 µL) for 15 minutes, followed by TMB solution (0.08 M, 40 µL). Thereafter, the absorbance spectrum of each set was tested with a UV-Vis spectrophotometer at room temperature. RESULTS AND DISCUSSION

Figure 2. The preparation of G4 hydrogel. (a) Schematic illustration of microgel formation using a microfluidic water-in-oil emulsion system. (b) Representative image of microgel droplets in flow after generation. Scale bar: 100 µm. (c) UV-Vis absorption spectra of the



TMB/H2O2 system in the presence of the AEH with different concentrations of K+. (d) The normalized absorbance at 652 nm. Before constructing the AEH-based sensor, it was necessary to confirm that the AEH was integral for gelation. Accordingly, we took advantage of a microfluidic water-in-oil emulsion approach to segment a pre-gel aqueous phase into uniform building blocks44, in which one flow contains G and borate and the other flow contains K+ ions (Figure 2a). The representative image in Figure 2b shows the formed AEH particles with a size of ∼40 µm (Figure 2b). Note that a range of microgel sizes with good dispersity can be created by regulating the flow rates and devising the geometry of the microfluidic channel45. Given the scalable production advantages offered by microfluidic devices46-47, including fast rates for thermal and mass transfer in a relatively small volume, low consumption of reagents, parallel synthetic channels, and continuous production, we expect that the AEH particles can also be potentially produced on larger scales. Circular dichroism (CD) spectroscopy, a useful tool to distinguish the configuration and conformation of asymmetric molecules48, was subsequently used to characterize the AEH. As shown in Figure S1a, the CD signals at 240 and 260 nm confirm the formation of the hydrogel, which is in good agreement with previous work37-38, 49. Taken together, we obtained an AEH using an easy preparation and low-cost approach. Next, the AEH was evaluated for its catalytic activity under different concentrations of K+, in which H2O2/TMB oxidation was chosen as a model reaction. By monitoring the oxidation of TMB, we found that the absorbance at 652 nm initially increased with increasing concentration of K+ (Figure 2c), suggesting not only that is the AEH is able to catalyze the H2O2/TMB oxidation but also that more AEH particles are formed. However, the continuous addition caused a decrease in absorbance when the concentration of K+ is greater than 50 mM (Figure 2d). This phenomenon is attributed to the increased formation of monoesters, rather


Page 8 of 25

Page 9 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


than diesters, favored by excess borate, resulting in the generation of fewer AEH particles37, wherein the catalytic activity of the AEH becomes lower. Stability and catalytic efficiency are two important factors for successful catalysts; therefore, assays of stability and catalytic efficiency in buffers with different pH values have been carried out. After storage for a long time at room temperature (e.g., 28 days), we found that the AEH still maintained its catalytic activity, while horseradish peroxidase (HRP) lost almost all activity (Figure S1b), indicating good stability of this AEH. Because the monoester crosslinks mainly depend on hydrogen bonds, both acid and alkali conditions may affect the formation of hydrogen bonds and thus induce the change in G4/hemin structure. Therefore, we hypothesized that the catalytic activity of the AEH is affected by pH. As shown in Figure S3a, we indeed observed a correlation between the catalytic activity of the AEH and the buffer pH Moreover, the catalytic efficiency reached a maximum when the buffer pH was 8, at which the hydrogen bonds are likely more stable. Notably, the optimized catalytic activity of the AEH under physiological conditions holds great promise for in vivo applications. Increasing evidence has shown that Pb2+-stabilized G4 with a more compact structure contributes to a higher efficiency of Pb2+ for stabilizing G4 compared with K+ 50-53. Moreover, 1 equivalent of Pb2+ can sufficiently stabilize G4, whereas millimolar amounts of K+ are required to obtain the same effect41. As shown in Figure 3a, K+ was substituted by Pb2+ once Pb2+ was added into the K+-stabilized G4 system, thus leading to a conformation change of the AEH. As shown in Figure S4, such responses to Pb2+ can be visually observed. The CD spectrum in Figure 3b shows a conformation transition after introducing Pb2+ into AEH, which is in good agreement with previous reports41. Notably, K+ favors hemin binding to DNAzyme, thus promoting DNAzyme activity, whereas Pb2+ triggers hemin release from DNAzyme, giving rise to the deactivation of the DNAzyme. Therefore, the conformational



interconversion discussed above certainly leads to the loss of AEH activity—namely, catalytic interconversion occurs.

