Colorimetric sensor for detecting lead ions and application of colorimetric sensor

Abstract

The invention relates to the technical field of lead ion detection, in particular to a lead ion detection colorimetric sensor and application thereof.The lead ion detection colorimetric sensor comprises hemin, a substrate chain S, an enzyme chain Dz and a DNA chain DS with three palindromic structures; in the absence of Pb < 2 + >, the substrate chain S and the enzyme chain Dz are hybridized to form a stable S-Dz double chain; when Pb is introduced, an enzyme chain Dz can be activated, and lead ions are specifically recognized and used as cofactors. Through an annealing process, the DNA chain DS is subjected to intramolecular hybridization and self-folding so as to be self-assembled into a definitely-defined and monodisperse DNA nanosphere; the substrate chain S and the enzyme chain Dz can be hybridized to form a stable S-Dz double chain; a plurality of S-Dz are concentrated on a limited nanosphere surface (DS-S-Dz), the catalysis direction is optimized, side reactions are reduced, and a signal DNAzyme can be quickly formed, so that a color signal is remarkably amplified, and lead ion detection with higher sensitivity, lower background and higher stability is realized.

Description

Technical Field

[0001] This invention relates to the field of lead ion detection technology, specifically to a lead ion detection colorimetric sensor and its application. Background Technology

[0002] Functional nucleic acids, due to their high specificity, high sensitivity, good stability, low cost, flexible design, and ease of synthesis and modification, have wide applications in gene detection, environmental monitoring, and food safety. Currently, functional nucleic acid-based biosensors are mainly used for lead ion detection, particularly for Pb. 2+ There are two types: specific DNAzyme and G4 DNAzyme. Pb 2+ Specific DNA enzymes consist of a substrate strand and an enzyme strand, and are a type of double-stranded DNA. In Pb... 2+ In the presence of enzymes, the substrate chain is cleaved into two segments at a cleavage site containing a single ribonucleotide. The discovery of the G4 DNAzyme and related research provides a good platform for developing excellent lead detection sensors. It is formed by the complex of hemin chloride and a G-quadruplex. The G-quadruplex is formed by G-rich nucleic acid sequences and is a functional nucleic acid capable of binding hemin chloride to form peroxidase-like activity, and contains various metal cations (such as K+). + Pb 2+ DNAzymes can enable specific nucleic acid sequences to form stable G-quadruplex structures. Compared with traditional proteases, DNAzymes have unique advantages such as high thermal stability, low cost, and ease of synthesis and modification. Therefore, they are widely used in the detection of heavy metal ions.

[0003] Combining DNAzymes with hard nanoparticles (gold, silver) has become an effective method for improving sensor performance. MEMON et al. designed a label-free colorimetric nanosensor that uses unmodified gold nanoparticles (AuNPs) as an indicator, achieving a detection limit as low as 0.2 nM under optimal conditions. This method enables visual detection of Pb. 2+ These nanoparticles exhibit excellent sensitivity and selectivity. While they provide a larger contact area for the DNAzyme detection system, improving stability and sensitivity compared to traditional methods, the addition of AuNPs introduces uncertainties. For example, the long preparation time and complex synthesis of AuNPs may lead to interference from complex sample matrices, resulting in reduced efficiency of metal ion-enzyme collision and binding, and decreased catalytic activity.

[0004] Therefore, sensors with higher sensitivity, lower background and stronger stability are needed to improve the stability, sensitivity and practical application of DNAzyme detection, and effectively reduce interference from other substances in complex sample matrices. Summary of the Invention

[0005] The purpose of this invention is to provide a lead ion detection colorimetric sensor with higher sensitivity, lower background and stronger stability.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention proposes a colorimetric sensor for lead ion detection, comprising heme chloride, substrate chain S, enzyme chain Dz, and a DNA chain DS having a three-palindromic structure; wherein the substrate chain S is in the absence of Pb 2+ Under certain conditions, substrate strand S and enzyme strand Dz will hybridize to form a stable S-Dz double strand; when Pb is introduced... 2+ At this time, enzyme chain Dz will be activated, specifically recognizing and utilizing lead ions as a cofactor; The nucleotide sequence of the substrate chain S is shown in SEQ ID No. 1; The nucleotide sequence of the enzyme chain Dz is shown in SEQ ID No. 2; The nucleotide sequence of the DNA chain DS is shown in SEQ ID No. 3.

