Journal Pre-proofs

Self-assembly of Copper Nanoclusters Using DNA Nanoribbon Templates for

Sensitive Electrochemical Detection of H2O2 in Live Cells

Lan Luo, Yukun Xing, Yue Fu, Le Li, Xinya Yang, Yumiao Xue, Jing Luo,
Huaiyu Bu, Fangfang Chen, Xiangyuan Ouyang

PII: S0021-9797(23)02531-6
DOI: https://doi.org/10.1016/j.jcis.2023.12.189
Reference: YJCIS 33868

To appear in: Journal of Colloid and Interface Science

Received Date: 1 November 2023

Revised Date: 11 December 2023
Accepted Date: 31 December 2023

Please cite this article as: L. Luo, Y. Xing, Y. Fu, L. Li, X. Yang, Y. Xue, J. Luo, H. Bu, F. Chen, X. Ouyang,
Self-assembly of Copper Nanoclusters Using DNA Nanoribbon Templates for Sensitive Electrochemical
Detection of H2O2 in Live Cells, Journal of Colloid and Interface Science (2024), doi: https://doi.org/10.1016/
j.jcis.2023.12.189

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 Self-assembly of Copper Nanoclusters Using DNA Nanoribbon

Templates for Sensitive Electrochemical Detection of H2O2 in

Live Cells

Lan Luo#a, Yukun Xing#a, Yue Fua, Le Lia, Xinya Yanga, Yumiao Xuea, Jing Luo b,
Huaiyu Bu b, Fangfang Chena*, Xiangyuan Ouyanga*

aXi'an Key Laboratory of Functional Supramolecular Structure and Materials, Key

Laboratory of Synthetic and Natural Functional Molecule of Ministry of Edu-cation,
College of Chemistry & Mate-rials Science, Northwest University, Xi’an, Shaanxi
710127, P. R. China
b KeyLaboratory of Resource Biology and Biotechnology in West-ern China (Ministry
of Educa-tion), College of Life Sci-ences, Northwest University, Xi’an, Shaanxi 710069,
PR China

*Corresponding authors. Fangfang Chen and Xiangyuan Ouyang

E-mail: [email protected] and [email protected]

1
1 Abstract

2 The excessive secretion of H2O2 within cells is closely associated with cellular
3 dysfunction. Therefore, high sensitivity in situ detection of H2O2 released from living
4 cells was valuable in clinical diagnosis. In the present work, a novel electrochemical
5 cell sensing platform by synthesizing copper nanoclusters (CuNCs) at room
6 temperature based on DNA nanoribbon (DNR) as a template (DNR-CuNCs). The tight
7 and ordered arrangement of nanostructured assemblies of DNR-CuNCs conferred the
8 sensor with superior stability (45 days) and electrochemical performance. The MUC1
9 aptamer extending from the DNR template enabled the direct capture MCF-7 cells on
10 electrode surface, this facilitated real-time monitoring of H2O2 release from stimulated
11 MCF-7 cells. While the captured MCF-7 cells on the electrode surface significantly
12 amplified the current signal of H2O2 release compared with the traditional
13 electrochemical detection H2O2 released signal by MCF-7 cells in PBS solution. The
14 approach provides an effective strategy for the design of versatile sensors and achieving
15 monitored cell release of H2O2 in long time horizon (10 h). Thereby expanding the
16 possibilities for detecting biomolecules from live cells in clinical diagnosis and
17 biomedical applications.

18

19

20

21

22 Keywords: DNA nanoribbon template; copper nanoclusters; real-time monitoring;

23 MCF-7 cells; H2O2; electrochemical

2
1 1. Introduction

2 Hydrogen peroxide (H2O2) serves as a vital cellular metabolite, exhibiting higher

3 levels in cancer cells compared to normal cells. The excessive generation of H2O2 can
4 give rise to severe diseases as it leads to oxidative changes that inflict cellular harm [1,
5 2]. Rapid and reliable measurement of H2O2 is of utmost importance because it is not
6 only a product of many highly selective oxidase-catalyzed reactions, but also serves as
7 a cellular metabolic biomarker and an important representative of reactive oxygen
8 species in biomedical diagnostics [3, 4]. Methods for detecting H2O2 encompass various
9 techniques, including colorimetry, fluorescence, chemiluminescence, and
10 electrochemical sensing. Electrochemical methods have several advantages, including
11 rapid response time, simple instrumentation, ease of miniaturization, and high
12 specificity and sensitivity, which was regarded as the most promising approach for
13 H2O2 detection in comparison to other available methods, improving the sensitivity and
14 long-term stability is on top of the list of requirements [5-13].

