CN117169205B - Detection method of hypoxanthine based on colorimetric biosensor - Google Patents
Detection method of hypoxanthine based on colorimetric biosensor Download PDFInfo
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Abstract
The invention discloses a detection method of hypoxanthine based on a colorimetric biosensor, and belongs to the field of analysis and detection. The method comprises the following steps: (1) Taking a solution sample to be detected, adding xanthine oxidase, and incubating for 15 minutes at 37 ℃ to obtain a primary incubation liquid; (2) Mixing the primary incubation liquid, TMB solution, acetic acid solution and NiPt NPs solution, and incubating for 30 minutes at 37 ℃ to obtain an incubation liquid; (3) Determining the maximum absorption value of the incubation liquid at 652nm by using a colorimetric method; (4) Substituting into an ultraviolet absorption standard curve of hypoxanthine, and calculating to obtain the concentration of hypoxanthine in the sample of the solution to be detected. The detection method of hypoxanthine based on the colorimetric biosensor can simply and rapidly detect the hypoxanthine content, has high accuracy and good economic value, and has important significance for monitoring the freshness of the aquatic products.
Description
Technical Field
The invention relates to a detection method of hypoxanthine based on a colorimetric biosensor, belonging to the field of analysis and detection.
Background
Under the conditions of death, exposure to temperature change, oxygen deficiency and the like, the activity of metabolic enzymes changes, and thus the accumulation of hypoxanthine is caused. Hypoxanthine generally accumulates through the following pathways: adenosine Triphosphate (ATP), adenosine Diphosphate (ADP), adenosine Monophosphate (AMP), inosinic acid (IMP), inosine (HxR), inosine (Hx), uric Acid (UA).
The production of hypoxanthine is closely related to the early freshness and deterioration degree of aquatic products. Different aquatic products have different accumulation pathways. Hypoxanthine may have a negative impact on human health in some cases. In certain aquatic products, such as fish and shellfish, the hypoxanthine content may be contaminated and grown by some bacteria, resulting in an increase. Therefore, the content and the source of hypoxanthine in aquatic products and factors related to food safety are studied, which are helpful for developing corresponding quality control and monitoring strategies.
In the prior art, the detection method of hypoxanthine mainly comprises capillary electrophoresis, high performance liquid chromatography and electrochemical methods. These methods have many limitations, such as the need for large instruments, the need for skilled technicians to perform the experiments, high costs, and few on-site analyses. Therefore, it is highly desirable to develop a simple, rapid, low cost method for detecting hypoxanthine.
Colorimetry (colorimetry) is a method of determining the content of a component to be measured by comparing or measuring the color depth of a solution of a colored substance. Colorimetry allows for target detection by macroscopic color change (qualitative analysis) and measurement of ultraviolet absorbance (quantitative analysis). Colorimetric methods generally require probe recognition and signal transduction. In recent years, a new material, nano-enzyme, has emerged in signal transduction. Since the enzyme-like effects of ferroferric oxide were reported in 2007, nanoezymes have attracted worldwide attention. As an emerging transduction medium, nanoezymes not only have the physical and chemical properties of nanomaterials, but also have catalytic functions similar to those of natural enzymes. In addition, catalytic activity can be modulated by modifying morphology and size, creating catalysts with multiple enzymatic activities, an important bridge across the fields of material science, biosensing, biochemistry and medical diagnostics.
Chinese patent application CN 109856102A discloses a biosensor for detecting hypoxanthine in aquatic products, and based on the catalytic activity of platinum nano (Pt NPs), a fluorescent biosensor is constructed for detecting the content of hypoxanthine Hx in aquatic products. However, the cost is high, the consumption of the nano material is high, the time is long, the operation is more complex, and the research on the related kinetic parameters of the nano material is not performed. More importantly, the invention is based on macroscopic color changes, because it does not require expensive fluorescent markers or excitation light sources, thus making the manufacturing cost much lower than fluorescent biosensors, and colorimetric changes are generally less sensitive to environmental factors, so colorimetric biosensors are more stable and reliable in some complex practical samples.