[0007] The present invention also includes the application of the aforementioned lead ion detection colorimetric sensor in the detection of lead ions.

[0008] Furthermore, the method for detecting lead ions is as follows: (1) Anneal the DNA strands using DS and cool to room temperature; (2) Add DNA chain DS solution, substrate chain S solution, enzyme chain Dz solution, Hepes buffer and the sample to be tested to the centrifuge tube, then add heme chloride, incubate to form heme chloride / G-tetramplex complex; (3) Cool to room temperature, add ABTS solution, H2O2 and water, and finally use a UV spectrophotometer to measure the absorbance at 420 nm.

[0009] Furthermore, the annealing temperature in step (1) is 90°C and the time is 3 minutes.

[0010] Furthermore, in step (2), the concentrations of the DNA chain DS solution, substrate chain S solution, and enzyme chain Dz solution are all 10 μM; the concentration of the Hepes buffer is 25 mM; and the concentration of the heme chloride is 100 μM.

[0011] Furthermore, the incubation temperature in step (2) is 37°C and the incubation time is 30 minutes.

[0012] Furthermore, the pH of the Hepes buffer solution in step (2) is 6.

[0013] Furthermore, in step (3), the concentration of the ABTS solution is 50 μM and the concentration of the H2O2 is 1 M.

[0014] Compared with existing technologies, the DNA strand DS undergoes intramolecular hybridization and self-folding through the annealing process, thereby self-assembling into a well-defined, monodisperse DNA nanosphere. This bottom-up DNA self-assembly process is highly predictable and reproducible, and can produce a biocompatible nanostructure with precisely controlled size and surface chemistry. The substrate chain S and the enzyme chain Dz can hybridize to form a stable S-Dz double strand; multiple S-Dz strands concentrate on a finite nanosphere surface (DS-S-Dz), resulting in a significant increase in local concentration and accelerating the formation of Pb. 2+ The cleavage reaction is triggered, and the conformational freedom of the DNA strand DS is restricted, optimizing the catalytic direction and reducing side reactions. This spatially confined microenvironment acts as a catalytic center, ensuring that once the G-rich fragment is released, a signal DNAzyme can be rapidly formed, thereby significantly amplifying the color signal and achieving lead ion detection with higher sensitivity, lower background and stronger stability. Attached Figure Description

[0015] Figure 1 The schematic diagram in (1A) illustrates the effect of DS-S-Dz on Pb. 2+ (1B) Detection principle; (1C) Atomic force microscopy characterization of DS-S-Dz; (1D) Representative UV spectra of different sample configurations; (1D) Summary of absorbance intensities at 420 nm corresponding to the systems in (1C), with insets showing the color changes of these samples; (1E) Native (12%) PAGE analysis confirming Pb 2+ Inducing DS-S-Dz cuts, where the solid symbol " "and hollow sign" "Indicates the presence and absence of a specific component, respectively, and the error bar represents the average value ±SD (n=3).

[0016] Figure 2 (2A)DS-S-Dz for different concentrations (0-720 nM) of Pb 2+ (2B) Ultraviolet spectrum; (2B) Maximum absorbance and Pb 2+ Linear relationship between concentrations (0-720 nM); (2C) DS-S-Dz versus Pb 2+ (480 nM) and potential interfering metal ions (Mg) 2 +Zn 2+ Ag + Hg 2+ Al 3+ Fe 2+ and Ca 2+ Selectivity testing of potential interfering cations and mixtures thereof, wherein the concentration of interfering metal ions was 2.5 μM; (2D) comparison of Pb 2+ Radar plot of absorbance of coexisting ions, with error bars representing the average value ±SD (n=3).

[0017] Figure 3 Application of the DS-S-Dz sensor in detecting lead ions in real samples; comparison of lead ion recovery rates in samples with 480 nM lead ions added in different environmental and food matrices (lake water, tap water, wastewater, and milk) using the DS-S-Dz sensor (3A) and the S-Dz sensor (3B); (3C) shows a geographical map of water sample collection points in Fuzhou City. (3D) Detection of endogenous lead ion levels in real water samples collected from different universities, watersheds, and parks using the DS-S-Dz sensor. Statistical significance is expressed as (*p<0.05 compared to the 4.8 nM standard; p>0.05 compared to the blank control); error bars in (3A) and (3B) represent the standard deviation of three repeated measurements (n = 3).