15 In recent years, metal nanoclusters (NCs) have attracted significant attention in the
16 fields of Electrochemical sensing [14, 15], Electrocatalysis, and Electrochemi-
17 luminescence, owing to their unique physical and chemical properties. Noble metals,
18 including Au, Ag, and Pt, are commonly used to construct NCs for multifunctional
19 modified electrodes, in which redox-active clusters act as electron conductors and
20 electron transfer mediators. However, the considerable expense of noble metals poses
21 a significant challenge to practical applications. A viable strategy for mitigating the
22 expenses associated with sensing applications involves the exploration of more
23 economically viable metal options. Copper (Cu), notable for its affordability and
24 widespread availability, has emerged as a promising substitute for noble metals like Au
25 and Ag. Simultaneously, there is a growing focus on utilizing copper nanoclusters
26 (CuNCs) due to their ability to achieve high yields under mild conditions. The appeal
27 of CuNCs is enhanced by their cost-effectiveness, water solubility, and well-defined
28 structures, making them highly interesting for electrochemical sensing ranging from
29 fundamental to analytical fields [16]. For instance, Lan et al. presented PtCu-rGO
30 bimetallic nanocomposite modified carbon fibers microelectrodes for detecting
31 hydrogen peroxide released from living cells [17]. Nevertheless, the incorporation of
32 noble metal materials is frequently required to attain enhanced catalytic activity,
33 thereby overcoming the sensitivity and detection range limitations observed in copper-
34 based electrochemical sensors [18-21]. Wang et al. projected a ligand-based strategy to
35 arrange the self-assembled CuNCs structure, and the assembled CuNCs nanoribbons
36 successfully showed excellent electrocatalytic properties for glucose, with detection
37 limits as low as 4.5 μM, but utilize ligands protected the synthesis process of CuNCs,
38 making the whole process troublesome and time-consuming [22]. In recent years,
39 CuNCs have received much attention due to their excellent physical and chemical
40 properties, their atomically precise structure and composition exhibit excellent catalytic
41 capabilities. However, challenges such as susceptibility to oxidation, difficulty in size
42 control, and irreversible aggregation of CuNCs during preparation have hindered its use
3
1 in sensing applications.

2 To address this issue, protective ligands such as DNA, amino acids, peptides, and
3 proteins have been introduced to improve the stability of CuNCs [23]. Since the
4 pioneering work of Rotaru et al, CuNCs have been synthesized on double-stranded
5 DNA (dsDNA) in solution [24]. Due to its programmable structure, controllable
6 transformation, easy synthesis and modification, and low cost, DNA holds promise as
7 a biological template for CuNCs fabrication. In addition, a "bottom-up" approach has
8 been reported for the synthesis of CuNCs using DNA nanoribbons as a template
9 structure [25]. This process is fast and simple, requiring only a few min of reaction time
10 at room temperature between Cu2+ and sodium ascorbate (SA). By utilizing DNA
11 template sequences, CuNCs can be stabilized and assembled onto the DNA nanoribbon
12 in an orderly manner (DNR-CuNCs), thus overcoming the problem of poor stability of
13 individual CuNCs. Compared to the synthesis of CuNCs using dsDNA or
14 polythymidine single-stranded templates, the DNA framework for assembling DNR
15 provides easy modification of base sequences, making DNA nanomaterials applicable
16 to a wider range of detection scenarios [26]. Additionally, most electrochemical sensors
17 for detecting hydrogen peroxide released by cells can only detect it in solution, lacking
18 the ability to capture cells on the electrode surface, resulting in lower sensitivity [27-
19 29]. Therefore, there is a pressing need to develop a cost-effective strategy that ensures
20 superior stability for the preparation of NCs sensitivity for constructing an
21 electrochemical detection platform.

22 In the present work, a simple electrochemical sensor modified with DNR-CuNCs

23 was employed for the electrocatalytic real-time detection of H2O2 released from living
24 cells. CuNCs were synthesized using DNR as a template structure at room temperature.
25 The surface of glass carbon electrode (GCE) was modified with p-Arg through
26 electropolymerization, and DNR-CuNCs solution was then applied to the surface of p-
27 Arg (DNR-CuNCs/p-Arg/GCE), where negatively charged DNR-CuNCs readily
28 assembled with positively charged p-Arg polymer through electrostatic interaction. The
29 DNR-CuNCs/p-Arg/GCE sensor demonstrated remarkable electrocatalytic activity
30 towards H2O2. Additionally, we exploited the programmability of the DNR framework
31 to effortlessly incorporate the MUC1 protein aptamer into the DNR structure and
32 captured MCF-7 cells on the electrode surface for real-time H2O2 detection, resulting
33 in the enhanced sensitivity of the fabricated biosensor for H2O2 detection while enabling
34 in-situ monitoring effectively [30]. The use of DNR-templated CuNCs in
35 electrochemical sensing represents a promising new approach, resulting in the sensor
36 with exceptional detection performance, as well as low cost, good stability, and signal
37 sensitivity advantages. Notably, it allows for the monitoring of long-term (10 h) release
38 of H2O2 from cells. The integration of these advantages in the design of DNR-templated
39 NCs sensors holds promising potential for future applications, particularly in the
40 sensitive monitoring of H2O2 secretion from live cancer cells.

41 2. Experimental section
4
1 2.1. Synthesis of DNR-CuNCs

2 The preparation of DNR was based on previously reported literature [25]. Initially,
3 1 μL of DNR-S1, DNR-S2, DNR-ST1, DNR-ST2, and DNR-ST3 with an initial
4 concentration of 100 μM was mixed with 95 μL of Tris buffer (20 mM Mg2+, 40 mM
5 Tris-HAc) with a pH of 7.5 and reacted for approximately 2 h in a PCR instrument to
6 prepare the DNR template. Subsequently, 0.4 μL of 100 mM CuSO4 solution was added
7 to 100 μL of the DNR solution, and reacted at room temperature for 5 min, followed by
8 the addition of 0.4 μL of 1000 mM SA solution to react for 10 min to form stable Cu
9 NCs (DNR-CuNCs). As a control, the CuNCs synthesized without the DNR template
10 were obtained instead of ultrapure water into the DNR solution while maintaining the
11 same buffer environment and other conditions.