Disclosure of Invention
Aiming at the prior art, the invention provides a detection method of hypoxanthine based on a colorimetric biosensor, and belongs to the field of analysis and detection. According to the method, the nano material NiPt NPs with high catalytic efficiency is obtained by a simple solvent mixing method, and then the hypoxanthine is detected by utilizing a biosensor formed by the nano material NiPt NPs and Xanthine Oxidase (XOD), so that the hypoxanthine content can be simply and quickly measured, and the method has good economic value. The invention also measures the dynamic parameters of the nano material, which provides reference for the application of the nano material in other fields.
The invention is realized by the following technical scheme:
a method for detecting hypoxanthine based on a colorimetric biosensor, comprising the steps of:
(1) Taking a solution sample to be detected, adding xanthine oxidase, and incubating for 15 minutes at 37 ℃ to obtain a primary incubation liquid;
(2) Mixing the preliminary incubation liquid, TMB (tetramethylbenzidine) solution, acetic acid solution and NiPt NPs solution, and co-incubating for 30 minutes at 37 ℃ to obtain an incubation liquid;
(3) Determining the maximum absorption value of the incubation liquid at 652nm by using a colorimetric method;
(4) Substituting into an ultraviolet absorption standard curve of hypoxanthine, and calculating to obtain the concentration of hypoxanthine in the sample of the solution to be detected.
Further, the solution sample to be detected is obtained by pretreatment of an aquatic product, wherein the aquatic product is selected from fresh living products such as fish, shrimp, crab, shellfish and the like, and can be specifically selected from picoshrimp, metapenaeus ensis, portunus trituberculatus, clams and abalones. The pretreatment method comprises the following steps: and (3) extracting hypoxanthine in the aquatic product by using a TCA (trichloroacetic acid) method (the method is a conventional method in the prior art) to obtain a solution sample to be detected.
Further, in the step (1), the concentration of xanthine oxidase is 0.2U/mL.
Further, the specific operation of the step (2) is as follows: mixing 20 mu L of the primary incubation liquid, 27 mu L of TMB solution, 93 mu L of acetic acid solution and 1 mu L of NiPt NPs solution, and incubating at 37 ℃ for 30 minutes to obtain an incubation liquid, wherein the concentration of the TMB solution is 8mM; the concentration of the acetic acid solution was 1M and the pH was 5.0; the particle concentration of the NiPt NPs solution was 6.8X10 -11 M。
Further, in the step (2), the NiPt NPs solution is prepared by the following method: 10mg of chloroplatinic acid, 7mg of nickel chloride, 80mg of polyvinylpyrrolidone and 10mL of tetraethylene glycol were added to 90mL of ultrapure water, and heated at 150℃for 30 minutes; then adding 1mL of the acetaldehyde-tetraethylene glycol mixed solution, and continuously keeping the temperature at 150 ℃ and heating for 30 minutes; cooling to room temperature to obtain NiPt NPs solution with particle concentration of 1.4X10 -9 M, storing at 4 ℃ in a dark place; the volume ratio of the acetaldehyde to the tetraethylene glycol mixed solution is 1:1.
Further, in the step (4), the ultraviolet absorption standard curve of hypoxanthine is established by the following method: preparing a gradient series concentration sample of hypoxanthine, adding xanthine oxidase, and incubating for 15 minutes at 37 ℃ to obtain a primary incubation liquid; mixing the primary incubation liquid, TMB solution, acetic acid solution and NiPt NPs solution, and incubating for 30 minutes at 37 ℃ to obtain an incubation liquid; determining the maximum absorption value of the incubation liquid at 652nm by using a colorimetric method; a standard curve of hypoxanthine concentration and absorbance was established.
Further, the specific method for preparing the gradient series concentration samples of hypoxanthine is as follows: weighing 0.0065g of hypoxanthine, adding into 1mL of 1M NaOH solution, adding 1mL of 1M hydrochloric acid, and using ultrapure water to fix the volume to 5mL to obtain hypoxanthine mother liquor; the hypoxanthine mother solution is diluted with ultrapure water to form a gradient series concentration sample with the concentration range of 0-0.6 mM.
Still further, the xanthine oxidase concentration was 0.2U/mL.
Further, in the incubation liquid, the amount of the preliminary incubation liquid is 20 mu L; the TMB solution had a concentration of 8mM and an amount of 27. Mu.L; the concentration of the acetic acid solution is 1M, the pH value is 5.0, and the dosage is 93 mu L; the particle concentration of the NiPt NPs solution was 6.8X10 -11 M was used in an amount of 1. Mu.L.
