Research Article Cite This: ACS Appl. Mater. Interfaces 2019, 11, 21859−21864
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Framework Nucleic Acid-Enabled Programming of Electrochemical Catalytic Properties of Artificial Enzymes Dongdong Zeng,‡ Lili San,† Fengyu Qian,‡ Zhilei Ge,§ Xiaohui Xu,‡ Bin Wang,‡ Qian Li,*,∥ Guifang He,† and Xianqiang Mi*,†,⊥ †
Shanghai Advanced Research Institute, Chinese Academy of Sciences, Shanghai 201210, China Shanghai Key Laboratory of Molecular Imaging, Shanghai University of Medicine & Health Sciences, Shanghai 201318, China § School of Chemistry and Chemical Engineering and ∥School of Chemistry and Chemical Engineering, Shanghai Jiao Tong University, Shanghai 200240, China ⊥ Key Laboratory of Functional Materials for Informatics, Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences, Shanghai 200050, China
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S Supporting Information *
ABSTRACT: The creation and engineering of artificial enzymes remain a challenge, especially the arrangement of enzymes into geometric patterns with nanometer precision. In this work, we fabricated a series of novel DNA-tetrahedronscaffolded-DNAzymes (Tetrazymes) and evaluated the catalytic activity of these Tetrazymes by electrochemistry. Tetrazymes were constructed by precisely positioning Gquadruplex on different sites of a DNA tetrahedral framework, with hemin employed as the co-catalyst. Immobilization of Tetrazymes on a gold electrode surface revealed horseradish peroxidase (HPR)-mimicking bioelectrocatalytic property. Cyclic voltammogram and amperometry were employed to evaluate the capability of Tetrazymes of different configurations to electrocatalyze the reduction of hydrogen peroxide (H2O2). These artificial Tetrazymes displayed 6- to 14-fold higher enzymatic activity than G-quadruplex/hemin (G4-hemin) without the DNA tetrahedron scaffold, demonstrating application potential in developing novel G-quadruplex-based electrochemical sensors. KEYWORDS: framework nucleic acid, DNA tetrahedral nanostructure, G-quadruplex, electrochemistry, enzymatic activity construction of complex biomolecular networks.14−19 The use of double helical DNA molecules for nanoscale engineering pursuit began with Seeman’s construction of artificial branched DNA tiles, where four rationally designed oligomeric nucleic acid strands self-assembled into an immobile four-way junction.20 Since then, structural DNA nanotechnology has enjoyed a rapid progress, and numerous complex nanostructures and different fabrication techniques have been introduced.21−23 The nanoscale addressability of DNA nanostructures makes it an intriguing approach for developing artificial enzyme complexes. To date, numerous examples of organizing chemical reactions with programmability by taking advantages of DNA nanotechnology have been reported.24−27 We have previously reported a rectangular DNA origami platform to anchor glucose oxidase (GOx) and horseradish peroxidase (HRP).24 We studied the distance effect on enzyme cascade efficiency and found out that the enzyme cascade efficiency can
1. INTRODUCTION To maintain complex metabolic pathways, nature uses compartmentalization and spatial organization of metabolically active units to separate specialized functions, control activity, and gain specificity. In cells, localization of enzymes at specific organelles has a profound effect on their spatial action and ultimately allows different metabolic pathways to operate simultaneously in close proximity but in different compartments.1−3 Artificial engineered enzymes have enormous potential in medical diagnosis and treatment as well as in industrial applications, albeit their creation and engineering remain a challenge, especially the arrangement of enzymes into geometric patterns with nanometer precision, as in natural systems.4−8 Molecular self-assembly is an elegant and powerful approach to pattern matter at the atomic scale, and there have been extensive studies on the development of self-assembling biomaterials.9−13 DNA is a self-assembling biopolymer that is directed by canonical Watson−Crick base pairing to form predictable, double helical secondary structures, which enables it to be one of the most promising biomolecules for the © 2019 American Chemical Society