12 2.2. Construction of DNR-CuNCs/p-Arg/GCE sensor

13 The surface of the GCE (3 mm in diameter) was cleaned by polishing it evenly with
14 a wet suede cloth containing 0.05 μm alumina, followed by ultrasonication in ultrapure
15 water for 5 seconds. In this work, a three-electrode system was then constructed using
16 the GCE as the working electrode, Ag/AgCl as the reference electrode, and platinum
17 wire as the counter electrode, and all the tests were carried out at room temperature.
18 The GCE surface was modified by electropolymerizing Arg (10 mM in 0.1 M PBS,
19 pH=7.0) twice cycles at a voltage range of -1.6 V to 2.5 V and a scanning speed of 100
20 mV/s (as shown in Figure S1) [31]. Subsequently, dropping 10 μL of DNR-CuNCs
21 solution on the modified electrode, followed by electrostatic adsorption between p-Arg
22 and DNR-CuNCs at room temperature for 30 min and washing of the residual solution
23 on the electrode surface with 0.1 M PBS at pH 7.5. Finally, all the electrochemical tests
24 were performed in 0.1 M PBS buffer at pH 7.5 containing H2O2, with a scanning rate
25 of 100 mV/s and the cyclic scan ranging from -1.0 V~0 V.

26 2.3. Real-time Detection of H2O2 in Living Cells

27 Breast cancer cells (MCF-7) were cultured in a Petri dish with 5% CO2 and 95%
28 air at 37 °C. The number of cells was estimated using the platelet count method, and
29 the cell was adjusted and verified to contain 2×108 MCF-7 living cells in the culture
30 media, the cells were digested with trypsin and then diluted to 5 mL with 0.1 M PBS
31 (pH 7.5) for testing.

32 3. Results and discussion

33 3.1. Preparation and Characterization of DNR-CuNCs/p-Arg/GCE

5
1

2 Scheme 1. (A) Preparation of DNA nanoribbons: design principles of self-assembly

3 DNA nanoribbons, and (B) the preparation process of DNR-CuNCs, (C) the
4 electrocatalytic application of DNR-CuNCs sensors in H2O2 detection.

6 Scheme 1 shows the construction process of DNR-CuNCs/p-Arg/GCE, which can

7 be divided into two steps as follows: (1) Scheme 1A illustrated the design of DNR,
8 which employed a tile self-assembly mode similar to Tetris, resulting in a simple and
9 efficient construction process. The structure diagram and base sequence of different
10 DNR can be found in the Supporting Information (Table S1). For the specific
11 localization of Cu2+ ions on the DNR, the same sequences used by Rotaru et al. were
12 selected and highlighted in orange in Scheme 1A. (2) In Scheme 1B, the process of
13 ultrasmall CuNCs self-assembly on DNR was presented. The process began by adding
14 CuSO4 to a mixed solution (pH=7.5) containing prepared DNR for 5 min. Next, sodium
15 ascorbate SA was added to the solution, which was then allowed to sit at room
16 temperature for 10 min, resulting in the assembly of CuNCs onto the DNR. The material
17 by electrostatic adsorption used to immobilize on a glassy carbon electrode, the
18 obtained DNR-CuNCs/p-Arg/GCE display good electronic conductivity and mimic the
19 catalase decomposition activity to produce H2O and O2 (Scheme 1C). Moreover, the
20 electrochemical current response exhibited a correlation with variations in the
21 concentration of H2O2. Utilizing the programmable attributes of DNR, we integrated
22 MUC1 protein aptamer sequences to immobilize MCF-7 cells onto electrodes, allowing
23 for direct in-situ detection of H2O2 secreted from live MCF-7 cells. In contrast to
24 detecting H2O2 with MCF-7 cells dispersed in a solution, capturing the cells on the
25 electrode surface eliminated the diffusion process of H2O2 from the solution to the
26 electrode surface, which led to a more noticeable current signal response, achieved with
6
1 a decreased MCF-7 cells quantity.

2 3.2. Characterization of DNR-CuNCs

4 Fig. 1. (A) AFM images of DNR, (B) DNR/Cu2+, (C) DNR/CuNCs. (D) The Statistical
5 distribution of height and width of DNR, DNR-Cu2+ and DNR-CuNCs. (E) XPS spectra
6 showing the full region of the CuNCs and (F) Cu 2p region.

