Method for simultaneously detecting two miRNAs (micro ribonucleic acids) by using double colorimetric biosensor based on colloidal gold and nano enzyme

Abstract

The invention relates to the technical field of biology, and particularly discloses a method for simultaneously detecting two miRNAs (micro Ribonucleic Acid) by a double colorimetric biosensor based on GNPs and nano-enzyme. The analysis method comprises the following steps: firstly, fully mixing the prepared DNA functionalized GNPs, DNA functionalized magnetic beads, hairpin DNA and a sample to be detected, and incubating; and carrying out magnetic separation to obtain an upper-layer red clear solution and a magnetic bead precipitate. And further, adding a catalytic substrate solution such as K < + >, Hemin and H2O2 / ABTS into the magnetic bead precipitate, mixing, carrying out incubation reaction, and carrying out magnetic separation to obtain a green clarified solution. Finally, the concentrations of the two tumor markers are obtained through naked eye observation or UV-vis determination of the two collected clarified solutions with different colors. The method does not need complex nanoprobe synthesis, does not need expensive precise instruments, and has the advantages of high accuracy based on multi-marker detection, low operation cost, simple detection, good selectivity and the like.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for simultaneously detecting two miRNAs by a dual colorimetric biosensor based on gold colloid and nanozyme. Background Art

[0002] Tumor markers are molecules related to tumors that appear in blood and tissues. Finding a method for early and sensitive detection of tumor markers is crucial for tumor diagnosis and treatment. In recent years, relevant studies have shown that miRNA is a biomarker for early screening of tumors, and their abnormal expression levels are closely related to the occurrence and development of tumors. In order to further improve the sensitivity of detection and achieve early diagnosis of tumors, a series of detection methods based on nucleic acid signal amplification have been established. According to whether the enzyme is involved, the signal amplification method can be divided into two categories: enzyme-mediated signal amplification and non-enzyme-mediated signal amplification. The enzymes used in enzyme-mediated signal amplification technology are expensive, have a short storage time, and the enzyme activity is easily affected by the environment. Signal amplification based on hybridization chain reaction (HCR) and catalytic hairpin self-assembly reaction (CHA) is widely used for sensitive detection of miRNAs because it does not require enzyme catalysis, professional technicians and amplification equipment, can react at room temperature, is easy to operate, has multiple selectivity, stable reaction, strong detection specificity and high sensitivity.

[0003] Although enzyme-free signal amplification strategies have significantly improved the sensitivity of detection, these detection technologies still require the use of complex and expensive instruments and equipment to output detection signals. Colorimetric biosensor technology does not require expensive and sophisticated instruments and equipment, and has the advantages of low cost, simplicity, and practicality. In addition, most current diagnostic methods often only target the detection of a single tumor marker, which may lead to the presence of false positive signals, resulting in low accuracy of tumor diagnosis. Therefore, it is of great significance to develop a simple colorimetric diagnostic method that can simultaneously detect multiple tumor-related miRNAs. Summary of the invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for simultaneously detecting two tumor markers using a dual colorimetric biosensor based on gold colloid (GNPs) and nanozymes. The present invention does not require complex nanoprobe synthesis and expensive precision instruments, and has the advantages of high accuracy based on multi-marker detection, low operating cost, simple detection, and good selectivity.

[0005] Technical solution: In order to solve the above technical problems, the present invention provides a method for simultaneously detecting two tumor markers using a dual colorimetric biosensor based on GNPs and nanozymes. The analytical method comprises the following steps: first, the prepared DNA functionalized GNPs, DNA functionalized magnetic beads, and hairpin DNA are fully mixed with the sample to be tested and incubated; after magnetic separation, an upper red clear solution and a magnetic bead precipitate are obtained; further, K is added to the magnetic bead precipitate. + , Hemin and H2O2 / ABTS catalytic substrate solutions, mixed and incubated, and a green clear solution was obtained after a magnetic separation step; finally, the solution was observed by naked eye or UV- vis The spectrometer measures the collected two clear solutions of different colors to obtain the concentrations of the two tumor markers.

[0006] Preferably, the GNPs concentration is 13 nM and the particle size is 10-50 nm.

