Framework Nucleic Acid-Enabled Programming of Electrochemical

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Functional Nanostructured Materials (including low-D carbon)

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 ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 22 May 2019 Downloaded from http://pubs.acs.org on May 22, 2019

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ACS Applied Materials & Interfaces

Framework

Nucleic

Acid-Enabled

Programming

of

Electrochemical Catalytic Properties of Artificial Enzymes Dongdong Zengba, Lili Sana, Fengyu Qianb, Zhilei Gec, Xiaohui Xub, Bin Wangb, Qian Lid*, Guifang Hea, Xianqiang Miae* aShanghai

Advanced Research Institute, Chinese Academy of Sciences, Shanghai 201210, China

bShanghai

Key Laboratory of Molecular Imaging, Shanghai University of Medicine & Health Sciences,

Shanghai 201318, China cSchool

of Chemistry and Chemical Engineering , Shanghai Jiao Tong University, Shanghai 200240,

China dSchool

of Chemistry and Chemical Engineering, Shanghai Jiao Tong University, Shanghai 200240,

China eKey

Laboratory of Functional Materials for Informatics, Shanghai Institute of Microsystem and

Information Technology, Chinese Academy of Sciences, Shanghai 200050, China

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-tetrahedron-scaffolded-DNAzyme (Tetrazyme), and evaluated the catalytic activity of these Tetrazymes by electrochemistry. Tetrazymes were constructed by precisely positioning G-quadruplex on different sites of DNA tetrahedral framework, with hemin employed as co-catalyst. Immobilization of Tetrazymes on gold electrode surface revealed horseradish peroxidase (HPR)-mimicking bioelectrocatalytic property. Cyclic voltammagram and amperametry 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 DNA tetrahedron scaffold, demonstrating

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application potential in developing novel G-quadruplex based electrochemical sensors.

Keywords: Framework nucleic acid; DNA tetrahedral nanostructure; G-quadruplex; Electrochemistry; Enzymatic activity

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 compartments1-3. 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 systems4-8. 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 biomaterials9-13. DNA is a self-assembling biopolymer that is directed by canonical Watson-Crick base paring to form predictable, double helical secondary structures, which enables it one of the most promising biomolecules for the construction of complex biomolecular networks14-19. 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 junction20. Since then, structural DNA nanotechnology has enjoyed a rapid progress, and numerous complex nanostructures and different fabrication techniques have been introduced21-22. 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 ACS Paragon Plus Environment

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nanotechnology have been reported24-27. 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 be further promoted by rolling the rectangular DNA origami into a three-dimensional DNA nanotube. As one kind of artificial enzyme, G-quadruplex/hemin (G4-hemin), formed by guanine-rich nucleic acid sequence fold into G-quadruplex comlexing tightly with hemin, has characteristics of high operational stability, insensitivity to environmental conditions, and low costs in preparation than natural enzymes28-30. Thus, it has been widely served as a stable catalytic label for various biosensors towards a variety of targets. However, the relatively low enzymatic acitivity of G4-hemin 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/hemin31. However, the electrochemical catalytic efficiency of this DNA tetrahedron- scaffolded artificial enzyme has not been explored. Herein,

we

evaluated

electrochemical

DNA-tetrahedron-scaffolded-DNAzyme

catalytic

(Tetrazyme)

and

property

of

constructed

an

electrochemical hydrogen peroxide (H2O2) biosensor using Tetrazyme as signal probes. Specifically, we constructed DNA tetrahedral framework as a scaffold to covalently attach 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 ACS Paragon Plus Environment


the spatial between activity center and electrode surface significantly affected the activity of Tetrazymes. Based on these studies, we developed a H2O2 biosensor with a linear range of 10-9000 H

and a detection limit of 3.0 H 8

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. The 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 Sigma-Aldrich (St. Louis, MO, USA). 30% H2O2, Tris (hydroxymethyl) aminomethane (Tris), Dimethyl Sulphoxide (DMSO) and other reagents were obtained from the 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 buffer saline (1X PBS, 10 mM Na2HPO4, 2 mM KH2PO4, 137 mM NaCl and 2.7 mM KCl, pH 7.4). The TCEP solution was 30mM in water. Hemin (100 H = was stored in Dimethyl Sulphoxide (DMSO) at -20 KC and diluted to the required concentration with TMK buffer. All solutions were prepared with Milli-Q water (18.2