Figure 3. Utilization of the AEH for colorimetric Pb2+ analysis in the TMB/H2O2 system. (a) Schematic of Pb2+-induced conformational interconversion for label-free colorimetric detection of Pb2+. (b) CD spectra of the AEH in the presence or absence of Pb2+. c) UV-Vis absorption spectra with different concentrations of Pb2+. d) Dependence of the absorbance at 652 nm (A652) on the Pb2+ concentration (10-12 M, 5×10-12 M, 10-11 M, 5×10-11 M, 10-10 M, 5×10-10 M, 10-9 M, 5×10-9 M, 10-8 M, 5×10-8 M, 10-7 M, 5×10-7 M, and 10-6 M). The inset shows a linear range from 10-12 to 5×10-8 M. Since AEH activity can be represented in the form of absorbance at 652 nm in the TMB/H2O2 oxidation system, we studied the AEH activity under different concentrations of Pb2+ by monitoring the absorbance at 652 nm. The UV-Vis spectra in Figure 3c display a gradual decrease in AEH activity with increasing concentration of Pb2+. In addition, we found that the absorbance at 652 nm shows a linear response over the range from 1 pM to 50 nM (R2 = 0.995; Figure 3d). The detection limit was calculated to be ~0.32 pM, which is 2~5 orders of magnitude lower than that of the previously reported G4-DNAzyme54-57 (Table 1). This improvement in sensitivity is likely caused by the increased freedom of the guanosines in the G-quartet to allow faster replacement of K+ and hemin by Pb2+ in the AEH compared


Page 10 of 25

Page 11 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


with guanines in the G4-DNAzyme that are bound by phosphodiester bonds and ribose. This is also demonstrated by the slightly higher enzyme efficiency of the AEH compared with G4-DNAzyme (Figure S2). Given these results, it is concluded that the presence of Pb2+ in the AEH is capable of inducing catalytic interconversion. Our constructed AEH-based sensor shows sensitive detection of Pb2+. Table 1. Comparison of G4-DNAzyme-based sensors for Pb2+ detection. Detection method

Linear range

LOD

Ref.

Colorimetric detection

1 pM to 50 nM

~0.32 pM

This work


20 to 200 nM

14 nM

58


0.1 µM to 10 µM

32 nM 55

Chemiluminescence detection

1 nM to ∼0.316 µM

1 nM

Electrochemical detection

1.0 nM to 1.0 µM

0.4 nM

59

Fluorescence

0 to 25 µM

3.4 µM

56

Fluorescence

5 nM to 1 µM

1 nM

60

Fluorescence

50 pM to 50 nM

22.8 pM

61

Fluorescence

20 nM to 1 µM

5 nM

54

Fluorescence

2 to 50 nM

400 pM

62

Fluorescence

0 to 1000 nM

0.4 nM

57

To assess our AEH-based sensor for the specific detection of Pb2+, a selectivity assay for Pb2+ analysis was carried out, in which other common metal ions (e.g. Na+, Ca2+, Mg2+, Li+, Zn2+, Hg2+, Fe3+, Cu2+, and Cr3+) were adopted in place of Pb2+ and added into the AEH-catalyzed TMB/H2O2 system (Figure 4a). It was found that these ions do not show an obvious change in absorbance, even at the 100 nM level. In comparison with other ions, the



presence of equal amounts of Pb2+ greatly decreased the absorbance (Figure 4b). It is noted that Hg2+ might inhibit the G4-DNAzyme activity as a result of the formation of T-Hg2+-T base pairs63. However, as the AEH has no T residues, it is impossible for Hg2+ to interact with the AEH and thus inhibit enzyme activity in our AEH-catalyzed TMB/H2O2 system. Taking these findings into consideration, we concluded that the AEH-based sensor has a high selectivity for Pb2+ over other metal ions. The high sensitivity and specificity of the AEH-based sensor may pave the way for practical sample analysis. To explore the feasibility of our AEH-based sensor in environmental analysis, we applied it to real lake water samples. Because no Pb2+ was detected in the lake water sample, the standard addition method was used to evaluate the practicality of the developed approach. All the water samples were spiked with Pb2+ at different concentration levels. As shown in Figure S5, the recoveries of Pb2+ were over the range of 91.7-98.3%, suggesting that the AEH-based sensor can be potentially applied to Pb2+ detection in environmental water samples.