[0007] Preferably, the preparation method of the DNA functionalized GNPs comprises the following steps: adding SH-10b DNA solution to GNPs, mixing and shaking and incubating for 10 to 30 min; next, adding PB buffer to the above solution, shaking and incubating for 10 to 30 min; then, adding 2 mol / L NaCl solution to the gold colloid solution every 20 min for aging reaction until the NaCl solution reaches 500 mM, continuing to place the mixed solution on a shaker for overnight reaction, and finally, using PBS buffer to centrifuge and wash the GNPs to obtain 10b-DNA functionalized GNPs.

[0008] Preferably, the PB buffer solution is: 10 mM NaH2PO4, 2.7 mM KCl, 2 mM KH2PO4, pH 7.0-7.5; the PBS buffer solution is: 137 mM NaCl, 10 mM NaH2PO4, 2.7 mM KCl, 2 mM KH2PO4, pH 7.0-7.5.

[0009] Preferably, the magnetic beads have a concentration of 0.2-1 mg / L and a particle size of 0.2-1 μm.

[0010] Preferably, the DNA concentration of the SH-10b is 25 nM to 100 nM.

[0011] Preferably, the method for preparing the DNA functionalized magnetic beads comprises the following steps: adding the DNA solution of Bio-HO and Bio-10b to the streptavidin modified magnetic bead solution, and placing the solution at 25 to 40 oC shaker and incubate for 15 to 60 minutes; finally, separate and remove the supernatant using magnetic separation technology, and wash the magnetic beads with Tris buffer to fully remove excess DNA, ultimately obtaining Bio-H0 and Bio-10b co-modified magnetic beads.

[0012] Preferably, the streptavidin-modified magnetic bead solution is: 10 mM Tris-HCl, pH 7.0-8.0, and contains 1 mM EDTA, 1 mol / L NaCl and 0.1% Tween-20.

[0013] Preferably, the concentrations of the DNA solutions of Bio-HO and Bio-10b are both 500 nM~1 μM.

[0014] Preferably, the Tris buffer is: 20 mM Tris-HCl, pH 7.0-8.0, 0.1% Tween-20.

[0015] Preferably, the sample to be tested described in the present invention is a miRNAs biomarker in tumor diagnosis.

[0016] Preferably, the incubation reaction of the present invention is carried out at a temperature of 25 to 40 o C, incubation time is 0.5~2 hours.

[0017] Preferably, the upper red clear solution of the present invention can be observed by naked eye or UV- vis The absorbance of the solution at 520 nm is measured to achieve semi-quantitative / quantitative detection of a miRNA in the sample.

[0018] Preferably, the catalytic reaction step of the present invention is: resuspending the magnetic bead precipitate after magnetic separation in MES buffer solution, adding Hemin aqueous solution to the magnetic bead mixture, and placing it at 37 o C for 0.5-1 h, then add H2O2 / ABTS aqueous solution and continue incubating the reaction for 10-30 min. Finally, separate the magnetic beads using magnetic separation technology and collect the upper green clear solution. Observe the color of the solution by naked eye or UV- vis The absorbance of the solution at 420 nm is measured to achieve semi-quantitative / quantitative detection of another miRNA in the sample.

[0019] Preferably, the MES buffer contains NaCl, KCl, DMSO and Tween-20. The concentration of the MES buffer is 10-100 mM, the pH is 4.0-60, the KCl concentration is 10-150 mM, the DMSO content is 1%, and the Tween-20 content is 0.1%.

[0020] Preferably, the final concentration of Hemin is 1-4 μM.

[0021] Preferably, the final concentration of H2O2 / ABTS is 1-5 mM. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a schematic diagram of the method of simultaneously detecting two tumor markers, miR-21 and miR-10b, using a dual-colorimetric biosensor based on nanozymes and gold nanoparticles; Figure 2 a is the UV-visible (UV- vis )Absorption spectrum; Figure 2 b is the TEM image of the synthesized GNPs; Figure 2 c is the UV-ray diffraction of SH-10b functionalized GNPs. vis Absorption spectrum; Figure 2 d is the UV-ray diffraction of GNPs functionalized with SH-10b at different concentrations. vis Absorption spectra and their corresponding photographs; Figure 3 The supernatant was incubated with 200 nM biotinylated DNA and different concentrations of streptavidin magnetic beads. vis Absorption spectrum; Figure 4 It is the agarose gel electrophoresis diagram of different DNA complexes; Figure 5 a~c show the UV- vis Absorption spectra spectra, photographs and linear relationship diagrams; Figure 5 d~f are UV- vis Absorption spectra spectra, photographs and linear relationship diagrams; Figure 6 It is the selectivity of the biosensor to different samples to be tested. DETAILED DESCRIPTION