The detection method of hypoxanthine based on colorimetric biosensor of the invention, the detection principle is: after incubation of XOD with Hx H is generated 2 O 2 In the nano material NiPt NPs and ethyl with high catalytic efficiencyHydroxyl free radicals (OH) are generated in an acid environment, colorless TMB can be oxidized into blue oxTMB, and the quantitative detection of Hx can be realized by detecting the ultraviolet absorption value. The nano material NiPt NPs have high catalytic efficiency and excellent stability, and are combined with the XOD to form the colorimetric biosensor.
Compared with CN 109856102A, the nano material of the invention is NiPt NPs, the dosage is extremely small, only 1 mu L is needed, the dosage is far less than the dosage of Pt NPs in CN 109856102A (50 mu L), the time is short, only 45 minutes is needed, and the time is far less than the time (90 minutes) used in CN 109856102A. In addition, the invention also measures the kinetic parameters of the nano material, which provides reference for the application of the nano enzyme in other fields. More importantly, the invention is based on macroscopic color changes, because it does not require expensive fluorescent markers or excitation light sources, thus making the manufacturing cost much lower than fluorescent biosensors, and colorimetric changes are generally less sensitive to environmental factors, so colorimetric biosensors are more stable and reliable in some complex practical samples.
The detection method of hypoxanthine based on the colorimetric biosensor can simply and rapidly detect the hypoxanthine content, has high accuracy and good economic value, and has important significance for monitoring the freshness of the aquatic products.
The various terms and phrases used herein have the ordinary meaning known to those skilled in the art.
Drawings
Fig. 1: standard curve schematic.
Fig. 2: schematic of absorption values at different pH systems.
Fig. 3: schematic of absorbance at different concentrations of TMB system.
Fig. 4: comparison of absorption values of the colorimetric biosensor for hypoxanthine and 15 interfering substances is shown in the schematic diagram.
Fig. 5: TEM scan and particle size distribution schematic of nanomaterial NiPt NPs.
Fig. 6: the EDS line scan of the nano material NiPt NPs is provided with a schematic diagram of element content distribution.
Fig. 7: comparison of catalytic activity of nanomaterial NiPt NPs with different storage times.
Fig. 8: schematic diagram of Mie curve of nanomaterial NiPt NPs versus TMB.
Fig. 9: a schematic of the double reciprocal curve of nanomaterial NiPt NPs versus TMB.
Fig. 10: nano material NiPt NPs vs H 2 O 2 Schematic diagram of Mie curve of (C).
Fig. 11: nano material NiPt NPs vs H 2 O 2 Is a double reciprocal plot of (2).
Detailed Description
The invention is further illustrated below with reference to examples. However, the scope of the present invention is not limited to the following examples. Those skilled in the art will appreciate that various changes and modifications can be made to the invention without departing from the spirit and scope thereof.
The instruments, reagents and materials used in the examples below are conventional instruments, reagents and materials known in the art and are commercially available. The experimental methods, detection methods, and the like in the examples described below are conventional experimental methods and detection methods known in the prior art unless otherwise specified.
EXAMPLE 1 establishment of ultraviolet absorption Standard Curve for hypoxanthine
The method comprises the following steps:
(1) weighing 0.0065g of hypoxanthine, adding into 1mL of 1M NaOH solution, adding 1mL of 1M hydrochloric acid, and using ultrapure water to fix the volume to 5mL to obtain hypoxanthine mother liquor;
(2) diluting hypoxanthine mother solution into gradient series concentration samples with the concentration range of 0-0.6 mM by using ultrapure water, taking 50 mu L, adding 50 mu L of 0.4U/mL xanthine oxidase solution, and incubating for 15 minutes at 37 ℃ to obtain a primary incubation liquid; the solvent of the xanthine oxidase solution is PB solution (phosphate buffer solution) with pH of 7.4 and 0.01M;
(3) mu.L of the preliminary incubation solution, 27. Mu.L of TMB solution, 93. Mu.L of acetic acid solution and 1. Mu.L of NiPt NPs solution were mixed and incubated at 37℃for 30 minutesObtaining an incubation liquid, wherein the concentration of the TMB solution is 8mM; the concentration of the acetic acid solution is 1M, and the pH value is 5.0; the particle concentration of the NiPt NPs solution was 6.8X10 -11 M；
(4) The maximum absorbance of the incubation at 652nm was measured by colorimetry, and a standard curve of hypoxanthine concentration versus absorbance was established as shown in fig. 1. The linear regression equation of the method for hypoxanthine is y=0.5409x+0.0584 (x is hypoxanthine concentration, y is absorption value at 652 nm), correlation coefficient (R 2 ) 0.9932. The linear range is 19 to 300. Mu.M, and the limit of detection (LOD) is 8.5. Mu.M.