8 To verify the successful formation of the DNR-CuNCs, the morphology of the

9 DNR-CuNCs was observed directly by AFM, and the DNR-CuNCs size was analyzed
10 on the basis of the height change of DNR nanoribbons. As shown in Figure 1, typical
11 AFM images of the continuous and discontinuous ribbons of DNR, DNR-Cu2+, and
12 DNR-CuNCs clearly reveal the morphology and the size of the as prepared DNR-
13 CuNCs. The dimensions of DNR were determined to be approximately 1.5 nm in height
14 and 15 nm in width (Figure 1A), while those of DNR-Cu2+ were approximately 1.75
15 nm and 15 nm (Figure 1B), respectively. Similarly, the dimensions of DNR-CuNCs
16 were found to be around 1.9 nm in height and 15 nm in width (Figure 1C). We
17 performed a statistical distribution of the 100 data points collected in the AFM image,
18 and the final results were shown in Figure 1D, the average widths of DNR, DNR-Cu2+,
19 and DNR-CuNCs were almost the same, indicating that the adsorption of Cu2+ ions by
20 DNR does not significantly change the structure of DNR scaffolds. The change in the
21 height of DNR-CuNCs compared to DNR or DNR-Cu2+ proved that CuNCs were
22 successfully assembled on DNR, and the DNR-CuNCs average size with 0.4 nm
23 indicating the number of atoms per cluster was N ≈ < 13 atoms. For XPS survey
24 spectrum of DNR-CuNCs in Figure 1E, the peaks containing all elements C, N, O, P,
25 and Cu were observed. The Cu 2p spectrum region proved two distinct peaks at 932.57
26 eV and 952.67 eV (Figure 1F), representing Cu 2p3/2 and 2p1/2, that belongs to Cu (0).

7
1 In addition, there was no observed characteristic peak at near 942 eV, which indicated
2 the absence of Cu2+ in DNR-CuNCs [31].

3 3.3. Characterization of DNR-CuNCs/p-Arg/GCE

4 Figure 2A describes the electrode surface modification with p-Arg to form a

5 conductive polymer with a positive charge. This modification not only improves the
6 electrode's ability to attract DNR-CuNCs through electrostatic interactions but also
7 enhances the stability of the sensing platform. Electrochemical impedance spectra (EIS)
8 and cyclic voltammetry (CV) measurements were conducted using 5.0 mM [Fe
9 (CN)6]3−/4− at each step of the fabrication process. As depicted in Figure 2A, the
10 electrode impedance underwent changes during each modification process. Compared
11 to the bare GCE, the p-Arg/GCE exhibited a smaller semicircle and a steeper slope,
12 indicating enhanced electron transfer properties of the redox probe at the electrode
13 surface. The reduced Ret value was attributed to the presence of p-Arg, which
14 facilitated electron transfer. Given that the guanidine moiety of Arg has a pKa of 12.1,
15 it is reasonable to assume that these groups will be protonated and positively charged
16 at pH 7.5, thereby attracting [Fe (CN)6]3-/4- to enhance the electrochemical performance
17 of GCE [32]. As DNA is known for its negative charge, it would repel [Fe (CN)6]3-/4-
18 from the surface of GCE. Subsequently, handling p-ARG/GCE with DNR-CuNCs,
19 resulted in an increased semicircle diameter, indicating successful attachment of DNR-
20 CuNCs on GCE. Meanwhile, as shown in Figure S2, the CV tests exhibited a similar
21 trend with EIS spectrum. Compared to the bare GCE, the peak current of p-Arg/GCE
22 electrode significantly increased. However, after incubating the p-Arg/GCE with DNR-
23 CuNCs, the signal correspondingly dropped. These results indicate that successful
24 preparation of electrode surface modification was achieved.

25 In order to further explain the mechanism of electrode surface modified by

26 electrostatic adsorption between p-Arg and DNR-CuNCs, the zeta potential values of
27 different substances were measured. The zeta potential of p-Arg was 30.10 mV (Figure
28 2B), indicating that p-Arg film was strongly positively charged. In contrast, DNR-
29 CuNCs were negatively charged -18.98 mV due to the hydrolysis of phosphate groups
30 in DNA structural framework. Negatively charged DNR-CuNCs were electrostatically
31 adsorbed onto the surface of p-Arg film, resulting in a positive zeta value of 25.50 mV
32 for DNR-CuNCs/p-Arg. XPS was also applied to further determine the atomic changes
33 of the electrode modification with p-Arg. The high-resolution C 1s spectrum of p-Arg
34 can be deconvoluted into four peaks, which correspond to C-C group (284.67 eV), C-
35 N group (285.9 eV), N-C(O)-C group (286.5 eV), and C=O group (288.8 eV) (Figure
36 2C). The peak observed at 286.5 eV was attributed to N-C(O)-C linkage during the
37 formation of p-Arg [33], as a consequence, the high-resolution C 1s spectrum of
38 arginine's C-NH2 group (288.0 eV) disappeared (Figure 2D), indicating the successful
39 polymerization of p-Arg [34].

8
1

2 Fig. 2. (A) Schematic diagram of electrode surface modification and corresponding

3 impedance spectra. (B) Zeta potential of p-Arg, DNR-CuNCs and DNR-CuNCs/p-Arg.
4 (C) XPS spectra of p-Arg C1s region, and (D) Arg C1s region. SEM images of carbon
5 cloth (E), p-Arg/C (F) and p-Arg/DNR-CuNCs/C (G).

7 In order to further demonstrate that DNR-CuNCs can be decorated through

8 electrostatic adsorption on the surface of GCE, carbon cloth was used instead of GCE,
9 and the surface morphologies were characterized by SEM. SEM with different
10 magnifications was conducted to characterize the surface morphologies of carbon cloth
11 (C), p-Arg/C and p-Arg/DNR-CuNCs/C. It could be clearly observed that bare carbon
12 cloth showed a smooth surface (Figure 2E), and the obvious rough substance appears
13 after the electropolymerization of Arg in Figure 2F. After the modification of DNR-
14 CuNCs on the surface, the presence of particles can be clearly seen on the enlarged
15 image in Figure 2G, indicating that the DNR-CuNCs had been successfully modified
16 on the surface via electrostatic adsorption. These results demonstrate the successful
17 modification of the biosensor based on Figure 2A.