Received: April 16, 2019 Accepted: May 20, 2019 Published: May 22, 2019 21859
DOI: 10.1021/acsami.9b06480 ACS Appl. Mater. Interfaces 2019, 11, 21859−21864
Research Article
ACS Applied Materials & Interfaces
outside, A1-inside, A1-outside, or V1-outside) was designed with an extended G-quadruplex structure sequence. The Tetrazyme was prepared by the following processes: equal amounts of oligonucleotides S1 (or S1-inside, S1-outside, A1-inside, A1-outside, V1-outside), S2, S3, and S4 were mixed in TMK buffer (20 mM Tris buffer, 50 mM MgCl2, and 50 mM KCl, pH 8.0) with 30 mM TCEP to reach a final concentration of 1 μM for each oligonucleotide. Annealing was performed by holding the mixture at 95 °C for 3 min followed by cooling to 4 °C for approximately 30 s using a BioRad thermal cycler PTC-100. The prepared DNA sample solutions were transformed into a freshly prepared 8% native polyacrylamide gel and was run at 80 V for 120 min in 1× TBE buffer. The gel was stained with GelRed and visualized under UV light. Circular dichroism (CD) measurements were performed as follows: oligonucleotides were diluted to 5 μM in potassium acetate (KAc) buffer (20 mM KAc, 70 mM KCl, pH 6.8). The spectra were obtained with a Chirascan CD Spectrometer (Applied Photophysics Ltd, UK). The wavelength was varied from 220 to 300 nm at 100 nm/min. The samples were measured at 20 °C with a square quartz cell with a 0.1 cm path length. The buffer spectrum was subtracted from each sample spectrum and smoothed. 2.3. Immobilization of Tetrazymes on Gold Electrodes. The gold electrodes (2 mm in diameter) were cleaned following a previous protocol.33 Tetrazymes (3 μL; 1 μM) were injected into cleaned gold electrodes and incubated overnight at room temperature for immobilization. Then, the electrodes with Tetrazymes were rinsed with 1× PBS washing buffer to remove nonspecifically adsorbed Tetrazymes and dried lightly with nitrogen gas and then immersed in 100 μL of 1 μM hemin solution for 30 min at 37 °C to obtain the Tetrazyme-based electrochemical sensor. For the control experiment, the cleaned gold electrodes were subjected to 1 μM of free G-quadruplex DNA in the TMK buffer overnight at room temperature, followed by treatment with MCH (1 mM) for 30 min to obtain well-aligned DNA monolayers. Eventually, the electrodes with free G-quadruplex DNA were rinsed with the washing buffer (1× PBS) to remove any remains of nonspecifically adsorbed and dried lightly with nitrogen gas and then immersed in 100 μL of 1 μM hemin solution for 30 min at 37 °C to obtain the G4hemin biosensor. Hexaammineruthenium(III) (Ruhex) was employed to quantify the surface density of free G-quadruplex immobilized on a gold electrode surface. Electrochemical characterization was performed in 10 mM Tris buffer (pH 7.4), which was continuously deoxygenated with nitrogen gas. 2.4. Electrochemical Characteristics of Tetrazymes. Electrochemical experiments were performed in deoxygenated TMK buffer containing various concentrations of H2O2 on a CHI760E Electrochemical Workstation (Chenhua Instrument Company of Shanghai, China). A three-electrode system was used in our system. It consisted of a modified gold electrode (2 mm in diameter) as the working electrode, an Ag/AgCl reference electrode, and a platinum wire auxiliary electrode. Cyclic voltammetry (CV) was conducted by monitoring the Tetrazymes at a scan rate of 100 mV/s ranging from −0.6 to 0.2 V, and the potential of amperometric current versus time was fixed at −0.4 V and the electroreduction current was measured at 100 s after the Tetrazymes redox reaction reached a steady state. The electrochemical measurements were carried out at room temperature.