L8 ' 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 report32. 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-outside, A1-inside, A1-outside or V1-outside) was designed with an extended G-quadruplex structure sequence. The Tetrazyme was prepared as the following processes: Equal amounts of oligonucleotides S1 (or S1-inside, S1-outside, ACS Paragon Plus Environment

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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 H

for each oligonucleotide. Annealing was performed by

holding the mixture at 95 KC for 3 minutes followed by cooling to 4 KC over approximately 30 seconds using a BioRad thermal cycler PTC-100. The prepared DNA sample solutions were transformed into the freshly prepared 8% native polyacrylamide gel and was run at 80 V for 120 minutes 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 H

in potassium acetate (KAc) buffer (20 mM KAc, 70 mM KCl,

pH 6.8). The spectral was obtained with a Chirascan CD Spectrometer (Applied Photophysics Ltd, UK). The wavelength was varied from 220 nm 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 protocol33. 3 H of Tetrazymes (1 H = were injected on 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 uL of 1 H

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 H

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 H of 1 H

hemin solution for 30 min at

37 °C to obtain the G4-hemin biosensor. Hexaammineruthenium(III) (Ruhex) was ACS Paragon Plus Environment


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). The 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 V 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.

3. RESULTS AND DISCUSSION 3.1 Design and fabrication of Tetrazymes We fabricated Tetrazymes by grafting G-quadruplex/hemin (G4-hemin) onto 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 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 ACS Paragon Plus Environment

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to form the tetrahedron. The formation of Tetrazyme was confirmd by circular dichroism (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

towards

the

electrocatalyzed reduction of H2O2. The bioelectrocatalytic cathodic currents generated by the tetrazyme-functionalized electrodes, upon the electrochemical reduction of H2O2, were directly correlated to the catalytic efficiency of the Tetrazyme (Figure 1A). A

B

1

-20

0 -1

5

3.8

4

A

-10

-30

-2

-40

-3

Curent

Current ( A)

0 Current ( A)

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


3 2 1

0.03

0.35

0

-0.6

-0.4

-0.2

0.0

0.2

0.4

-0.6

-0.4

-0.2

0.0

0.2

0.4

Potential (V vs.Ag/AgCl)

Potential (V vs.Ag/AgCl)

Figure 1. (A) Scheme of electrochemical reaction of Hemin (left), G4-Hemin (middle) and Tetrazyme-bottom-out on gold electrode catalyzing the reduction of hydrogen peroxide(H2O2), whereby the complexed hemin is oxidized. (B) Cyclic voltammograms (CVs) of Hemin (grey),



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G4-hemin DNAzyme (blue), Tetrazyme-bottom-out (red) in response to 6 mM of H2O2. Scan rate: 100 mV/s. Insert: Comparison of current response of hemin, G4-hemin and Tetrazyme-bottom-out. The potential was held at -0.4V (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 H

hemin.

3.2 Electrochemical response of Tetrazyme against H2O2 We investigated the electrocatalytic reduction of H2O2 of the Tetrazyme by cyclic voltammetry (CV) at a scan rate of 100 mV/s ranging from -0.6 V to 0.2 V (Figure 1B). The results indicated that the H2O2 was catalyzed only when both G-quadruplex structure and hemin were in presence. When G4-hemin was grafted on 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 microenviroment. The electric current of Tetrazyme-bottom-out was 5.0 ± 0.48 H

which increased 14.2 fold compared to

G4-hemin DNAzyme (0.35 ± 0.03 H =8 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 method34, while the surface density of DNA tetrahedron was about (3.0 ± 0.4) × 1012 molecules/cm2 according to previous research35. 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 HM, which decreased to be 0.34 fold of G4-hemin (2.2 ± 0.3 HM), indicating that the binding affinity of G-quadruplex for hemin was improved by the DNA tetrahedron scaffold (Figure 2B). Interestingly, the electric current of Tetrazyme-bottom-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 ACS Paragon Plus Environment

Page 9 of 19

catalytic activity of Tetrazyme-bottom-out was around 70 fold higher than the G4-hemin DNAzyme without DNA tetrahedron scaffold (Table S2). The observed increases in catalytic activities of Tetrazyme were attributed to be the following reasons. First, the affinity of the G-quadruplex for hemin increases with DNA tetrahedron scaffold assisted, which is in agreement of previous report31. Second, 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. And 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.