[0023] The present invention is further described below through specific embodiments and drawings. It should be pointed out that, for ordinary technicians in this field, several modifications and improvements can be made without departing from the principle of the present invention, and these should also be regarded as belonging to the protection scope of the present invention.

[0024] Reagents and instruments used in this experiment: Sodium chloride, potassium chloride, tris(hydroxymethylaminomethane) (Tris), disodium ethylenediaminetetraacetate (EDTA), Triton X-100, N,N-dimethylformamide (DMF), disodium hydrogen phosphate, potassium dihydrogen phosphate, anhydrous magnesium chloride, 2-morphine ethanesulfonic acid (MES), N-hydroxysuccinimide (NHS), Tween-20, dimethyl sulfoxide (DMSO), hydrogen peroxide, streptavidin (SA), hemin, 2, 2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS) were purchased from Tianjin Xiensi Biochemical Technology Co., Ltd. Trisodium citrate and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Streptavidin magnetic beads were purchased from Taizhou Bio-Tech Co., Ltd. DNA / RNA was synthesized by Shanghai Bioengineering Co., Ltd.

[0025] The coupling buffer in the embodiment of the present invention is: 10 mM Tris-HCl, pH 7.0-8.0, and contains 1 mM EDTA, 1 mol / L NaCl and 0.1% Tween-20.

[0026] The washing buffer in the embodiment of the present invention is: 20 mM Tris-HCl, pH 7.0-8.0, 0.1% Tween-20.

[0027] The PBS buffer in the embodiment of the present invention includes: 137 mM NaCl, 10 mM NaH2PO4, 2.7 mM KCl, 2 mM KH2PO4, pH 7.0-7.5.

[0028] The catalytic buffer in the embodiment of the present invention includes: 10-100 mM MES, pH 4.0-60, 10-150 mM KCl, 1% DMSO, and 0.1% Tween-20.

[0029] Example 1: Synthesis of gold colloid (GNPs) First, take 500 μL of 50 mM HAuCl4 aqueous solution and place it in a double-necked flask, then add 100 mL of ultrapure water and stir to obtain a 0.01% HAuCl4 aqueous solution. Then, set the temperature of the thermal constant temperature magnetic stirrer to 160 o C, heat reflux 0.01% HAuCl4 aqueous solution to boiling. Next, while keeping the speed unchanged, immediately and quickly add 4 mL of freshly prepared 1% trisodium citrate solution. The reaction solution changes from purple to wine red within 2 minutes. Keep the stirring speed unchanged, continue the reaction for 15 minutes, then remove it from the reaction device and continue stirring and cooling at room temperature for 2 hours. Finally, pour the prepared gold glue solution into a brown bottle and place it for 2-8 hours.oC Store in a dark place for future use.

[0030] Figure 2 a shows the UV-visible (UV- vis ) absorption spectrum, Figure 2 b shows the TEM image of synthesized GNPs. Figure 2 As can be seen in a, the prepared GNPs have a strong characteristic absorption peak at 519 nm, and its particle size is roughly about 16 nm. Figure 2 b It can be seen that GNPs have good monodispersity, spherical structure, and relatively uniform particle size with an average diameter of 20 nm. The above results together indicate the successful synthesis of GNPs.

[0031] Example 2: Preparation of DNA functionalized gold colloid That is, 1 mL of gold colloid solution was added to SH-10b with a final concentration of 25 nM and SH-DNA solution with a final concentration of 100 nM, and then placed at 37 o Then, 20 μL of 100 mM PB buffer containing 0.1% SDS and pH 7.2 was added to the above solution and incubated at 37 oC Incubate with shaking for 20 min. Then, add 25 μL of 2 mol / L NaCl solution to the gold colloid solution every 20 min until the NaCl concentration in the gold colloid solution reaches 500 mM. Then place the gold colloid mixed solution at 37 oC The mixture was shaken overnight. Finally, the gold colloid solution was centrifuged and washed twice with PBS to remove excess DNA. The washed gold colloid was redispersed in 500 μL of PBS solution containing 0.05% Tween-20 and stored at 2-8 °C. oC spare.