be further promoted by rolling the rectangular DNA origami into a three-dimensional DNA nanotube. As one kind of artificial enzyme, G-quadruplex/hemin (G4hemin), formed by guanine-rich nucleic acid sequence fold into G-quadruplex complexing tightly with hemin, has characteristics of high operational stability, insensitivity to environmental conditions, and low costs in preparation than natural enzymes.28−30 Thus, it has been widely served as a stable catalytic label for various biosensors toward a variety of targets. However, the relatively low enzymatic activity of G4hemin enzyme due to weak binding affinity to substrates restricted the improvement of their catalytic efficiency. As a consequence, it is urgent to fabricate a new methodology with simplicity and convenience to enhance the enzymatic activities of hemin/G-quadruplex artificial enzyme. Recently, researchers have fabricated an artificial enzyme by grafting G-quadruplex/ hemin onto DNA tetrahedral nanostructures to increase the catalytic efficiency of G-quadruplex/hemin.31 However, the electrochemical catalytic efficiency of this DNA tetrahedronscaffolded artificial enzyme has not been explored. Herein, we evaluated the electrochemical catalytic property of DNA-tetrahedron-scaffolded-DNAzyme (Tetrazyme) and constructed an electrochemical hydrogen peroxide (H2O2) biosensor using Tetrazyme as signal probes. Specifically, we constructed a DNA tetrahedral framework as a scaffold to covalently attach the G4-hemin complex with atom solution spatial control on the gold electrode surface. Taking advantages of the steady DNA nanostructure and consistently favorable orientation of DNA tetrahedral scaffold, we were able to control the distance and orientation between the activity center and electrode surface. We further evaluated the electrochemical activity of different configurations of Tetrazymes. Our results show clear evidence of improved electrochemical reactivity of Tetrazymes, in which G4-hemin is immobilized on DNA tetrahedron carrier, and the spatial between activity center and electrode surface significantly affected the activity of Tetrazymes. On the basis of these studies, we developed a H2O2 biosensor with a linear range of 10−9000 μM and a detection limit of 3.0 μM.
2. EXPERIMENTAL PART 2.1. Materials and Apparatus. All oligonucleotides were synthesized and purified by Sangon Biological Engineering Technology & Services Co. Inc (Shanghai, China) with their sequences listed in Table S1. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), hemin, amino acid 4-(2-hydroxyethyl)-1-piperazineëthanesulfonic acid (HEPES), 6-mercaptohexanol (MCH), and hexaammineruthenium(III) (Ruhex) were purchased from SigmaAldrich (St. Louis, MO, USA). H2O2 (30%), Tris(hydroxymethyl)aminomethane (Tris), dimethyl sulfoxide (DMSO), and other reagents were obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). All reagents were used without further processing. The DNA tetrahedral nanostructure forming buffer was TMK buffer (20 mM Tris buffer, 50 mM MgCl2, and 50 mM KCl, pH 8.0). The washing buffer for gold electrodes was phosphate-buffered saline (1× PBS, 10 mM Na2HPO4, 2 mM KH2PO4, 137 mM NaCl, and 2.7 mM KCl, pH 7.4). The TCEP solution was 30 mM in water. Hemin (100 μM) was stored in DMSO at −20 °C and diluted to the required concentration with TMK buffer. All solutions were prepared with Milli-Q water (18.2 MΩ·cm resistivity) from a Millipore system. 2.2. Self-Assembly and Characterization of Tetrazymes. The design and fabrication of Tetrazymes were performed similarly to the previous report.32 Four strands were used to form the DNA tetrahedral structure. Three of them (S2, S3, and S4) were modified with thiol group at their 5-terminals. The other strand (S1-inside, S1-
3. RESULTS AND DISCUSSION 3.1. Design and Fabrication of Tetrazymes. We fabricated Tetrazymes by grafting G-quadruplex/hemin (G4hemin) onto the DNA tetrahedral scaffold. As the 7 nm sides of the tetrahedral scaffold are composed of double-stranded DNA and thus are not free to rotate, the position of each oligonucleotide in the helical strands and the position of the G4-hemin on the tetrahedral scaffold are fixed (Figure S1). We can site-specifically position the 3 nm G4-hemin on the DNA tetrahedral scaffold with near-angstrom precision, with the helical turn of the anchoring site determine placement. We 21860
DOI: 10.1021/acsami.9b06480 ACS Appl. Mater. Interfaces 2019, 11, 21859−21864
Research Article
ACS Applied Materials & Interfaces