[0018] Figure 4 (4A) Atomic force image of DS; (4B) Atomic force image of S-Dz.

[0019] Figure 5 (5A), (5B), and (5C) are DLS diagrams of DS, S-Dz, and DS-S-Dz, respectively; (5D) are the zeta potentials of DS, S-Dz, and DS-S-Dz.

[0020] Figure 6 The effects of different parameters on the absorbance of DS-S-Dz: (6A) incubation time, (6B) incubation temperature, (6C) different ratios of DS, Dz and S, (6D) heme chloride concentration, (6E) pH, and (6F) K + Concentration; maximum absorbance wavelength is 420 nm, and error bars represent mean ± standard deviation (n=3).

[0021] Figure 7 (7A) Pb at different concentrations (0 - 720 nM) 2+ The ultraviolet spectrum of S-Dz exists; (7B) maximum absorbance and Pb 2+Linear relationship between concentrations (0 - 720 nM), error bars represent mean ± standard deviation (n = 3).

[0022] Figure 8 (8A) The reaction of DS-S-Dz to different metal ions in tap water; (8B) A diagram illustrating how DS-S-Dz distinguishes metal ions in tap water.

[0023] Figure 9 (9A) After storage at 4 °C for 1 to 9 days, DS-S-Dz showed positive results for Pb. 2+ Stability of detection; (9B) at different Pb 2+ Repeatability of DS-S-Dz at concentrations (0, 240, and 480 nM), with error bars representing mean ± standard deviation (n = 3).

[0024] Figure 10 To recover lead ions (Pb) from different environmental samples (lake water, milk, wastewater, tap water) using DS-S-Dz and S-Dz. 2+ In the case of Pb 2+ The concentration is 240 nM, and the error bars represent the mean ± standard deviation (n = 3).

[0025] Figure 11 This is an actual sample detected by inductively coupled plasma mass spectrometry.

[0026] Figure 12 (12A) Design and fabrication process of S-Dz; (12B) Assembly process of DNA nanospheres; (12C) Detection principle of DS-S-Dz sensor. Detailed Implementation

[0027] The technical solution of the present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0028] The oligonucleotides of DNA used in this invention (see Table 1) were all synthesized by Shanghai Sangon Biotech Co., Ltd. (Shanghai, China).

[0029] Table 1

[0030] Unless otherwise specified, all water used in this invention is ultrapure water (impedance value 18.25MΩ).

[0031] This invention provides a colorimetric sensor for lead ion detection with higher sensitivity, lower background and stronger stability, consisting of two modules, including heme chloride, substrate chain S, enzyme chain Dz and a DNA chain DS with three palindromic structures. like Figure 12 As shown, the first module is the core molecular switch and signal generation system. The nucleotide sequence of the substrate chain S is shown in SEQ ID No. 1. The substrate chain S has a single adenosine ribonucleic acid (rA) site as a specific cleavage site, and a G-rich sequence as a precursor to the signal catalyst; the substrate chain S in the absence of Pb 2+ In this case, the substrate chain S and the enzyme chain Dz (nucleotide sequence as shown in SEQ ID No. 2) hybridize to form a stable S-Dz double strand. This hybridization effectively “closes” the G-rich sequence, locking it in a linear conformation, thereby inhibiting its folding into a functional G-quadruplex. This locked “off” state is crucial for minimizing background signal and ensuring a low baseline signal. When Pb is introduced 2+ When activated, the enzyme chain Dz specifically recognizes and utilizes lead ions as a cofactor to catalyze the hydrolytic cleavage of substrate chain S at the rA site. This cleavage triggers the release of G-rich fragments, switching the system to the "on" state. The released fragments then fold into their thermodynamically stable G-quadruplex structure. Subsequently, this G-quadruplex captures a heme chloride cofactor, forming a highly active heme chloride / G-quadruplex (G4 DNAzyme). In the presence of H2O2, it catalyzes the oxidation of the substrate ABTS, producing a bright blue-green color, thus providing quantitative and visual readings. The second module is the amplification and stabilization module. The nucleotide sequence of the DNA chain DS is shown in SEQ ID No. 3. This chain contains three palindromic structures (self-complementary structures) interspersed with flexible linking segments, and a single-stranded "tail" on each side. These tails can serve as programmable anchors. Through the annealing process, the DNA chain DS undergoes intramolecular hybridization and self-folds to assemble into a well-defined, monodisperse DNA nanosphere. Compared with the cumbersome and often unstable synthesis methods of "hard" inorganic nanoparticles, this bottom-up DNA self-assembly process is highly predictable and reproducible, and can produce a biocompatible nanostructure with precisely controlled size and surface chemistry. By programming the tail end to concentrate multiple S-Dz particles onto a finite nanosphere surface (DS-S-Dz), the local concentration is significantly increased, accelerating the formation of Pb. 2+The cleavage reaction is triggered, and the conformational freedom of the DNA strand DS is restricted, optimizing its catalytic direction and reducing side reactions. This spatially confined microenvironment acts as a catalytic center, ensuring that once the G-rich fragment is released, a signal DNAzyme can be rapidly formed, thereby significantly amplifying the color signal.