Page 12 of 25

Page 13 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. The selectivity of the AEH-based Pb2+ sensor with various metal ions. (a) The absorbance at 652 nm with different metal ions within range from 1 pM to 50 nM. (b) Normal absorbance at 652 nm of the AEH-based Pb2+ sensor with 50 nM ions. On the basis of the above results, we built two logic gates with the AEH (Figure 5). As mentioned above, K+ favors hemin binding over DNAzyme, which promotes DNAzyme activity. In contrast, the presence of Pb2+-substituted K+ causes hemin release from DNAzyme, resulting in the deactivation of the DNAzyme. That is, the catalytic interconversion can be exploited to switch the AEH into two states: “active” and “inactive”. The design is in accord with a two-input INHIBIT logic gate behavior41. Because the absorbance at 652 nm (Abs652) can reflect enzyme activity, we defined a threshold of A652 = 0.3 for output 1 or 0. Figure 5a shows that the output was 1 when K+ was added alone; otherwise, it was 0, implying that the K+−Pb2+-switched AEH indeed works as a two-input INHIBIT logic gate. According to the defined threshold, the AEH remains active between pH 6 and 9 but is inactive outside this range. As a result, the K+−pH-switched AEH could also operate as a two-input INHIBIT logic gate (Figure 5b). These observations shows that our AEH system has great potential for biomolecular computation.



Figure 5. Operation of the AEH as a two-input INHIBIT logic gate. (a) K+−Pb2+-switched AEH INHIBIT logic gate. Absorbance changes at 652 nm in the form of a bar representation with a threshold of A652 = 0.3 for output 1 or 0 and truth table for the two-input INHIBIT logic gate. Four input models: 1. AEH without K+, hemin, H2O2 and TMB; 2. AEH without K+, Pb2+, hemin, H2O2 and TMB; 3. AEH with K+, hemin, H2O2 and TMB; 4. AEH with K+, Pb2+, hemin, H2O2 and TMB. All the reactions were carried out at pH 8. (b) K+−pH-switched AEH INHIBIT logic gate. Absorbance changes at 652 nm in the form of a bar representation with a threshold of A652 = 0.3 for output 1 or 0 and truth table for the two-input INHIBIT logic gate. Four input models: 1. AEH without K+, hemin, H2O2 and TMB, pH 8; 2. AEH without K+, hemin, H2O2 and TMB, pH 2; 3. AEH with K+, hemin, H2O2 and TMB, pH 8; 4. AEH with K+, hemin, H2O2 and TMB, pH 10. CONCLUSION In summary, we have developed an AEH-based sensor for Pb2+ detection with high sensitivity and selectivity based on catalytic interconversion. Compared with G4-DNAzyme 14 Environment ACS Paragon Plus