18 In order to improve the sensitivity of H2O2 detection, the electropolymerization of

19 Arg on GCE was optimized by varying the concentration of Arg and the number of
20 electropolymerization cycles. The results (Figure S3) indicated that the maximum
21 current signal was obtained when the Arg concentration was 10 mM and the number of
9
 1 electropolymerization cycles was 2. However, the application of CV test in detecting
2 low concentrations of H2O2 can be influenced by various factors, leading to inadequate
3 current signal. To overcome this limitation, a steady-state current-time (i-t) method was
4 established to measure the ampere signal response of the DNR-CuNCs/p-Arg/GCE
5 sensor within the low concentration range of H2O2. To optimize the current response
6 performance, several parameters potentially affecting the current signal were
7 systematically investigated using i-t test, including the concentrations of DNR and
8 DNR-CuNCs. Based on the results in Figure S4, a DNR concentration of 1.0 μM was
9 selected as the optimal concentration for preparing DNR-CuNCs, while a CuNCs
10 concentration of 400 μM resulted in maximum i-t current values. Additionally, the
11 selection of the working potential played a crucial role in the analysis system, and -0.8
12 V was chosen as the optimal potential to achieve the highest current response with a
13 lower background, as illustrated in Figure S5, thus ensuring the superior analytical
14 performance of this sensing system. These optimal modification and testing conditions
15 were utilized for subsequent experiments involving the DNR-CuNCs/p-Arg/GCE
16 sensor.

17 3.4. Electrocatalytic Performance of DNR-CuNCs/p-Arg/GCE

18

19 Fig. 3. (A) CV curves of DNR-CuNCs modified electrode with or without adding 10

20 mM H2O2. (B) CV curves of different modified electrodes in 10 mM H2O2 solution. (C)
21 CV curves of DNR-CuNCs modified electrode at different scan rates (10~200 mV/s) in
22 10 mM H2O2 solution, (D) and linear plot of peak current vs ν1/2.

23

24 To investigate the electrochemical performance of DNR-CuNCs, CV was

25 performed to characterize the as prepared electrode. The electrocatalytic behaviors of

10
 1 DNR-CuNCs/p-Arg/GCE for H2O2 reduction were investigated by CV method in 0.1
2 M PBS (pH=7.5) at a scan rate of 100 mV/s. The CV curves in Figure 3A clearly
3 showed that no obvious reduction or oxidation peaks could be observed at DNR-
4 CuNCs/p-ARG/GCE in the absence of H2O2 (as indicated by the grey line). However,
5 when 10 mM H2O2 was introduced into the PBS solution, a distinct reduction peak
6 current at -0.6 V was observed (as indicated by the orange line), thus providing
7 compelling evidence of H2O2 reduction on DNR-CuNCs. As a big comparison, the
8 electrocatalytic reduction of H2O2 during the GCE modification steps was also
9 investigated for bare GCE, p-Arg/GCE and DNR-CuNCs/p-Arg/GCE. The CV curves
10 in Figure 3B indicated that all of the modification processes exhibit no electro-chemical
11 reduction peak compared with the well-defined reduction peak observed in DNR-
12 CuNCs/p-Arg/GCE, which demonstrated that CuNCs served as the active catalytic
13 center for H2O2 reduction and DNR-CuNCs/p-Arg/GCE exhibited good electrocatalytic
14 current.

15 Furthermore, the electron transport behavior of DNR-CuNCs/p-Arg/GCE was

16 investigated by analyzing the effect of scan rates (υ) using CV studies. As shown in
17 Figure 3C, the results indicated that an augment in scan rates from 10 to 200 mV/s was
18 accompanied by a corresponding negative shift from −0.5 to −0.6 V in peak potential,
19 indicating irreversible electrochemical redox reactions. Moreover, it was observed in
20 Figure 3D that the peak currents (Ipc) exhibited a good exponential correlation with the
21 square root of the scan rate (ν), as evidenced by the equation Ipc(μA) = −5.9859 ν1/2-
22 61.3, identical with the previous report, which suggests that the reaction is a typical
23 surface-controlled electrode process [35].

24 3.5. Electrochemical Detection of H2O2

11
1

2 Fig. 4. (A) Signal response of the DNR-CuNCs/p-Arg/GCE sensor for the detection of
3 10~50 mM H2O2 and (B) corresponding calibration curves. (C) Current-time response
4 plots of continuous dropwise addition of H2O2 in PBS (0.1 M, pH 7.5) solution at -0.8
5 V (vs. Ag/AgCl)) and (D) corresponding calibration curves. (E) Selectivity of the DNR-
6 CuNCs/p-Arg/GCE sensor. (F) Stability of the DNR-CuNCs/p-Arg/GCE sensor. 300
7 rpm, 0.1 M PBS, pH 7.5, 25 °C