The NiPt NPs solution is prepared by the following method: 10mg of chloroplatinic acid, 7mg of nickel chloride, 80mg of polyvinylpyrrolidone and 10mL of tetraethylene glycol were added to 90mL of ultrapure water, placed in a three-necked flask, and heated at 150℃for 30 minutes; then adding 1mL of the acetaldehyde-tetraethylene glycol mixed solution, and continuously keeping the temperature at 150 ℃ and heating for 30 minutes; cooling to room temperature to obtain NiPt NPs solution with particle concentration of 1.4X10 -9 M, storing at 4 ℃ in a dark place; the volume ratio of the acetaldehyde to the tetraethylene glycol mixed solution is 1:1. When in use, the particles are diluted with ultrapure water to a particle concentration of 6.8X10 -11 M。
Example 2 parameter Condition optimization
The influence of different pH systems and TMB final concentrations on a color development system formed by NiPt NPs, TMB and acetic acid is examined.
The influence of different pH systems on a color development system formed by NiPt NPs, TMB and acetic acid is examined: acetic acid buffers were prepared at pH 2.0, 3.0, 4.0, 5.0 and 6.0, respectively, and the procedure was followed in example 1. Detection of H 2 O 2 (concentration of 1 mM) absorbance at 652nm, and as a result, as shown in FIG. 2, the color development system was best when pH=5.0.
The effect of different TMB final concentrations on the color development system consisting of NiPt NPs, TMB and acetic acid was examined: h was detected by following the procedure of example 1, with TMB final concentrations of 0.25, 0.5, 1.0, 1.5, 2.0mM, respectively 2 O 2 (concentration of 1 mM) absorbance at 652nm, and the result is shown in FIG. 3, the effect of the color development system is optimal when the final concentration of TMB is 1.5 mM.
Example 3 specificity experiments
With reference to example 1, the colorimetric biosensor of the present invention was studied for common 15 interfering substances such as urea, vc, na + 、K + 、Zn 2+ 、Mg 2+ The initial concentration of hypoxanthine in the reaction system was 300. Mu.M, the initial concentration of interfering substances was 500. Mu.M, and the absorbance at 652nm was measured after the completion of the reaction.
The results are shown in FIG. 4. The results show that only the hypoxanthine has visible blue after reaction, and other interfering substances do not interfere with the color development to a great extent, so that the specificity of the colorimetric biosensor is acceptable.
Example 4 partial characterization of nanomaterial NiPt NPs
FIG. 5 is a transmission electron microscope image of nanomaterial NiPt NPs, showing that the average particle size of the nanomaterial is 2.45.+ -. 0.52nm.
Fig. 6 is an X-ray spectrometer line scan of the nanomaterial NiPt NPs, showing that the content of Pt element in the nanomaterial is higher than Ni element, consistent with the original additive results.
EXAMPLE 5 investigation of catalytic Activity of nanomaterial NiPt NPs
After obtaining the nanomaterial NiPt NPs according to the procedure of example 1, the nanomaterial NiPt NPs were stored in a dark place at 4℃and the change in catalytic activity of the nanomaterial NiPt NPs (H in the reaction system) was examined for different storage times 2 O 2 1 mM).
The results are shown in FIG. 7. The result shows that the nano material has good catalytic activity within two weeks of synthesis. The operation is performed during this period of time, and as a result, the activity of the nanomaterial is not affected.
Example 6 determination of catalytic kinetic parameters of nanomaterial NiPt NPs
To quantify substrate affinity and catalytic efficiency of nanomaterial NiPt NPs, mies was calculated using the double reciprocal methodConstant (K) m ) And maximum initial velocity (V max ) And calculating the catalytic efficiency K after measuring the concentration of the nanomaterial by ICP-MS cat 。K m Smaller indicates that the higher the affinity of the nanomaterial for the substrate, K cat The larger the nanomaterial, the higher the catalytic efficiency of the nanomaterial towards the substrate.