10

0.4

3.0

0

0.14

0.8 2

1

0.2 0.0

G4-hemin DNAzyme Tetrzyme-bottom-out

2.2

0

0.6 0.76

0.34

0.4

6

3

0.0

0

8 6

2 1

12 10

3.8

4

0.2


10.9

5

4

0.35

1.0

Fold Change

0.6

1.0

A

15

3

C

1.2 1.0

Curent

0.8

4

Fold Change

1.0

20

5

B

1.2

1.0 22

)

25

Kd (

30

Fold Change

A

Surface Coverage (1012 molecules/cm2)

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


2 0


Figure 2. Comparison of surface coverage (A), dissociation constant (B) and current response (C) between G4-hemin and Tetrazyme-bottom-out. The corresponding values of surface coverage, dissociation constant and current response of G4-hemin DNAzyme were 0.14, 0.34 and 10.9 fold of G4-hemin, respectively.

3.3 Configuration dependent electrochemical activities of Tetrazymes To further explore the effect of G4-hemin placement and orientation on DNA tetrahedaral scaffold, we synthesized and characterized Tetrazymes with the G-quadruplex/hemin complex grafted on different sites of 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



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the side edge fixed outside the tetrahedron (Tetrazyme-side-out); 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 gave an amperometric signal of 5.0 ± 0.48 H

showing the best catalytic activity. Tetrazyme-side-in came in second place

with an amperometric current of 4.5 ± 0.10 H

which was approximately 12.7-fold

higher than the conventional G4-hemin DNAzyme. Tetrazyme-top gave the lowest amperometric current (2.3 ± 0.34 H = which was still 6.7-fold higher than the bare G4-hemin DNAzyme. The electrocatalytic efficiency order of these five types Tetrazymes is: Tetrazyme-bottom-in > Tetrazyme-side-in > Tetrazyme-bottom-out > Tetrazyme-side-out > Tetrazyme-top. The detailed data were collected and 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 H = and Tetrazyme-side-in (4.5 ± 0.10 H = compared to Tetrazyme-bottom-out (3.8 ± 0.24 H = Tetrazyme-side-out (3.5 ± 0.49 H = and Tetrazyme-top (2.4 ± 0.34 H = indicating the 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 Tetrazyme-side-in, with an amperometric current of 5.0 ± 0.48 H

compared to 4.5 ± 0.10 H 8 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

H =

>

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Tetrazyme-side-out (3.5 ± 0.49 H = > Tetrazyme-top (2.4 ± 0.34 H = was observed. These results indicated that the electrocatalytic efficiency is higher when the distance between the enzyme active center and gold electrode is shorter, probably because the shorter distance resulted in faster charge transfer between the enzyme active center and gold electrode surface. When the G-quadruplex/hemin located on the bottom of the tetrahedral framework, a higher catalytic efficiency was observed for Tetrazyme-bottom-in (5.0 ± 0.48 H = than Tetrazyme-bottom-out (3.8 ± 0.24 H =8 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 H

than Tetrazyme-side-out (3.5 ± 0.49 H =8 These

observations could be ascribed to the enhanced stability and more uniform distribution of G4-hemin in the DNA tetrahedral framework. Based on these results, we propose 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 RCA (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 electron shuttle.



A

B7

16

6

12.7 10.9

5

10.0

4

3.5

5.0

3.8

0

10 8 6

2.3

2 1

12

4.5

6.7

3

14

4 1.0 0.35 G4-hemin

Fold Change

14.2

Current ( A)

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

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2 top

side-out bottom-out side-in bottom-in

0

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; G4-hemin located on the top of the tetrahedron (Tetrazyme-Top); G4-hemin 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); 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.