Page 14 of 25

Page 15 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


for Pb2+ detection, this AEH-based sensor possesses several distinctive advantages. First, it provides a facile method to construct AEH-based sensors by simply incorporating hemin into G4-quartets during the cation-templated self-assembly between G and KB(OH)4. Second, because K+ favors hemin binding to DNAzyme, the AEH-based sensor exhibits higher catalytic activity than G4-DNAzyme and shows excellent long-term stability (e.g., 28 days). Third, substituting K+ with Pb2+ causes hemin release from DNAzyme, resulting in catalytic interconversion that contributes to the high sensitivity and selectivity of this AEH sensor. Furthermore, by switching the G4 self-assembly induced by K+ and Pb2+, or K+ and pH, two two-input INHIBIT logic gates have been constructed. Given its versatility, it is envisioned that this AEH-based system can provide a novel platform for applications in sensing, biomolecular computation, and drug delivery. ASSOCIATED CONTENT Supporting Information. This material is available free of charge on the ACS Publications website. CD spectra of the AEH and catalytic efficiency change of the AEH and HRP during 28 d storage; kinetics curves for the TMB/H2O2 reactions catalyzed by the AEH and G4-DNAzyme in the presence of different H2O2 concentrations; UV-Vis absorption spectra of the AEH in different pH buffers with the TMB/H2O2 system and the normalized A652 of the AEH in different pH buffers; photographs of the TMB/H2O2 oxidation system catalyzed by the AEH with and without Pb2+; recovery rate of Pb2+ in the lake water sample (PDF). AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; [email protected]. Tel.: (+86) 021-54345484. Author Contributions



†

Page 16 of 25

These authors contributed equally.

Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the National Science Foundation of China (grant numbers 21722502, 21605026, 21505045, 21705048, 81672720), the Shanghai Pujiang Talent Project (16PJ1402700),

and

the

China

Postdoctoral

Science

Foundation

(2015M581565,

2017T100283). REFERENCES (1) Berglund, G. I.; Carlsson, G. H.; Smith, A. T.; Szoke, H.; Henriksen, A.; Hajdu, J. The Catalytic Pathway of Horseradish Peroxidase at High Resolution. Nature 2002, 417, 463-468. (2) Nath, I.; Chakraborty, J.; Verpoort, F. Metal Organic Frameworks Mimicking Natural Enzymes: A Structural and Functional Analogy. Chem. Soc. Rev. 2016, 45, 4127-4170. (3) Hudson, S.; Cooney, J.; Magner, E. Proteins in Mesoporous Silicates. Angew. Chem. Int. Edit. 2008, 47, 8582-8594. (4) Chen, Z.; Xu, L.; Liang, Y.; Zhao, M. pH-Sensitive Water-Soluble Nanospheric Imprinted Hydrogels Prepared as Horseradish Peroxidase Mimetic Enzymes. Adv. Mater. 2010, 22, 1488-1492. (5) Ikeda, M.; Tanida, T.; Yoshii, T.; Kurotani, K.; Onogi, S.; Urayama, K.; Hamachi, I. Installing Logic-Gate Responses to A Variety of Biological Substances in Supramolecular Hydrogel–Enzyme Hybrids. Nat. Chem. 2014, 6, 511-518.


Page 17 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(6) Hsu, C.-L.; Lien, C.-W.; Wang, C.-W.; Harroun, S. G.; Huang, C.-C.; Chang, H.-T. Immobilization of Aptamer-Modified Gold Nanoparticles on BiOCl Nanosheets: Tunable Peroxidase-Like Activity by Protein Recognition. Biosens. Bioelectron. 2016, 75, 181-187. (7) Chen, P.; Pan, D.; Fan, C.; Chen, J.; Huang, K.; Wang, D.; Zhang, H.; Li, Y.; Feng, G.; Liang, P.; He, L.; Shi, Y. Gold Nanoparticles for High-Throughput Genotyping of Long-Range Haplotypes. Nat. Nanotechnol. 2011, 6, 639-644. (8) Rebilly, J.-N.; Colasson, B.; Bistri, O.; Over, D.; Reinaud, O. Biomimetic Cavity-Based Metal Complexes. Chem. Soc. Rev. 2015, 44, 467-489. (9) Kirkorian, K.; Ellis, A.; Twyman, L. J. Catalytic Hyperbranched Polymers as Enzyme Mimics; Exploiting the Principles of Encapsulation and Supramolecular Chemistry. Chem. Soc. Rev. 2012, 41, 6138-6159. (10) Yao, G.; Li, J.; Chao, J.; Pei, H.; Liu, H.; Zhao, Y.; Shi, J.; Huang, Q.; Wang, L.; Huang, W.; Fan, C. Gold-Nanoparticle-Mediated Jigsaw-Puzzle-like Assembly of Supersized Plasmonic DNA Origami. Angew. Chem. Int. Edit. 2015, 54, 2966-2969. (11) Chao, F.-A.; Morelli, A.; Iii, J. C. H.; Churchfield, L.; Hagmann, L. N.; Shi, L.; Masterson, L. R.; Sarangi, R.; Veglia, G.; Seelig, B. Structure and Dynamics of A Primordial Catalytic Fold Generated by in Vitro Evolution. Nat. Chem. Biol. 2013, 9, 81-83. (12) Qi, L.; Xiao, M.; Wang, X.; Wang, C.; Wang, L.; Song, S.; Qu, X.; Li, L.; Shi, J.; Pei, H. DNA-Encoded Raman-Active Anisotropic Nanoparticles for microRNA Detection. Anal. Chem. 2017, 89, 9850-9856. (13) Zhao, Y.; Chen, F.; Zhang, Q.; Zhao, Y.; Zuo, X.; Fan, C. Polymerase/nicking enzyme synergetic isothermal quadratic DNA machine and its application for one-step amplified biosensing of lead (II) ions at femtomole level and DNA methyltransferase. NPG Asia Mater. 2014, 6, e131.