[0032] Figure 2 c shows the UV-Vis of SH-10b functionalized GNPs. vis Absorption spectrum, Figure 2 d shows the UV-Vis of GNPs functionalized with SH-10b at different concentrations. vis Absorption spectra and photos after salt aging, from Figure 2 As can be seen in c, the maximum UV- vis The absorption peak position shifted by 4 nm, and with the increase of SH-10b addition, GNPs no longer showed peak position shift and agglomeration phenomenon. These results collectively indicate the successful preparation of SH-10b functionalized GNPs.

[0033] Example 3: Preparation of DNA functionalized magnetic beads Take 1 mL of 1 mg / mL streptavidin-modified magnetic beads (dissolved in coupling buffer), add Biotin-H0 and Biotin-10b at a final concentration of 500 nM, and place at 37 o C shaker incubation for 30 min. Next, the magnetic bead mixture was separated by magnetic separation technology and the supernatant was removed. The magnetic beads were washed twice with washing buffer to fully remove excess DNA. Finally, the magnetic beads were dispersed in 1 mL washing buffer to prepare DNA functionalized magnetic beads and stored at 2-8 oC spare.

[0034] Figure 3 The UV-Vis spectra of the supernatant after incubation of 200 nM biotinylated DNA with different concentrations of streptavidin-modified magnetic beads are shown. vis Absorption spectrum. As can be seen from the figure, with the increase of the concentration of streptavidin-modified magnetic beads, the residual biotinylated DNA in the supernatant gradually decreased, indicating the successful preparation of DNA functionalized magnetic beads.

[0035] Example 4: Feasibility verification of targeting miR-21 and miR-10b to activate HCR and CHA Agarose gel electrophoresis was used to characterize whether the target miR-21 could initiate the HCR reaction of the three designed hairpin DNAs H0, H1 and H2, and whether the target miR-10b could initiate the CHA reaction of the designed SH-10b and Bio-10b. Different DNA complexes were prepared to verify the feasibility of the target miR-21 activating the HCR reaction. That is, 50 μL of the mixed solution of miR-21, Bio-H0, H1, H2, Bio-H0+H1, Bio-H0+H1+H2, and miR-21+Bio-H0+H1+H2 with a final concentration of 1 μM was prepared using pH 8.0 Tris-HCl (20mM, containing 50 mM MgCl2) buffer. The above 7 groups of solutions were placed at 95 o C for 5 min, then place at 4 o C refrigerator for 2 h, and different DNA complexes with a concentration of 1 μM were obtained. Similarly, different DNA complexes were prepared to verify the feasibility of target miR-10b activating CHA. That is, 50 μL of miR-10b, SH-10b, Bio-10b, SH-10b+Bio-10b, miR-10b+Bio-10b, and miR-10b+Bio-10b+SH-10b solutions with a final concentration of 1 μM were prepared using pH 8.0 Tris-HCl (20 mM, containing 50 mM MgCl2) buffer. The above 6 groups of solutions were placed at 95 oC for 5 min, then place at 4 o C refrigerator for 2 h to obtain different DNA complexes with a concentration of 1 μM.

[0036] For agarose gel electrophoresis analysis, the steps are as follows: Preparation of gel: Weigh 1.20 g agarose into a 100 mL conical flask, slowly pour in 30 mL of 1×TAE buffer, and heat in a microwave until the agarose is completely dissolved. Continue to add 3 μL of 10000×4SRed Plus nucleic acid stain into the bottle, and shake it gently. Quickly pour the agarose solution into the gel mold, and then insert the comb at the appropriate position. The gel can be used only after it is completely solidified at room temperature.

[0037] Loading: Take an appropriate amount of sample and mix it with 10× Loading buffer, then carefully add it to the sample tank using a micropipette.