3.4 H2O2 detection with Tetrazymes The bioelectrocatalytic functions of the DNAzyme have been used to develop electrochemical sensors. H2O2 sensor has great importance in biological, clinical and environmental fields35-37. Based on the configuration dependent electrochemical activities of Tetrazymes, we herein evaluated their H2O2 sensing activities.


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We first selected Tetrazyme-bottom-in with relatively higher emzymatic 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 rate (10 mV/s to 190 mV/s) in nitrogen saturated 6 mM of H2O2 solution (Figure S6

. Electrocatalytic cathodic currents corresponding to the reduction of H2O2 were

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 mV/s to 190 mV/s (Figure S6A), and the currents at -0.4V (vs. Ag/AgCl ) showed a linear dependence with function of scan rate (Figure S6B), indicating the electrochemical reaction of Tetrazyme is a surface-cotrolled process. We

then

compared

emzymatic

activities

of

Tetrazyme-bottom-in

with

Tetrazyme-bottom-out. The electrochemical signal was increased with the increase of H2O2 concentration for both of the Tetrazymes (Figure S7). Amperometries 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, 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 Tetrzymes for the different concentration additions of H2O2. The amperometric signal increased monotonically with the concentration of H2O2 across the range from 1 H

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 H

to 6 mM for

Tetrazyme-bottom-in, Tetrazyme-bottom-out and Tetrzyme-side-in; 10 H for Tetrazyme-top; 10 H

to 8 mM

to 9 mM for Tetrazyme-side-out (Figure 4A). The linear

regression equations of Tetrazymes were listed in Table S3. The LODs were calculated to be 4.6 H

for Tetrazyme-bottom-out, 8.9 µM for Tetrazyme-side-in, 3.0 ACS Paragon Plus Environment



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Corresponding Authors Xianqiang Mi) Email: [email protected].

*(

*(Qian

Li) Email: [email protected]

Acknowledgment 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 KFJ-EW-STS-140, KFJ-EW-STS-096), Shanghai Municipal Science and Technology Commission (Grant 15441905000,16DZ1930700), Youth Innovation Promotion Association CAS (No. 2017356) and Shanghai University of Medicine & Health Sciences (Grant SPCI-17-19-002).

Supporting Information Synthetic oligonucleotide probes employed in this study, assembly of Tetrazymes, native PAGE gel analysis, circular dichroism (CD) spectra, the dissociation constant (Kd) measurements, cyclic voltammogram (CVs) at differnt scan rates and in response to variable concentrations of hydrogen peroxide, the limit of detection (LOD), linear range and linear regression equation of Tetrazymes with different configurations. REFERENCES (1) Xu, J. Y.; Xu, Y.; Chu, X.; Tan, M.; Ye, B. C. Protein Acylation Affects the Artificial Biosynthetic Pathway for Pinosylvin Production in Engineered E. coli. Acs Chem. Biol. 2018, 13 (5), 1200-1208, DOI: 10.1021/acschembio.7b01068. (2) Einfalt, T.; Witzigmann, D.; Edlinger, C.; Sieber, S.; Goers, R.; Najer, A.; Spulber, M.; Onaca-Fischer, O.; Huwyler, J.; Palivan, C. G. Biomimetic Artificial Organelles with in Vitro and in Vivo Activity Triggered by Reduction in Microenvironment. Nat. Commun. 2018, 9 (1), 1127, DOI: 10.1038/s41467-018-03560-x. (3) Elani, Y.; Law, R. V.; Ces, O. Vesicle-based Artificial Cells as Chemical Microreactors with Spatially Segregated Reaction Pathways. Nat. Commun. 2014, 5, 5305, DOI: 10.1038/ncomms6305. (4) Najafpour, M. M.; Madadkhani, S.; Zand, Z.; Holynska, M.; Allakhverdiev, S. I. Engineered



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Framework Nucleic Acid-Enabled Programming of Electrochemical

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