(14) Zhu, X.; Lin, Z.; Chen, L.; Qiu, B.; Chen, G.; A sensitive and specific electrochemiluminescent sensor for lead based on DNAzyme. Chem. Commun. 2009, 6050-6052. (15) Zhao, Y.; Liu, H.; Chen, F.; Bai, M.; Zhao, J.; Zhao, Y. Trifunctional molecular beacon-mediated quadratic amplification for highly sensitive and rapid detection of mercury(II) ion with tunable dynamic range Biosens. Bioelectron. 2016, 86, 892-898. (16) Lewis, J. C. Artificial Metalloenzymes and Metallopeptide Catalysts for Organic Synthesis. ACS Catal. 2013, 3, 2954-2975. (17) Pei, H.; Lu, N.; Wen, Y. L.; Song, S. P.; Liu, Y.; Yan, H.; Fan, C. H. A DNA Nanostructure-based Biomolecular Probe Carrier Platform for Electrochemical Biosensing. Adv. Mater. 2010, 22, 4754-4758. (18) Dydio, P.; Key, H.; Nazarenko, A.; Rha, J.-E.; Seyedkazemi, V.; Clark, D.; Hartwig, J. An Artificial Metalloenzyme with the Kinetics of Native Enzymes. Science 2016, 354, 102-106. (19) Qiu, Y.; Park, K. Environment-Sensitive Hydrogels for Drug Delivery. Adv. Drug. Deliv. Rev. 2001, 53, 321-339. (20) Nan, J. Y.; Chen, Y.; Li, R. T.; Wang, J. F.; Liu, M. H.; Wang, C. P.; Chu, F. X. Polymeric Hydrogel Nanocapsules: A Thermo and pH Dual-responsive Carrier for Sustained Drug Release. Nano-Micro Lett. 2014, 6, 200-208. (21) Holtz, J. H.; Asher, S. A. Polymerized Colloidal Crystal Hydrogel Films as Intelligent Chemical Sensing Materials. Nature 1997, 389, 829-832. (22) Lutolf, M. P.; Hubbell, J. A. Synthetic Biomaterials as Instructive Extracellular Microenvironments for Morphogenesis in Tissue Engineering. Nat. Biotechnol. 2005, 23, 47-55. 18 Environment ACS Paragon Plus