8 Under optimal conditions, the DNR-CuNCs/p-Arg/GCE sensor demonstrated a

9 distinct stepwise increase in voltammetry current responses upon increasing
10 concentrations of H2O2. Due to the irreversible nature of the redox reaction, the peak
11 current potential shifts to negative potential with increasing concentration of H2O2, as
12 shown in Figure 4A. Furthermore, a superior linear correlation between voltammetry
13 current and H2O2 concentration was established within the concentration range of 1
14 mM~10 mM (Figure S6) and 10 mM~50 mM (S/N=3), as illustrated in Figure 4B. The
15 regression equations were I (mA) = 10.886 C𝐻2𝑂2 (mM) -5.518 (R2=0.9896) and I (mA)

16 = 17.943 C𝐻2𝑂2 (mM) -74.211 (R2=0.9935), respectively, indicating that DNR-

17 CuNCs/p-Arg/GCE sensor exhibits excellent catalytic activity towards H2O2 reduction

18 and can be utilized for detecting H2O2 in a wide concentration range.
12
1 Furthermore, the steady-state i-t method was employed to detect the ampere signal
2 response of the DNR-CuNCs/p-Arg/GCE sensor for the detection of low concentrations
3 of H2O2 in the concentration range of 10 μM to 1 mM. Under optimal conditions, the
4 DNR-CuNCs/p-Arg/GCE sensor demonstrated a clear and stepwise increase in
5 amperometric current responses immediately after the addition of varying
6 concentrations of H2O2, as depicted in Figure 4C. The obtained calibration curve
7 (current versus H2O2 concentration) from Figure 4D with a regression equation of I (μA)
8 = 39.694 C𝐻2𝑂2 (mM) + 1.0698 (R2=0.9889), demonstrated a remarkable sensitivity of

9 567.057 μA cm−2 mM−1 with a linear range of 10 μM to 1000 μM, and a detection limit
10 of 0.56 μM (S/N=3). Comparatively, the DNR-CuNCs/p-Arg/GCE H2O2 sensor
11 demonstrated superior sensing performance, compared with the previously reported
12 Cu-based H2O2 sensors, the detection limit was lower and the linear range was
13 significantly extended, as shown in Table S2.

14 The electrocatalytic activity of DNR-CuNCs/p-Arg/GCE towards H2O2 was

15 investigated through a steady-state i-t response experiment with continuous addition of
16 H2O2 into a stirring PBS solution at the optimized conditions. As illustrated in Figure
17 S7a, the catalytic current exhibited an initial linear dependence on the H2O2
18 concentration, followed by saturation at high substrate concentrations, which is
19 consistent with the saturation curve of the Michaelis-Menten reaction scheme. The
20 calibration plot of the electrocatalytic current versus H2O2 concentration closely
21 matched this curve from 10 μM to 5 mM (Figure S7b), indicating the efficient
22 electrocatalytic activity of DNR-CuNCs in mediating H2O2 reduction.

23 To further investigate the electrocatalytic reduction of H2O2 mediated by DNR-

24 CuNCs, the effect of different potentials on the current signals were assessed. The plots
25 of DNR-CuNCs/p-Arg/GCE electrocatalytic currents in response to H2O2 concentration
26 at different potentials were obtained (Figure S8) and analyzed using the Michaelis-
27 Menten equation [36]:

𝑖·𝐶𝐻2𝑂2
28 𝑖𝑐𝑎𝑡 = (1)
𝑘𝑚𝑎𝑥 + 𝐶𝐻2𝑂2

29 Where icat is the maximum electrocatalytic current for H2O2 reduction and Km is
30 the Michaelis constant. The resulting double-reciprocal graphs of normalized catalytic
31 current vs. H2O2 concentration (Figure S9). The dependence of the catalytic reduction
32 rate on the applied potential was observed in the same H2O2 concentration range,
33 manifest that the electrocatalytic rate for H2O2 reduction is limited by the applied
34 potential. Based on the above experimental findings, a reliable mechanism for the
35 electrocatalytic reduction of H2O2 by the electrogenerated Cu (1) was proposed [37-
36 40]. Firstly, DNR-Cu(0)NCs undergo electrochemical oxidation to form the
37 catalytically active Cu(1) oxidation state (reaction 2). Mechanistically, the saturation of
38 the electrocatalytic current at sufficiently high H2O2 concentrations indicated the
13
1 formation of an adduct between the electrogenerated Cu (1) centers and the H2O2
2 molecule (reaction 3) [36]. Subsequently, reduction of the H2O2 bound to Cu (1) may
3 involve heterogeneous electron transfer (reaction 4), completing the mechanism. These
4 results revealed an efficient electrocatalytic activity of the DNR-CuNCs in mediating
5 the H2O2 reduction through the redox transformation of the metal cluster, and the
6 CuNCs as transition metal nanozymes, exhibit activity similar to that of noble metal
7 nanozymes.

8 DNR ― Cu(0)NCs = Cu(1) + + e ―

9 (2)

10 Cu(1) + + H2O2 = Cu(1) + . O2H2 (3)

11 Cu(1) + . O2H2 = DNR ― Cu(0)NCs + O2 + 2H + + e ―

12 (4)

13 Moreover, comparative experiments conducted under different pH values (Figure

14 S10) demonstrated the stable existence of CuNCs prepared by the DNR template in
15 neutral to weakly alkaline environments. This is in contrast to most sensors that can
16 only function effectively within a specific pH range. The DNR-CuNCs/p-Arg/GCE
17 sensor exhibited a wider range of applications and overcomes the disadvantage of
18 natural enzymes' susceptibility to easy inactivation. Subsequently, we choose the pH
19 7.5 0.1M PBS solution as the electrolyte to detect H2O2 released by cells in order to
20 maintain the biological activity of the cells in an environment that closely resembles
21 physiological pH conditions.