DNA tetrahedral nanostructure, its catalytic activity was enhanced. We chose Tetrazyme-bottom-out as a model to compare with free G4-hemin because they are in the most similar situations, considering the distance between the active center to gold electrode surface and the surrounding microenvironment. The electric current of Tetrazymebottom-out was 5.0 ± 0.48 μA, which increased 14.2-fold compared to G4-hemin DNAzyme (0.35 ± 0.03 μA). To better understand this phenomenon, we evaluated and compared the surface coverage, dissociation constant, and current response of G4-hemin and Tetrazyme-bottom-out (Figure 2). The surface coverage of free G-quadruplex was determined to be (2.2 ± 0.2) × 1013 molecules/cm2 according to Tarlov’s method,34 while the surface density of DNA tetrahedron was about (3.0 ± 0.4) × 1012 molecules/cm2 according to previous research.35 Therefore, the surface density of Tetrazymes immobilized on gold surface was ∼0.14-fold of free G-quadruplex (Figure 2A). The dissociation constant (Kd) value of Tetrazyme-bottom-out was determined to be 0.76 ± 0.15 μM, which decreased to be 0.34-fold of G4-hemin (2.2 ± 0.3 μM), indicating that the binding affinity of G-quadruplex for hemin was improved by the DNA tetrahedron scaffold (Figure 2B). Interestingly, the electric current of Tetrazymebottom-out increased 10.9-fold compared to G4-hemin DNAzyme (Figure 2C). By calculating the catalytic activity of one single molecule, we found that the catalytic activity of Tetrazyme-bottom-out was around 70-fold higher than that of the G4-hemin DNAzyme without the DNA tetrahedron scaffold (Table S2). The observed increases in catalytic activities of Tetrazyme were attributed to the following reasons. First, the affinity of the G-quadruplex for hemin increases with the DNA tetrahedron scaffold assisted, which is in agreement of previous report.31 Second, the DNA tetrahedron scaffold can prevent oligomerization of the hemin/G-quadruplex that may decrease catalytic activity. Third, the active center of Tetrazyme is in a more stable environment because of protection by DNA tetrahedron. As a result, the stability of enzyme was enhanced, which probably also improved the catalytic activity. Last but not least, the active center of G4-hemin is uniformly distributed on spatially isolated DNA tetrahedral nanostructures that minimize interactions between catalytic sites and thus facilitate catalysis. 3.3. Configuration-Dependent Electrochemical Activities of Tetrazymes. To further explore the effect of G4hemin placement and orientation on the DNA tetrahedral scaffold, we synthesized and characterized Tetrazymes with the G-quadruplex/hemin complex grafted on different sites of the DNA tetrahedral scaffold (Figure 3A). In addition to the Tetrazyme-bottom-out structure described above, we designed four other Tetrazymes of different configurations: G4-hemin extended from the bottom edge fixed inside the tetrahedron (Tetrazyme-bottom-in); G4-hemin extended from the top of the tetrahedron (Tetrazyme-top); G4-hemin extended from the side edge fixed outside the tetrahedron (Tetrazyme-sideout); and G4-hemin extended from the side edge fixed inside the tetrahedron (Tetrazyme-side-in). Native polyacrylamide gel electrophoresis (PAGE) analysis confirmed different configurations of Tetrazymes (Figure S3). The enzymatic activities of Tetrazymes were characterized by CV and amperometric current. An average of 10-fold higher enzymatic activity than conventional G4-hemin DNAzyme was obtained, as shown in Figure 3B. Among these, Tetrazyme-bottom-in
assembled Tetrazymes from stoichiometric equivalents of three thiolated 62-nt DNA oligonucleotide strands and one 80-nt DNA strand with appended G-quadruplex DNA sequence. These four oligonucleotide strands with partially complementary sequences can self-assemble into a tetrahedron (Figure S2). The six edges are 20 base pairs in length and are connected by single unpaired nucleotides at the vertex that ensure sufficient flexibility to form the tetrahedron. The formation of Tetrazyme was confirmed by CD. The appearance of a specific peak at ∼263 nm confirms that the G-quadruplex, either in free form or located on DNA tetrahedron, retains the parallel conformation adopted by the isolated quadruplex domain (Figure S3). To explore the electrochemical activity of Tetrazyme, the Tetrazyme was designed with three thiol groups which readily attached to a gold electrode via well-established self-assembled chemistry. In the presence of hemin, the HRP-mimicking Tetrazyme complex is formed on the electrode. Here, hemin is as an active cofactor of Tetrazyme, which is crucial in donating electron density to the ion to be oxidized. We investigated the bioelectrocatalytic functions of Tetrazymes toward the electrocatalyzed reduction of H2O2. The bioelectrocatalytic cathodic currents generated by the Tetrazyme-functionalized electrodes, upon the electrochemical reduction of H2O2, were directly correlated with the catalytic efficiency of the Tetrazyme (Figure 1A).