[0032] The sensor detects lead ions using the following method: (1) Anneal the DNA strand DS at 90°C for 3 minutes and cool it to room temperature. Store it in a refrigerator at 4°C for later use. (2) Add 1 µL of DNA strand DS solution, 1 µL of substrate strand S solution, 1 µL of enzyme strand Dz solution, and 20 µL of Hepes buffer (25 mM HEPES, 100 mM NaCl, 5 mM KCl, 0.05% Triton) to the centrifuge tube. TM X-100, pH=6) and the sample to be tested were added, and then 2 µL of hemin chloride (100 μM) was added and incubated at 37°C for 30 minutes to form a hemin chloride / G-tetrachain complex. The DNA chain DS solution, substrate chain S solution, and enzyme chain Dz solution are solutions in which the DNA chain DS, substrate chain S, and enzyme chain Dz are each dissolved in 1×TE buffer, and the concentration of each solution is 10 μM. (3) Cool to room temperature, add 2 µL of ABTS solution (50 μM), 2 µL of H2O2 (1 M) and water to 350 µL, and finally use a UV spectrophotometer to measure the absorbance at 420 nm. Example 1:

[0033] Feasibility verification like Figure 1 As shown in (1A), the sensor provided by the present invention is related to Pb 2+ After incubation, the substrate chain S is cleaved, releasing a G-rich sequence that binds to heme chloride to form a heme chloride / G-tetrachain complex, which catalyzes the oxidation of the chromogenic substrate ABTS, resulting in a noticeable color change and enhanced absorbance.

[0034] The method based on this invention will be compared with the traditional S-Dz-based Pb method. 2+ The detection methods were compared, and both were validated using AFM, dynamic light scattering (DLS), UV-Vis spectroscopy, and polyacrylamide gel electrophoresis (PAGE).

[0035] like Figure 1 As shown in (1B), DS-S-Dz exhibits a dispersed spherical structure with a height of approximately 10 nm and a diameter of 500 nm, combined with Figure 4Atomic force microscopy images of DS and S-Dz further confirmed the formation of DS-S-Dz.

[0036] like Figure 5 As shown, dynamic light scattering (DLS) reveals that the average hydrodynamic diameter of DS-S-Dz is 696.9 nm, indicating a uniform size distribution. Zeta potential analysis shows that the value of DS is -7.8 mV, the value of S-Dz is -15.2 mV, and the value of DS-S-Dz is -25.1 mV, further confirming the formation of DS-S-Dz.

[0037] Ultraviolet-visible spectrum such as Figure 1 As shown in (1C-1D), the sensor's performance in the absence of Pb is further verified. 2+ In the presence of Pb, the substrate chain S is not cleaved, and both DS-S-Dz (line a) and regular S-Dz (line c) have very low absorbance. 2+ Subsequently, the substrate chain S is cleaved, and the G-rich sequence binds to heme chloride to form a heme chloride / G-quadruplex complex, which catalyzes the oxidation of ABTS by H₂O₂, generating a colorimetric signal with significantly enhanced absorbance and a noticeable color change. Compared to the traditional S-Dz, DS-S-Dz exhibits a lower background signal, thereby improving sensor sensitivity.