Page 18 of 25

Page 19 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(23) Ryu, J.-H.; Chacko, R. T.; Jiwpanich, S.; Bickerton, S.; Babu, R. P.; Thayumanavan, S. Self-Cross-Linked Polymer Microgels: A Versatile Nanoscopic Drug Delivery Platform. J. Am. Chem. Soc. 2010, 132, 17227-17235. (24) Zhang, X.; Malhotra, S.; Molina, M.; Haag, R. Micro- and Microgels with Labile Crosslinks-From Synthesis to Biomedical applications. Chem. Soc. Rev. 2015, 44, 1948-1973. (25) Qu, X.; Wang, S.; Ge, Z.; Wang, J.; Yao, G.; Li, J.; Zuo, X.; Shi, J.; Song, S.; Wang, L.; Li, L.; Pei, H.; Fan, C. Programming Cell Adhesion for On-Chip Sequential Boolean Logic Functions. J. Am. Chem. Soc. 2017, 139, 10176-10179. (26) Pei, H.; Liang, L.; Yao, G.; Li, J.; Huang, Q.; Fan, C. Reconfigurable Three-Dimensional DNA Nanostructures for the Construction of Intracellular Logic Sensors. Angew. Chem. Int. Edit. 2012, 124, 9154-9158. (27) Singh, S.; Topuz, F.; Hahn, K.; Albrecht, K.; Groll, J. Embedding of Active Proteins and Living Cells in Redox-Sensitive Hydrogels and Microgels through Enzymatic Cross-Linking. Angew. Chem. Int. Edit. 2013, 52, 3000-3003. (28) Qu, X.; Zhu, D.; Yao, G.; Su, S.; Chao, J.; Liu, H.; Zuo, X.; Wang, L.; Shi, J.; Wang, L.; Huang, W.; Pei, H.; Fan, C. An Exonuclease III-Powered, On-Particle Stochastic DNA Walker. Angew. Chem. Int. Edit. 2017, 56, 1855-1858. (29) Zhu, D.; Song, P.; Shen, J. W.; Su, S.; Chao, J.; Aldalbahi, A.; Zhou, Z.; Song, S. P.; Fan, C. H.; Zuo, X. L.; Tian, Y.; Wang, L. H.; Pei, H. PolyA-Mediated DNA Assembly on Gold Nanoparticles for Thermodynamically Favorable and Rapid Hybridization Analysis. Anal. Chem. 2016, 88, 4949-4954. (30) Yang, F.; Zuo, X.; Li, Z.; Deng, W.; Shi, J.; Zhang, G.; Huang, Q.; Song, S.; Fan, C. A Bubble-Mediated Intelligent Microscale Electrochemical Device for Single-Step Quantitative Bioassays. Adv. Mater. 2014, 26, 4671-4676.



(31) Yang, X.; Fang, C.; Mei, H.; Chang, T.; Cao, Z.; Shangguan, D. Characterization of G-Quadruplex/Hemin Peroxidase: Substrate Specificity and Inactivation Kinetics. Chem-Eur. J. 2011, 17, 14475-14484. (32) Li, W.; Li, Y.; Liu, Z.; Lin, B.; Yi, H.; Xu, F.; Nie, Z.; Yao, S. Insight into G-Quadruplex-Hemin DNAzyme/RNAzyme: Adjacent Adenine as the Intramolecular Species for Remarkable Enhancement of Enzymatic Activity. Nucleic Acids Rev. 2016, 44, 7373-7384. (33) Qu, X.; Yang, F.; Chen, H.; Li, J.; Zhang, H.; Zhang, G.; Li, L.; Wang, L.; Song, S.; Tian, Y.; Pei, H. Bubble-Mediated Ultrasensitive Multiplex Detection of Metal Ions in Three-Dimensional DNA Nanostructure-Encoded Microchannels. ACS Appl. Mater. Inter. 2017, 9, 16026-16034. (34) Zhu, X.; Gao, X.; Liu, Q.; Lin, Z.; Qiu, B.; Chen, G. Pb2+-introduced activation of horseradish peroxidase (HRP)-mimicking DNAzyme. Chem. Commun. 2011, 47, 7437-7439. (35) Shen, J. L.; Xu, L. F.; Wang, C. P.; Pei, H.; Tai, R. Z.; Song, S. P.; Huang, Q.; Fan, C. H.; Chen, G. Dynamic and Quantitative Control of the DNA-Mediated Growth of Gold Plasmonic Nanostructures. Angew. Chem. Int. Edit. 2014, 53, 8338-8342. (36) Pei, H.; Zuo, X. L.; Zhu, D.; Huang, Q.; Fan, C. H. Functional DNA Nanostructures for Theranostic Applications. Acc. Chem. Res. 2014, 47, 550-559. (37) Peters, G. M.; Skala, L. P.; Plank, T. N.; Oh, H.; Manjunatha Reddy, G. N.; Marsh, A.; Brown, S. P.; Raghavan, S. R.; Davis, J. T. G4-Quartet·M+ Borate Hydrogels. J. Am. Chem. Soc. 2015, 137, 5819-5827. (38) Peters, G. M.; Skala, L. P.; Plank, T. N.; Hyman, B. J.; Manjunatha Reddy, G. N.; Marsh, A.; Brown, S. P.; Davis, J. T. A G4·K+ Hydrogel Stabilized by an Anion. J. Am. Chem. Soc. 2014, 136, 12596-12599.