Figure 1. (A) Scheme of electrochemical reaction of hemin (left), G4hemin (middle), and Tetrazyme-bottom-out on the gold electrode catalyzing the reduction of hydrogen peroxide (H2O2), whereby the complexed hemin is oxidized. (B) Cyclic voltammograms of hemin (gray), G4-hemin DNAzyme (blue), and Tetrazyme-bottom-out (red) in response to 6 mM of H2O2. Scan rate: 100 mV/s. Inset: Comparison of current response of hemin, G4-hemin, and Tetrazymebottom-out. The potential was held at −0.4 V (vs Ag/AgCl) and the reduction current was recorded at 100 s. All measurements were performed in deoxygenated TMK buffer solution (pH 8.0) containing 6 mM H2O2 and 1 μM hemin.
3.2. Electrochemical Response of Tetrazyme against H2O2. We investigated the electrocatalytic reduction of H2O2 of the Tetrazyme by CV at a scan rate of 100 mV/s ranging from −0.6 to 0.2 V (Figure 1B). The results indicated that the H2O2 was catalyzed only when both G-quadruplex structure and hemin were present. When G4-hemin was grafted on the 21861
DOI: 10.1021/acsami.9b06480 ACS Appl. Mater. Interfaces 2019, 11, 21859−21864
Research Article
ACS Applied Materials & Interfaces
Figure 2. Comparison of surface coverage (A), dissociation constant (B), and current response (C) between G4-hemin and Tetrazyme-bottomout. The corresponding values of surface coverage, dissociation constant, and current response of G4-hemin DNAzyme were 0.14, 0.34, and 10.9fold of G4-hemin, respectively.
position of G-quadruplex/hemin in and out of the tetrahedral framework played a major role on affecting catalytic efficiency. When the G4-hemin active centers located in the DNA tetrahedral framework, the observed electrocatalytic efficiency of Tetrazyme-bottom-in was higher than that of Tetrazymeside-in, with an amperometric current of 5.0 ± 0.48 μA compared to 4.5 ± 0.10 μA. And when the G4-hemin active centers located out of the DNA tetrahedral framework, an order of electrocatalytic efficiency of Tetrazyme-bottom-out (3.8 ± 0.24 μA) > Tetrazyme-side-out (3.5 ± 0.49 μA) > Tetrazyme-top (2.4 ± 0.34 μA) was observed. These results indicated that the electrocatalytic efficiency is higher when the distance between the enzyme active center and the gold electrode is shorter, probably because the shorter distance resulted in faster charge transfer between the enzyme active center and the gold electrode surface. When the Gquadruplex/hemin located on the bottom of the tetrahedral framework, a higher catalytic efficiency was observed for Tetrazyme-bottom-in (5.0 ± 0.48 μA) than Tetrazymebottom-out (3.8 ± 0.24 μA). When the G-quadruplex/hemin located on the side of the tetrahedral framework, a higher electrocatalytic efficiency was also observed for the structure with G-quadruplex/hemin in the cage, with a higher amperometric current for Tetrazyme-side-in (4.5 ± 0.10 μA) than Tetrazyme-side-out (3.5 ± 0.49 μA). These observations could be ascribed to the enhanced stability and more uniform distribution of G4-hemin in the DNA tetrahedral framework. On the basis of these results, we propose that there are options to further enhance the electrocatalytic efficiency of Tetrazymes. For example, we can construct Tetrazymes containing more G-quadruplex/hemin units, such as grafting G-quadruplex/hemin onto all edges of DNA tetrahedral framework, or employing signal amplification method like rolling circle amplification. Another option is to improve the electron transfer between the active center and the gold electrode surface, for example, by employing methylene blue as the electron shuttle. 