[0038] like Figure 1 As shown in (1E), the corresponding DNA assembly process was also verified by 12% non-denaturing polyacrylamide gel electrophoresis, with Pb 2+ The addition of [substrate name] resulted in the appearance of some new bands at the lower position of channel 5, indicating that the substrate chain S was cleaved, releasing the G-rich sequence. After anchoring S and Dz onto DNA nanospheres, slower-migrating bands were observed, indicating the formation of DS-S-Dz (6 channels); some new bands with lower molecular weights also appeared in channel 7, representing the cleavage products of this sequence. These results demonstrate the successful construction of the colorimetric sensor of this invention, validate the working principle, and exhibit a significant signal amplification effect compared to traditional free solution methods. Example 2:

[0039] Sensor detection performance optimization like Figure 6 As shown in (6A), in the analysis of Pb at 480 nM 2+ When, A / A o It reaches its maximum value at 30 minutes; like Figure 6 As shown in (6B), enzyme chain cleavage experiments were performed at five different temperatures (4℃, 16℃, 25℃, 37℃ and 55℃), A / A o It reaches its maximum value at 37℃; like Figure 6 As shown in (6C), the background signal is minimized when the molar ratio of substrate chain S, enzyme chain Dz, and DNA chain DS is 1:1:1.

[0040] like Figure 6 As shown in (6D), when the concentration of heme chloride increases from 0.1 µM to 200 µM, the A / A ratio... o The value increases accordingly until it reaches a stable value at 100 µM, indicating that the saturation concentration of heme chloride is 100 µM; like Figure 6 As shown in (6E), the optimal pH value for Hepes buffer is 6; like Figure 6 As shown in (6F), the optimal K + The concentration is 5 mM. Example 3:

[0041] Performance Analysis of the Colorimetric Sensor of the Invention like Figure 2 As shown in (2A), at a wavelength of 420 nm, the absorbance systematically increases with increasing lead ion concentration from 0 to 720 nM. This quantitative change is accompanied by a distinct color transition from light green to deep blue-green, confirming the practicality of this sensor in spectrophotometry and qualitative analysis. Figure 2 As shown in (2B), the absorbance of the sensor exhibits a strong linear relationship with the lead ion concentration in the range of 0.96 nM to 720 nM: ΔA = 0.0008c + 0.4004(R). 2 =0.9917). The lowest limit of detection (LOD) is 0.96 nM, which is comparable to the traditional non-stented S-Dz system (LOD = 48 nM, Figure 7 Compared to [previous model], the sensitivity is improved by 50 times, providing clear evidence for the signal amplification effect inherent in DNA nanosensor design. Notably, this detection limit is significantly lower than the 72 nM detection level for lead in drinking water set by the U.S. Environmental Protection Agency (EPA), indicating that the sensor of this invention is well-suited for detecting environmentally relevant lead ion concentrations. Selectivity is a crucial characteristic for practical sensors.

[0042] like Figure 2 As shown in (2C), the DS-S-Dz colorimetric sensor of the present invention was tested with a group of common metal ions (e.g., magnesium ions, zinc ions, mercury ions, iron ions, calcium ions) at a concentration (480 nM) exceeding the concentration of lead ions by more than 40 times. Only lead ions elicited a significant colorimetric response. Figure 2As shown in (2D), even in a complex mixture containing all interfering ions, the signal is statistically not significantly different from that of individual lead ions, which indicates that the sensor has excellent selectivity and strong anti-interference ability.

[0043] To further verify its practical applicability, the sensor of this invention was tested on 30 independent tap water samples (collected from the Minjiang University campus). These samples were randomly supplemented with lead ions or one of five different interfering metal ions. The detection results are as follows: Figure 8 As shown, the sensor accurately identified pre-labeled lead-containing samples, demonstrating its reliability and effectiveness in real-world sample environments.