Page 20 of 25

Page 21 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(39) Qu, X.; Li, M.; Zhang, H.; Lin, C.; Wang, F.; Xiao, M.; Zhou, Y.; Shi, J.; Aldalbahi, A.; Pei, H.; Chen, H.; Li, L. Real-Time Continuous Identification of Greenhouse Plant Pathogens Based on Recyclable Microfluidic Bioassay System. ACS Appl. Mater. Inter. 2017, 9, 31568-31575. (40) Kotch, F. W.; Fettinger, J. C.; Davis, J. T. A Lead-Filled G-Quadruplex: Insight into the G-Quartet's Selectivity for Pb2+ over K+. Org. Lett. 2000, 2, 3277-3280. (41) Li, T.; Wang, E.; Dong, S. Potassium−Lead-Switched G-Quadruplexes: A New Class of DNA Logic Gates. J. Am. Chem. Soc. 2009, 131, 15082-15083. (42) Pei, H.; Wan, Y.; Li, J.; Hu, H. Y.; Su, Y.; Huang, Q.; Fan, C. H. Regenerable Electrochemical Immunological Sensing at DNA Nanostructure-Decorated Gold Surfaces. Chem. Commun. 2011, 47, 6254-6256. (43) Bhattacharyya, T.; Kumar, Y. P.; Dash, J. Supramolecular Hydrogel Inspired from DNA Structures Mimics Peroxidase Activity. ACS Biomater. Sci. Eng. 2017, 3, 2358-2365. (44) Qu, X.; Zhang, H.; Chen, H.; Aldalbahi, A.; Li, L.; Tian, Y.; Weitz, D. A.; Pei, H. Convection-Driven Pull-Down Assays in Nanoliter Droplets Using Scaffolded Aptamers. Anal. Chem. 2017, 89, 3468-3473. (45) Griffin, D. R.; Weaver, W. M.; Scumpia, P. O.; Carlo, D. D.; Segura, T. Accelerated Wound Healing by Injectable Microporous Gel Scaffolds Assembled from Annealed Building Blocks. Nat. Mater. 2015, 14, 737-744. (46) Kim, Y. H.; Zhang, L.; Yu, T.; Jin, M.; Qin, D.; Xia, Y. Droplet-based microreactors for continuous production of palladium nanocrystals with controlled sizes and shapes. Small 2013, 9, 3462-3647. (47) Zhang, L.; Wang, Y.; Tong, L.; Xia, Y. Synthesis of colloidal metal nanocrystals in droplet reactors: the pros and cons of interfacial adsorption. Nano Lett. 2014, 14, 4189-4194. 21 Environment ACS Paragon Plus