In the catalytic kinetics experiments, the relevant parameters of TMB were first determined: h 2 O 2 The final concentration was 0.14mM, the TMB concentration was varied and the kinetics of the enzyme was determined using an enzyme-labeled instrument at 37 ℃. The reaction rate was found by using the absorbance value, and then the Miq constant (K m ) And maximum initial velocity (V max ). Catalytic constant (K) cat ) Is calculated by using the formula K cat ＝V max /[E],[E]Is the concentration of the particles. Next, H is measured 2 O 2 Is a related parameter of (a): TMB final concentration 1mM, remain unchanged, H 2 O 2 Concentration was varied, and the procedure was as described above.
The Mies curve of nanomaterial NiPt NPs versus TMB is shown in FIG. 8, and the double reciprocal curve of nanomaterial NiPt NPs versus TMB is shown in FIG. 9. Nano material NiPt NPs vs H 2 O 2 The Mies curve of (a) is shown in FIG. 10, and the NiPt NPs of the nanomaterial is shown as H 2 O 2 The double reciprocal curve of (2) is shown in figure 11. Through calculation, the Mie constant (K) of the nano material NiPt NPs to TMB m ) Is 1.0X10 -3 M, maximum initial velocity (V max ) Is 5.9X10 -7 M s -1 Catalytic constant (K) cat ) Is 1.2X10 6 s -1 K of nanomaterials (NiPt NPs) cat Is 279.07 times that of natural horseradish peroxidase. Nano material NiPt NPs vs H 2 O 2 Miq constant (K) m ) 4.7X10 -5 M, maximum initial velocity (V max ) Is 3.1X10 × 10 -7 M s -1 。
Example 7 establishment of method for detection of hypoxanthine based on colorimetric biosensor
The method comprises the following steps:
(1) Taking 50 mu L of a solution sample to be detected, adding 50 mu L of 0.4U/mL xanthine oxidase solution, and incubating for 15 minutes at 37 ℃ to obtain a primary incubation liquid;
(2) Mixing 20 mu L of the primary incubation liquid, 27 mu L of TMB solution, 93 mu L of acetic acid solution and 1 mu L of NiPt NPs solution, and incubating at 37 ℃ for 30 minutes to obtain an incubation liquid, wherein the concentration of the TMB solution is 8mM; the concentration of the acetic acid solution is 1M, and the pH value is 5.0; the particle concentration of the NiPt NPs solution was 6.8X10 -11 M；
(3) Determining the maximum absorption value of the incubation liquid at 652nm by using a colorimetric method;
(4) Substituting into an ultraviolet absorption standard curve of hypoxanthine, and calculating to obtain the concentration of hypoxanthine in the sample of the solution to be detected.
Example 8 detection example of hypoxanthine based on colorimetric biosensor
Taking the skin shrimp, the base shrimp, the portunus trituberculatus, the clams and the abalones as samples, taking the content of hypoxanthine contained in the samples which are placed for 0 hour at room temperature as a reference, extracting hypoxanthine by a TCA method to obtain a solution sample to be detected, respectively adding 37.5 mu M and 75 mu M hypoxanthine standard substances, measuring the absorption value at 652nm according to the operation of the embodiment 1, substituting the absorption value into the hypoxanthine standard curve established in the embodiment 1 to obtain the hypoxanthine measured concentration in the samples, measuring three times for each sample, and calculating the RSD and the recovery rate.
The specific operation of extracting hypoxanthine by the TCA method is as follows: 10g of meat was taken per sample, hx was extracted with TCA at a concentration of 0.1g/mL, centrifuged at 4000rpm for 20 minutes at 4℃using a centrifuge, and the supernatant was pH-adjusted to 6.0 and then filtered through a 0.45 μm filter.
The information about the standard recovery rate of the real samples of the picoshrimp, the base shrimp, the portunus trituberculatus, the clams and the abalones is shown in table 1. As can be seen from Table 1, the recovery rate of the colorimetric biosensor of the invention to the measurement results of the five samples is within the range of 83.69% -118.0%, the RSD is less than or equal to 8.75%, the accuracy is high, and the colorimetric biosensor has a good application prospect.