[0044] In addition, such as Figure 9 As shown, the sensor exhibits excellent operational stability, maintaining the integrity of its response for over 9 days, and demonstrates high reproducibility with a relative standard deviation (RSD) of less than 4% for repeated measurements. Compared with other reported Pb... 2+ The comparison of detection methods shows that the DNA nanosphere-based sensor of this invention has competitive advantages in terms of detection limit, assembly, and rapid detection time, making it a very effective analytical tool. Example 4:

[0045] Applicability of the colorimetric sensor of this invention The present invention detects lead ions (Pb). 2+ The accuracy of the study was assessed using a variety of challenging real-world samples, including lake water, milk, industrial wastewater, and tap water. For example... Figure 3 As shown in (3A), the accuracy of the sensor is demonstrated by the spiked recovery rate. For samples with 480 nM lead ions added, the colorimetric sensor consistently achieved excellent recoveries ranging from 90% to 110%. Figure 10 As shown, for the sample with 240 nM lead ions added, all relative standard deviations (RSDs) were below 8%, which confirms the reliability of the sensor at different concentrations.

[0046] In comparison, such as Figure 3 As shown in (3B), conventional non-supported S-Dz systems exhibit poor and unstable recovery rates, ranging from 65% to 202% under the same conditions. This contrast highlights the enhanced stability and robust resistance to matrix effects of the colorimetric sensor of the present invention.

[0047] To further demonstrate the potential of the colorimetric sensor of this invention in environmental monitoring, it was used to analyze lead ions (Pb) in nine water samples collected from different locations in Fuzhou City (including parks, lakes, and the Minjiang River). 2+ Pollution was screened; sampling map is available. Figure 3(3C). For example... Figure 3 As shown in (3D), preliminary screening results indicated no significant lead ion contamination at any of the collected sites, and the sensor signal was not statistically significantly different from the blank control group (p>0.05). To confirm the sensor's ability to detect low levels of contamination in these real matrices, these samples were subsequently supplemented with an environmentally relevant lead ion concentration (4.8 nM). The resulting signal was highly statistically significant (p<0.05). Crucially, as... Figure 11 As shown, these findings were independently verified and are consistent with the results of the standard method—inductively coupled plasma mass spectrometry (ICP-MS).

[0048] While specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments described are merely illustrative and not intended to limit the scope of the invention. Modifications and variations made by those skilled in the art in accordance with the spirit of the invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A colorimetric sensor for lead ion detection, characterized in that: It includes heme chloride, substrate chain S, enzyme chain Dz, and a DNA chain DS having a three-palindromic structure; said substrate chain S is in the absence of Pb 2+ Under certain conditions, substrate strand S and enzyme strand Dz will hybridize to form a stable S-Dz double strand; when Pb is introduced... 2+ At this time, enzyme chain Dz will be activated, specifically recognizing and utilizing lead ions as a cofactor; The nucleotide sequence of the substrate chain S is shown in SEQ ID No. 1; The nucleotide sequence of the enzyme chain Dz is shown in SEQ ID No. 2; The nucleotide sequence of the DNA chain DS is shown in SEQ ID No.

3.

2. The application of the lead ion detection colorimetric sensor as described in claim 1 in the detection of lead ions.

3. The application according to claim 2, characterized in that: The method for detecting lead ions is as follows: (1) Anneal the DNA strands using DS and cool to room temperature; (2) Add DNA chain DS solution, substrate chain S solution, enzyme chain Dz solution, Hepes buffer and the sample to be tested to the centrifuge tube, then add heme chloride, incubate to form heme chloride / G-tetramplex complex; (3) Cool to room temperature, add ABTS solution, H2O2 and water, and finally use a UV spectrophotometer to measure the absorbance at 420 nm.

4. The application according to claim 3, characterized in that: The annealing temperature in step (1) is 90°C and the time is 3 minutes.

5. The application according to claim 3, characterized in that: In step (2), the concentrations of the DNA strand DS solution, substrate strand S solution, and enzyme strand Dz solution are all 10 μM; the concentration of the Hepes buffer is 25 mM; and the concentration of the heme chloride is 100 μM.

6. The application according to claim 3, characterized in that: The incubation temperature in step (2) is 37°C and the incubation time is 30 minutes.

7. The application according to claim 3, characterized in that: The pH of the Hepes buffer solution in step (2) is 6.

8. The application according to claim 3, characterized in that: In step (3), the concentration of the ABTS solution is 50 μM and the concentration of the H2O2 is 1 M.

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