3.4. H2O2 Detection with Tetrazymes. The bioelectrocatalytic functions of the DNAzyme have been used to develop electrochemical sensors. The H2O2 sensor has great importance in biological, clinical, and environmental fields.35−38 Based on the configuration-dependent electrochemical activities of Tetrazymes, we herein evaluated their H2O2 sensing activities. We first selected Tetrazyme-bottom-in with relatively higher enzymatic activities to investigate their electrochemical response to H2O2. Cyclic voltammograms were obtained upon the treatment of Tetrazyme-bottom-in on gold electrodes at various scan rates (10 to 190 mV/s) in nitrogen-saturated 6 mM of H2O2 solution (Figure S6). Electrocatalytic cathodic currents corresponding to the reduction of H2O2 were
Figure 3. Comparison of electrocatalytic activities of Tetrazymes of different configurations. (A) Schematic illustration of Tetrazymes of different configurations, from left to right: G4-hemin DNAzyme; G4hemin located on the top of the tetrahedron (Tetrazyme-Top); G4hemin located on the side edge and outside the DNA tetrahedron (Tetrazyme-side-out); G4-hemin located on the bottom edge and outside the DNA tetrahedron (Tetrazyme-bottom-out); G4-hemin located on the side edge and inside the DNA tetrahedron (Tetrazyme-side-in); and G4-hemin located on the bottom edge and inside the DNA tetrahedron (Tetrazyme-bottom-in). (B) Current−time responses comparison of Tetrazymes of different configurations in deoxygenated TMK buffer solution containing 6 mM H2O2 and 1 μM hemin. Error bars show the standard deviations of measurements collected from at least three independent experiments.
gave an amperometric signal of 5.0 ± 0.48 μA, showing the best catalytic activity. Tetrazyme-side-in came in second place with an amperometric current of 4.5 ± 0.10 μA, which was approximately 12.7-fold higher than that of the conventional G4-hemin DNAzyme. Tetrazyme-top gave the lowest amperometric current (2.3 ± 0.34 μA), which was still 6.7-fold higher than that of the bare G4-hemin DNAzyme. The electrocatalytic efficiency order of these five types of Tetrazymes is as follows: Tetrazyme-bottom-in > Tetrazyme-side-in > Tetrazyme-bottom-out > Tetrazyme-side-out > Tetrazyme-top. The detailed data were collected and are shown in Table S2. There are a few factors affecting the enzymatic activities of Tetrazymes, such as the space distance between the active center of Tetrazyme and gold electrode, the diffusion efficiency of H2O2 and the stability of enzyme. The observed enzymatic activities of Tetrazymes were results of the combination of all of these effects. In the presence of 6 mM H2O2, which is a high concentration, a relatively higher catalytic current was observed for both Tetrazyme-bottom-in (5.0 ± 0.48 μA) and Tetrazyme-side-in (4.5 ± 0.10 μA) compared to Tetrazymebottom-out (3.8 ± 0.24 μA), Tetrazyme-side-out (3.5 ± 0.49 μA), and Tetrazyme-top (2.4 ± 0.34 μA), indicating the 21862
DOI: 10.1021/acsami.9b06480 ACS Appl. Mater. Interfaces 2019, 11, 21859−21864
Research Article
ACS Applied Materials & Interfaces
Figure 4. Dynamic range (A) and LOD (B) comparison of Tetrazyme-based H2O2 biosensors of different configurations.
4. CONCLUSIONS In summary, a novel artificial DNAzyme (Tetrazyme) was fabricated by grafting a G4-hemin on three-dimensional DNA tetrahedral frameworks. We have demonstrated that Tetrazyme exhibited high electrocatalytic ability using electrochemical method. Compared to free G4-hemin DNAzyme, the Tetrazyme exhibited higher electrocatalytic ability for the reduction of H2O2. Furthermore, we found Tetrazyme-bottomin, in which G4-hemin was extended from the bottom edge and fixed inside the DNA tetrahedron, exhibited the strongest electrocatalytic ability. This enabled the potential use of the Tetrazyme as a versatile electrocatalytic label for developing electrochemical enzyme-based sensors.