[0038] Electrophoresis: After loading, adjust the gel position to ensure that the sample migration direction is consistent with the electrophoresis direction. Close the electrophoresis tank cover and turn on the power. Control the voltage to maintain 110 V. When the band moves to 1 / 3 of the distance from the bottom edge of the gel (about 1 h), stop the experiment. Take out the gel block and place it in the gel imaging system for photography and analysis.

[0039] Figure 4 a. Figure 4 b shows the agarose gel electrophoresis of different DNA complexes. Figure 4 As shown in a, each synthesized DNA chain has a high purity. As the number of DNA involved in self-assembly increases, no band migration occurs in lanes 5 to 6, indicating that there is almost no cross-reaction between these DNA sequences; however, when miR-21 is present, the band mobility in lane 7 is significantly reduced and multiple new bands appear, indicating that the target miR-21 can successfully activate H0, H1 and H2 to undergo HCR reaction. Figure 4 As shown in a, each synthesized DNA chain has a high purity. Lane 4 has no obvious band migration, indicating that there is no reaction between SH-10b and Bio-10b. Lane 5 has migration compared to lane 2, indicating that miR-10b and SH-10b have hybridized. When Bio-10b is added (lane 6), the band mobility decreases more significantly, indicating that the target miR-10b can successfully activate SH-10b and Bio-10b to react with CHA.

[0040] Example 5: Detection and analysis of different concentrations of miR-21 and miR-10b 100 μL of DNA functionalized gold colloid prepared in Example 2 was mixed with 100 μL of DNA functionalized magnetic beads prepared in Example 3, and 5 μL of different concentrations of the test sample (containing miR-21, miR-10b), H1 (10 μM) and H2 (10 μM) were added respectively, and the mixed solution was placed at 37 o C and incubate for 2 h. Next, the reaction mixture was magnetically separated, and the upper layer of gold colloid solution and the lower layer of magnetic bead precipitate were retained separately. On the one hand, the color depth of the red gold colloid solution was observed by naked eyes, or the ultraviolet absorbance value of the solution at 520 nm was recorded. On the other hand, the magnetic bead precipitate after magnetic separation was resuspended in 100 μL pH 5.0 MES buffer solution (containing 200 mM NaCl, 0.1% Tween-20, 1% DMSO and 50 mM KCl). Subsequently, 1 μL of 200 mM Hemin aqueous solution was added to the magnetic bead mixture and placed at 37 o Finally, add 2 μL of 50 mM ABTS aqueous solution and 1 μL of 100 mM H2O2 aqueous solution and place at 37 o C for 20 min. Finally, separate the supernatant solution by magnetic separation, and observe the color depth of the solution by naked eyes, or record the ultraviolet absorbance value of the solution at 420 nm.

[0041] Figure 5 a~c show the UV- vis Absorption spectrum, photos and linear relationship diagrams show that in the range of 2 nM~250 nM, the reduction ratio of UV absorbance value and the logarithmic value of miR-21 concentration show a good linear relationship, and the detection limit is 1.5 nM. Figure 5 d~f show the UV- vis Absorption spectrum, spectra, photos and linear relationship diagrams show that in the range of 2 nM~500 nM, the logarithm of the UV absorbance value and the logarithm of the miR-21 concentration show a good linear relationship, and the detection limit is 2.2 nM.

[0042] Example 6: Selectivity analysis of miR-10b and miR-21 For the selective analysis of miRNA-10b, 100 μL of the DNA functionalized gold colloid prepared in Example 2 was mixed with 100 μL of the DNA functionalized magnetic beads prepared in Example 3, and 5 μL of different samples to be tested were added respectively. The mixed solution was placed at 37 o C for 2 h. Next, the reaction mixture was magnetically separated and the supernatant was retained. The color depth of the upper red gold colloid solution was observed by naked eyes, or the ultraviolet absorbance value of the solution at 520 nm was recorded.