22 To verify the selectivity of the DNR-CuNCs/p-Arg/GCE sensor against

23 interferences such as glucose, epinephrine, AA, and DA that may exist in biological
24 samples, current-time responses of the electrode were tested with 20-fold concentrated
25 glucose, epinephrine, AA, and DA in 0.1 mM H2O2, as shown in Figure 4E. The results
26 indicated that interferences were almost negligible, confirming the excellent selectivity
27 of the DNR-CuNCs catalyst as a biomimetic sensor. The stability of the DNR-
28 CuNCs/p-Arg/GCE was assessed with the addition of 1 mM H2O2, and the current
29 response remained stable for 1600 s, as shown in Figure S11, indicating the stability of
30 the proposed sensor.

31 In order to assess the reproducibility and storage stability of the sensor, five
32 independent DNR-CuNCs/p-Arg/GCE electrodes were examined using the CV
33 technique under identical conditions, as depicted in Figure S12. The relative standard
34 deviation (RSD) for electrode-to-electrode reproducibility was found to be less than
35 4.09%.

36 Furthermore, as illustrated in Figure 4F, the electrode maintained a strong signal

37 even after being stored at 4 °C for a period of 45 days, CuNCs synthesized using DNA
38 nanoribbon templates exhibit enhanced stability time about 2~3 times in comparison to
39 CuNCs prepared with other templates in Table S3. DNA serves as a ligand for the
14
1 protection of CuNCs, whereby ds DNA tightly assembles to form well-organized DNA
2 nanoribbons. The resultant nanoribbons exhibit remarkable structural rigidity
3 surpassing that of dsDNA and DNA nanowires. Consequently, copper clusters
4 encapsulated within DNA nanoribbons demonstrate enhanced stability, enabling
5 prolonged storage. demonstrating the exceptional storage stability of DNR-CuNCs and
6 the reproducibility of the proposed sensor.

7 3.6. In Situ Detection of H2O2 Secreted from Live MCF-7 Cells

9 Fig. 5. (A) Schematic of detection H2O2 released from MCF-7 cells in solution. (B)
10 Current-time response plots of the DNR-CuNCs/p-Arg/GCE sensor in different
11 solution with addition 15 μL PMA (6.25 μg/mL) at -0.8 V. (C) Continuous dropwise
12 addition 15 μL PMA (6.25 μg/mL) stimulated MCF-7 cells (1×103) captured on the
13 electrode surface. (D) Schematic of detection H2O2 released from MCF-7 cells captured
14 on the electrode surface. (E) Detection H2O2 response signals of schematic A (MCF-7
15 cells in solution) and schematic D (MCF-7 cells on electrode surface). (F) Monitored
16 H2O2 concentration at different time intervals.

17

18 H2O2 is an essential biomarker secreted by cells, holds immense potential for

19 disease detection. Consequently, real-time monitoring of H2O2 release from cells has
20 promising clinical diagnostic and pathophysiology applications. Currently, the majority
21 of studies on electrochemical detection of H2O2 released by cells primarily concentrate
22 on cells in solution. Previous studies have reported the rapid release of H2O2 from living
23 cells upon the stimulation of PMA [9], PMA can activate protein kinase C and lead to
24 the release of detectable levels of H2O2 from cells [41]. In order to verify the capability
25 of DNR-CuNCs/p-Arg/GCE sensors to detect the release of H2O2 by MCF-7 cells in
26 solution, PMA stimulation induced the release of H2O2 by MCF-7 cells (Figure 5A),
27 which undergoes electrocatalytic reduction on the CuNCs of the DNR-CuNCs/p-
28 Arg/GCE sensor, resulting in corresponding change in the generated current signal.

15
 1 Through a series of experiments, we confirmed that the observed current response
2 signals originated from H2O2 released by MCF-7 cells and further investigated the
3 effects of PMA stimulation and dosage on cellular H2O2 release. In both 5 mL PBS
4 solutions with and without 2×106 MCF-7 cells, the injection of 15 μL PMA (6.25 μg/mL)
5 induced temporary changes in the current response curve exclusively in the solution
6 containing MCF-7 cells. Conversely, the response curves of the solution without MCF-
7 7 cells and the solution without PMA showed no discernible current fluctuations (Figure
8 5B). These findings suggested that the observed variations in the current signal were
9 specifically attributed to the release of H2O2 from stimulated MCF-7 cells in the
10 presence of PMA. Increasing the addition amount to 15 μL significantly enhanced the
11 current signal and expedited the response speed (Figure S13).