observed, indicating that Tetrazymes were captured on the gold electrode and acted as efficient electrocatalysts for H2O2. The redox peak currents increased upon increasing the scan rate from 10 to 190 mV/s (Figure S6A), and the currents at −0.4 V (vs Ag/AgCl) showed a linear dependence with function of scan rate (Figure S6B), indicating that the electrochemical reaction of Tetrazyme is a surface-controlled process. We then compared enzymatic activities of Tetrazymebottom-in with Tetrazyme-bottom-out. The electrochemical signal was increased with the increase of H2O2 concentration for both of the Tetrazymes (Figure S7). Amperometric results showed the electrocatalytic cathodic currents for the detection of different concentrations of H2O2 were discriminated. The background current was as low as ∼60 nA, while amperometric current of Tetrazyme-bottom-in for 1 mM H2O2 was ∼1032 nA, and amperometric current of Tetrazyme-bottom-in for 1 mM H2O2 was ∼1000 nA. This high signal-to-background ratio further confirmed the catalyzing ability of Tetrazyme. Finally, we evaluated and compared the detection sensitivity of all the five Tetrazymes against H2O2 by employing the amperometric I−t method. Figure S8 displays the amperometric current response of various Tetrazymes for the different concentration additions of H2O2. The amperometric signal increased monotonically with the concentration of H2O2 across the range from 1 μM to 10 mM, spanning a response region of at least 4 orders of magnitude. The response of the sensor based on Tetrazyme was linear over the concentration of H2O2 from 10 μM to 6 mM for Tetrazyme-bottom-in, Tetrazyme-bottom-out and Tetrazyme-side-in; 10 μM to 8 mM for Tetrazyme-top; 10 μM to 9 mM for Tetrazyme-sideout (Figure 4A). The linear regression equations of Tetrazymes are listed in Table S3. The limit of detections (LODs) were calculated to be 4.6 μM for Tetrazyme-bottomout, 8.9 μM for Tetrazyme-side-in, 3.0 μM for Tetrazyme-sideout, and 9.8 μM for Tetrazyme-top, which are much lower than that of G4-hemin enzyme (199 μM) (Figure 4B). At low H2O2 concentration, the efficiency of enzyme reaction is mainly dependent on the diffusion of H2O2. The diffusion of H2O2 in Tetrazyme-side-out-based sensor is easier than that of Tetrazyme-bottom-in, because the enzyme active center is located out of the DNA tetrahedron cage. Therefore, it was easier to detect lower concentration of H2O2 by the Tetrazyme-side-out-based H2O2 sensor than Tetrazymebottom-in. These results show that our Tetrazymes can be used as advanced artificial enzymes for sensitive and low-level detection of H2O2.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.9b06480. Synthetic oligonucleotide probes employed in this study; assembly of Tetrazymes; native PAGE gel analysis; CD spectra; dissociation constant (Kd) measurements; cyclic voltammograms at different scan rates and in response to variable concentrations of hydrogen peroxide; and LOD, linear range, and linear regression equation of Tetrazymes with different configurations (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected] (Q.L.). *E-mail:
[email protected] (X.M.). ORCID
Zhilei Ge: 0000-0001-7184-7565 Qian Li: 0000-0002-1166-6583 Xianqiang Mi: 0000-0001-7111-6818 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are thankful for support from the National Key Research and Development Program of China (grant 2016YFC0100600), National Key Technology Research and Development Program of the Ministry of Science and Technology of China (grant 2015BAI02B02), Chinese National Natural Science Foundation (grant 21605152), Science and Technology Service Network Initiative, CAS (grant KFJEW-STS-140, KFJ-EW-STS-096), Shanghai Municipal Science and Technology Commission (grant 15441905000, 16DZ1930700, 19ZR1474300), Youth Innovation Promotion 21863
DOI: 10.1021/acsami.9b06480 ACS Appl. Mater. Interfaces 2019, 11, 21859−21864
Research Article
ACS Applied Materials & Interfaces
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Association CAS (no. 2017356), and Shanghai University of Medicine & Health Sciences (grant SPCI-17-19-002).
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DOI: 10.1021/acsami.9b06480 ACS Appl. Mater. Interfaces 2019, 11, 21859−21864