[0043] For the selective analysis of miRNA-21, 100 μL of the DNA functionalized gold colloid prepared in Example 2 was mixed with 100 μL of the DNA functionalized magnetic beads prepared in Example 3, and 5 μL of different samples to be tested, H1 (10 μM) and H2 (10 μM) were added respectively. The mixed solution was placed at 37 o C for 2 h. Next, the reaction mixture was magnetically separated and the magnetic bead precipitate was retained. The magnetic bead precipitate was resuspended in 100 μL of pH 5.0 MES buffer solution (containing 200 mM NaCl, 0.1% Tween-20, 1% DMSO and 50 mM KCl). Subsequently, 1 μL of 200 mM Hemin aqueous solution was added to the magnetic bead mixture and placed at 37 o C for 1 h. Finally, add 2 μL of 50 mM ABTS aqueous solution and 1 μL of 100 mM H2O2 aqueous solution, and continue to incubate the reaction for 20 min. Finally, separate the supernatant solution by magnetic separation, observe the color depth of the solution by naked eyes, or record the ultraviolet absorbance value of the solution at 420 nm.

[0044] Figure 6 a and 6b show the selectivity of the biosensor for miR-10b and miR-21, respectively, which shows good selectivity for miR-10b and miR-21.

[0045] The above embodiments are only preferred technical solutions of the present invention and should not be regarded as limitations of the present invention. The protection scope of the present invention should be the technical solutions recorded in the claims, including equivalent replacement solutions of the technical features in the technical solutions recorded in the claims, that is, equivalent replacement improvements within this scope are also within the protection scope of the present invention.

Claims

1. A method for simultaneously detecting two miRNAs using a dual colorimetric biosensor based on GNPs and nanozymes, characterized in that: This paper includes a dual colorimetric biosensor based on gold colloid (GNPs) and nanozymes for the simultaneous detection of two miRNAs.

2. The method for simultaneously detecting two miRNAs using a dual colorimetric biosensor based on GNPs and nanozymes according to claim 1, characterized in that: The prepared DNA functionalized GNPs, DNA functionalized magnetic beads, and hairpin DNA were fully mixed with the sample to be tested and incubated. After magnetic separation, an upper red clear solution and a magnetic bead precipitate were obtained. Further, K + , Hemin and H2O2 / ABTS and other catalytic substrate solutions are mixed and incubated for reaction, and a green clear solution is obtained through a magnetic separation step.

3. The method for simultaneously detecting two miRNAs using a dual colorimetric biosensor based on GNPs and nanozymes according to claim 2, characterized in that: The GNPs particle size is 10-50 nm.

4. The method for simultaneously detecting two miRNAs using a dual colorimetric biosensor based on GNPs and nanozymes according to claim 2, characterized in that: The DNA functionalized GNPs are prepared by coupling thiolated DNA with GNPs; the coupling reaction temperature is 25-40 o C; the reaction time is 8 to 24 hours.

5. The method for simultaneously detecting two miRNAs using a dual colorimetric biosensor based on GNPs and nanozymes according to claim 2, characterized in that: The particle size of the magnetic beads is 0.2~1 μm.

6. The method for simultaneously detecting two miRNAs using a dual colorimetric biosensor based on GNPs and nanozymes according to claim 2, characterized in that: The DNA functionalized magnetic beads are prepared by coupling biotinylated DNA with streptavidin-modified magnetic beads; the coupling buffer is 10 mM Tris-HCl, pH 7.0-8.0, and contains 1 mM EDTA, 1 mol / L NaCl and 0.1% Tween-20; the coupling reaction temperature is 25-40 o C; reaction time is 15~60 minutes.

7. The method for simultaneously detecting two miRNAs using a dual colorimetric biosensor based on GNPs and nanozymes according to claim 2, characterized in that: The samples to be tested are biomarkers of two miRNAs in tumor diagnosis.

8. The method for simultaneously detecting two miRNAs using a dual colorimetric biosensor based on GNPs and nanozymes according to claim 2, characterized in that: The incubation temperature is 25-40 o C, incubation time is 0.5~2 hours.

9. The method for simultaneously detecting two miRNAs using a dual colorimetric biosensor based on GNPs and nanozymes according to claim 2, characterized in that: The key component of the catalytic substrate solution is MES buffer, which contains NaCl, KCl, Hemin, H2O2, ABTS, DMSO and Tween-20.

10. The method for simultaneously detecting two miRNAs using a dual colorimetric biosensor based on GNPs and nanozymes according to claim 2, characterized in that: The catalytic substrate solution has a MES buffer concentration of 10-100 mM, a pH of 4.0-60, a KCl concentration of 10-150 mM, a Hemin concentration of 1-4 μM, and a H2O2 / ABTS concentration of 1-5 mM.

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