12 However, the H2O2 released by cells is diluted by the buffer solution, leading to a
13 substantial decrease in signal magnitude upon reaching the electrode surface,
14 consequently lowering the sensitivity of the detection. To enhance the detection
15 sensitivity, the programmable properties of DNR was utilized to introduce MUC1
16 protein aptamer sequences for efficient capture of MCF-7 cells onto the electrode
17 surface. This approach enabled real-time detection and in-situ monitoring of H2O2 re-
18 leased by the cells, as depicted in Figure 5D. To demonstrate that DNR-CuNCs/p-
19 Arg/GCE sensors can be captured MCF-7 cells on electrode surface, we used ITO
20 electrodes instead of GCE to acquired fluorescence confocal images [42, 43], Figure
21 S14 indicated that MCF-7 cells can be captured on DNR-CuNCs /p-Arg/GCE sensors.
22 Compared to H2O2 detection in solution, capturing the cells on the electrode surface
23 eliminated the diffusion process of H2O2 from the solution to the electrode surface,
24 resulting in a larger current signal response. The fabricated DNR-CuNCs/p-Arg/GCE
25 sensor was immersed in an MCF-7 cells solution (1×103 cells) for 1.5 h, followed by
26 the injection of 15 μL PMA (6.25 μg/mL) into a 5 mL PBS solution, and the captured
27 MCF-7 cells loaded electrode was placed in it. The results demonstrated a significantly
28 increased current signal response upon capturing MCF-7 cells on the electrode surface,
29 with a calculated value of 5.71 μA (n=3), corresponding to an approximate H2O2
30 concentration of 116.89 μM (Figure 5C). Furthermore, continuous stimulation of the
31 captured MCF-7 cells on the electrode surface resulted in H2O2 generation, but the
32 current signal response decreased after three stimulations, indicating a decline in cell
33 viability upon repeated stimulation and further confirming the origin of the current
34 signal from H2O2 released by MCF-7 cells (Figure 5C) [44]. In comparison, direct
35 detection of H2O2 released by MCF-7 cells in solution, with an increased cell count of
36 4×107 cells, only generated a current signal response of 0.87 μA (n=3), corresponding
37 to an H2O2 concentration of approximately 6.59 μM (Figure 5E). These findings
38 demonstrated that utilizing the programmable characteristics of DNR and introducing
39 aptamer sequences allow for the flexible capture of cell specificity on the electrode
40 surface, enabling in-situ monitoring and significantly enhancing the sensitivity of the
41 fabricated biosensor for H2O2 detection.

42 Furthermore, the performance of the developed DNR-CuNCs//p-Arg/GCE sensor

43 was investigated for long-term monitoring of H2O2 released by MCF-7 cells. The DNR-
16
1 CuNCs/p-Arg/GCE electrode was co-cultured with the cells in a cell incubator, and the
2 changes in H2O2 concentration were monitored at different time intervals (Figure S15).
3 As illustrated in Figure 5F, the current signal corresponding to H2O2 release from MCF-
4 7 cells exhibited a gradual increase over the 0~10 h period, demonstrating the long-time
5 detection ability of the DNR-CuNCs/p-Arg/GCE sensor in detecting H2O2 [45]. These
6 results highlight the exceptional tunability of the developed DNR-CuNCs/p-Arg/GCE
7 sensor, enabling highly sensitive detection of in situ H2O2 release from cancer cells.

8 4. Conclusion

9 By utilizing the electrocatalytic platform consisting of positively charged p-Arg to

10 immobilize DNR-CuNCs, we successfully constructed a sensor for the detection of
11 H2O2. The specific binding sites on DNR were exploited to anchor Cu2+ ions, which
12 were subsequently reduced by SA to generate CuNCs. This facile method offers the
13 benefits of straightforward preparation, low cost, and mild conditions, while the
14 synthesized DNR-CuNCs exhibited great catalase-like activity and stability, resulting
15 in long-time efficient electrocatalytic reduction of H2O2, ranking the best stability
16 reported CuNCs sensors. Moreover, we exploited the programmability of the DNR
17 framework to utilize the MUC1 protein aptamer into the DNR structure and captured
18 MCF-7 cells on the electrode surface, in contrast to detecting H2O2 with MCF-7 cells
19 dispersed in a solution, capturing the cells on the electrode surface enable eliminated
20 the diffusion process of H2O2 from the solution to the electrode surface, and enhanced
21 sensitivity of the fabricated biosensor for H2O2 detection. The DNR-CuNCs/p-
22 Arg/GCE sensor was successfully employed to detect H2O2 in situ and first realized to
23 monitor the long-time horizon (10 h) release of H2O2 from MCF-7 living cells. It is our
24 belief that this method of synthesizing CuNCs by exploiting DNA folding structures
25 holds tremendous promise for a wide range of applications and can utilization aptamer
26 to fabricate electrochemical sensors for the detection of various analytes.

27

28 Declaration of Competing Interest

29 The authors declare that there are no known competing financial interests or
30 personal relationships that could have potentially influenced the work presented in this
31 paper.

32

33 Acknowledgments

34 This work is supported by grants 22105159 and 22374118 from the National
35 Natural Science Foundation of China, 2022JZ-08 from The Natural Science Foundation
17
1 of Shaanxi Province, Xi'an Key Laboratory of Functional Supramolecular Structure and
2 Materials (CFZKFKT23004), the Open Fund of Key Laboratory of Synthetic and
3 Natural Functional Molecules, Ministry of Education (KLSNFM2020009), the “Top-
4 rated Discipline” construction scheme of Shaanxi higher education and “Tang scholar”.

6 Appendix A. Supplementary data

7 Supplementary data to this article can be found online at https://doi...

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2 Declaration of Competing Interest

3 The authors declare that there are no known competing financial interests or
4 personal relationships that could have potentially influenced the work presented in this
5 paper.

24