(48) Greenfield, N. J. Using Circular Dichroism Spectra to Estimate Protein Secondary Structure. Nat. Protoc. 2006, 1, 2876-2890. (49) Chen, L.; Chao, J.; Qu, X.; Zhang, H.; Zhu, D.; Su, S.; Aldalbahi, A.; Wang, L.; Pei, H. Probing Cellular Molecules with PolyA-Based Engineered Aptamer Nanobeacon. ACS Appl. Mater. Inter. 2017, 9, 8014-8020. (50) Stefan, L.; Xu, H.-J.; Gros, C. P.; Denat, F.; Monchaud, D. Harnessing Nature’s Insights: Synthetic Small Molecules with Peroxidase-Mimicking DNAzyme Properties. Chem-Eur. J. 2011, 17, 10857-10862. (51) Qi, L.; Xiao, M.; Wang, F.; Wang, L.; Ji, W.; Man, T.; Aldalbahi, A.; Naziruddin Khan, M.; Periyasami, G.; Rahaman, M.; Alrohaili, A.; Qu, X.; Pei, H.; Wang, C.; Li, L. Poly-Cytosine-Mediated Nanotags for SERS Detection of Hg2+. Nanoscale 2017, 9, 14184-14191. (52) Ye, D. K.; Zuo, X. L.; Fan, C. H. DNA Nanostructure-Based Engineering of the Biosensing Interface for Biomolecular Detection. Prog Chem 2017, 29, 36-46. (53) Ge, Z. L.; Pei, H.; Wang, L. H.; Song, S. P.; Fan, C. H. Electrochemical Single Nucleotide Polymorphisms Genotyping on Surface Immobilized Three-Dimensional Branched DNA Nanostructure. Sci. China Chem. 2011, 54, 1273-1276. (54) Li, T.; Dong, S. J.; Wang, E. K. A Lead(II)-Driven DNA Molecular Device for Turn-On Fluorescence Detection of Lead(II) Ion with High Selectivity and Sensitivity. J. Am. Chem. Soc. 2010, 132, 13156-13157. (55) Li, T.; Wang, E.; Dong, S. Lead(II)-Induced Allosteric G-Quadruplex DNAzyme as a Colorimetric and Chemiluminescence Sensor for Highly Sensitive and Selective Pb2+ Detection. Anal. Chem. 2010, 82, 1515-1520.


Page 22 of 25

Page 23 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(56) Xiang, Y.; Tong, A.; Lu, Y. Abasic Site-Containing DNAzyme and Aptamer for Label-Free Fluorescent Detection of Pb2+ and Adenosine with High Sensitivity, Selectivity, and Tunable Dynamic Range. J. Am. Chem. Soc. 2009, 131, 15352-15357. (57) Li, C.; Liu, K.; Lin, Y.; Chang, H. Fluorescence Detection of Lead(II) Ions Through Their Induced Catalytic Activity of DNAzymes. Anal. Chem. 2011, 83, 225-230. (58) Zhang, Q.; Cai, Y.; Li, H.; Kong, D.-M.; Shen, H.-X. Sensitive Dual DNAzymes-Based Sensors Designed by Grafting Self-Blocked G-Quadruplex DNAzymes to the Substrates of Metal Ion-Triggered DNA/RNA-Cleaving DNAzymes. Biosens. Bioelectron. 2012, 38, 331-336. (59) Li, F.; Feng, Y.; Zhao, C.; Tang, B. Crystal Violet as A G-Quadruplex-Selective Probe for Sensitive Amperometric Sensing of Lead. Chem. Commun. 2011, 47, 11909-11911. (60) Guo, L.; Nie, D.; Qiu, C.; Zheng, Q.; Wu, H.; Ye, P.; Hao, Y.; Fu, F.; Chen, G. A G-Quadruplex Based Label-Free Fluorescent Biosensor for Lead Ion. Biosens. Bioelectron. 2012, 35, 123-127. (61) Zhao, Y.; Zhang, Q.; Wang, W.; Jin, Y. Input-Dependent Induction of G-Quadruplex Formation for Detection of Lead (II) by Fluorescent Ion Logic Gate. Biosens. Bioelectron. 2013, 43, 231-236. (62) Li, X.; Wang, G.; Ding, X.; Chen, Y.; Gou, Y.; Lu, Y. A "Turn-on" Fluorescent Sensor for Detection of Pb2+ Based on Graphene Oxide and G-Quadruplex DNA. Phy. Chem. Chem. Phy. 2013, 15, 12800-12804. (63) Li, T.; Dong, S.; Wang, E. Label-Free Colorimetric Detection of Aqueous Mercury Ion (Hg2+) Using Hg2+-Modulated G-Quadruplex-Based DNAzymes. Anal. Chem. 2009, 81, 2144-2149.




Page 24 of 25

Page 25 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table of Contents


Logic Catalytic Interconversion of G-Molecular Hydrogel - ACS

Recommend Documents