Table 1 information on the recovery of labeled samples
The foregoing examples are provided to fully disclose and describe how to make and use the claimed embodiments by those skilled in the art, and are not intended to limit the scope of the disclosure herein. Modifications that are obvious to a person skilled in the art will be within the scope of the appended claims.
Claims (7)
1. A method for detecting hypoxanthine based on a colorimetric biosensor, comprising the steps of:
(1) Taking a solution sample to be detected, adding xanthine oxidase into the solution sample to be detected, enabling the concentration of xanthine oxidase to be 0.2U/mL, and incubating the mixture for 15 minutes at 37 ℃ to obtain a primary incubation liquid;
(2) Mixing 20 mu L of the primary incubation liquid, 27 mu L of TMB solution, 93 mu L of acetic acid solution and 1 mu L of NiPt NPs solution, and incubating at 37 ℃ for 30 minutes to obtain an incubation liquid, wherein the concentration of the TMB solution is 8mM; the concentration of the acetic acid solution is 1M, and the pH value is 5.0; the particle concentration of the NiPt NPs solution was 6.8X10 -11 M；
The NiPt NPs solution is prepared by the following method: 10mg chloroplatinic acid, 7mg nickel chloride, 80mg polyvinylpyrrolidone and 10mL tetraethylene glycol were added to 90mL ultrapure water and heated at 150℃for 30 minutes; then adding the acetaldehyde-tetraethylene glycol mixed solution of 1mL, and continuously heating at 150 ℃ for 30 minutes; cooling to room temperature to obtain NiPt NPs solution with particle concentration of 1.4X10 -9 M, diluted with ultrapure water to a particle concentration of 6.8X10 -11 M; the volume ratio of the acetaldehyde to the tetraethylene glycol mixed solution is 1:1;
(3) Colorimetric determination of the maximum absorbance of the incubation at 652 nm;
(4) Substituting into an ultraviolet absorption standard curve of hypoxanthine, and calculating to obtain the concentration of hypoxanthine in the sample of the solution to be detected.
2. The colorimetric biosensor-based detection method of hypoxanthine according to claim 1, wherein: the solution sample to be detected is obtained by pretreatment of aquatic products, wherein the aquatic products are selected from fish, shrimp, crab and Bei Xianhuo products; the pretreatment method comprises the following steps: and extracting hypoxanthine in the aquatic product by using a TCA method to obtain a solution sample to be detected.
3. The method for detecting hypoxanthine based on a colorimetric biosensor according to claim 2, wherein: the aquatic product is selected from the group consisting of picopenaeus monodon, metapenaeus ensis, portunus trituberculatus, clam and abalone.
4. The colorimetric biosensor-based detection method of hypoxanthine according to claim 1, wherein: in the step (4), the ultraviolet absorption standard curve of hypoxanthine is established by the following method: preparing a gradient series concentration sample of hypoxanthine, adding xanthine oxidase, and incubating for 15 minutes at 37 ℃ to obtain a primary incubation liquid; mixing the primary incubation liquid, TMB solution, acetic acid solution and NiPt NPs solution, and incubating for 30 minutes at 37 ℃ to obtain an incubation liquid; colorimetric determination of the maximum absorbance of the incubation at 652 nm; a standard curve of hypoxanthine concentration and absorbance was established.
5. The method for detecting hypoxanthine based on the colorimetric biosensor according to claim 4, wherein the specific method for preparing the hypoxanthine gradient series concentration samples is as follows: weighing 0.0065g hypoxanthine, adding into 1M NaOH solution of 1mL, adding 1M hydrochloric acid of 1mL, and fixing volume to 5mL with ultrapure water to obtain hypoxanthine mother liquor; the hypoxanthine mother solution is diluted into gradient series concentration samples with the concentration range of 0 to 0.6 and mM by ultrapure water.
6. The method for detecting hypoxanthine based on a colorimetric biosensor according to claim 4, wherein: the concentration of xanthine oxidase was 0.2U/mL.
7. The method for detecting hypoxanthine based on a colorimetric biosensor according to claim 4, wherein: in the incubation liquid, the consumption of the preliminary incubation liquid is 20 mu L; the TMB solution had a concentration of 8mM in an amount of 27. Mu.L; the concentration of the acetic acid solution is 1M, the pH value is 5.0, and the dosage is 93 mu L; the particle concentration of the NiPt NPs solution was 6.8X10 -11 M was used in an amount of 1. Mu.L.
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