CN110646417A - Rapid pyridoxal phosphate determination method taking nanogold as chromogenic probe - Google Patents

Rapid pyridoxal phosphate determination method taking nanogold as chromogenic probe Download PDF

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CN110646417A
CN110646417A CN201911019849.2A CN201911019849A CN110646417A CN 110646417 A CN110646417 A CN 110646417A CN 201911019849 A CN201911019849 A CN 201911019849A CN 110646417 A CN110646417 A CN 110646417A
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nanogold
pyridoxal phosphate
mercaptoethylamine
concentration
reaction
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CN110646417B (en
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邓豪华
陈伟
黄开源
彭花萍
何少斌
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Fujian Medical University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/314—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry with comparison of measurements at specific and non-specific wavelengths
    • G01N21/3151—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry with comparison of measurements at specific and non-specific wavelengths using two sources of radiation of different wavelengths
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/314—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry with comparison of measurements at specific and non-specific wavelengths
    • G01N2021/3155—Measuring in two spectral ranges, e.g. UV and visible

Abstract

The invention discloses a method for rapidly determining pyridoxal phosphate by using nanogold as a color development probe, which is characterized in that the pyridoxal phosphate concentration is determined by utilizing the action of the pyridoxal phosphate and the nanogold to cause the nanogold to be aggregated to show the change of the solution color and the ultraviolet-visible absorption spectrum characteristic, when the color characteristic is observed visually, the color of mercaptoethylamine-nanogold is changed from wine red → pink → purple → blue along with the gradual increase of the pyridoxal phosphate concentration (0 ~ 0.5 mu mol/L), and when the absorbance ratio A is determined700/A525When the concentration of pyridoxal phosphate is changed within the range of 0.25 ~ 0.5.5 mu mol/L, the absorbance ratio A of mercaptoethylamine to nanogold700/A525Increases with increasing pyridoxal phosphate concentration.

Description

Rapid pyridoxal phosphate determination method taking nanogold as chromogenic probe
Technical Field
The invention relates to a method for rapidly determining pyridoxal phosphate content by taking nanogold as a chromogenic probe, and belongs to the field of analytical chemistry and nanotechnology.
Background
Gold nanoparticles have received much attention due to their ease of preparation and biofunctionalization, excellent biostability and unique spectral characteristics. The surface plasmon absorption band of the gold nanoparticles is located in the visible region of the electromagnetic spectrum and is influenced by the morphology of the nano-aggregates. Typical colloidal nanogold particles are wine red, while their aggregates appear purple or blue due to the shift of the surface plasmon absorption band of nanogold toward long wavelengths. The method based on the principle can be used for detecting various analytes such as cells, proteins, DNA, metal ions and the like.
Pyridoxal phosphate, also known as pyridoxal-5' -phosphate, is vitamin B6The coenzyme form is involved in transamination in amino acid metabolism, and is mainly used for biochemical research, structural modification of progesterone characteristics, enzyme inhibitors and the like. In medicine, pyridoxal phosphate is vitamin B6The main raw materials of the medicine are. Therefore, the development of a method for rapidly and sensitively detecting pyridoxal phosphate is of great significance. Heretofore, various techniques including electrochemical methods, fluorescence spectroscopy and high performance liquid chromatography have been applied to the detection of pyridoxal phosphate. Although most of the above techniques allow for sensitive, selective detection of pyridoxal phosphate, the need for sophisticated instrumentation and trained personnel limits their use in routine assays.
The invention provides a novel method for detecting pyridoxal phosphate, which is rapid, simple, convenient and sensitive by taking mercaptoethylamine-nanogold as a chromogenic probe.
Disclosure of Invention
The invention aims to provide a rapid, simple and sensitive pyridoxal phosphate determination method by using mercaptoethylamine-nanogold as a color development probe and utilizing the change of the dispersion state of the nanogold after the interaction with pyridoxal phosphate.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for rapidly determining pyridoxal phosphate by using nanogold as a color development probe is characterized in that the pyridoxal phosphate and the nanogold are reacted to cause the nanogold to be aggregated to show the change of solution color and ultraviolet-visible absorption spectrum characteristics to measureDetermining pyridoxal phosphate concentration; judging the concentration of pyridoxal phosphate by using the color change of the nanogold solution; the used nano-gold is prepared by the following steps: first, 400. mu.L of mercaptoethylamine hydrochloride having a concentration of 213 mmol/L and 2.23 mL of HAuCl having a concentration of 10mg/mL were taken4Adding into a beaker containing 37.5 mL of water, and stirring at room temperature for reaction for 20 min; after the reaction, 10. mu.L of NaBH of 10 mmol/L concentration was added to the reaction solution4Then, continuously stirring and reacting at room temperature for 30 min to obtain a mercaptoethylamine-nanogold crude product; purifying the prepared mercaptoethylamine-nanogold crude product by using a dialysis bag with the molecular weight cutoff of 7000 to obtain a mercaptoethylamine-nanogold pure product; sealing the obtained pure mercaptoethylamine-nanogold product in a dark place, and storing the product in a refrigerator at 4 ℃ for later use.
A method for rapidly determining pyridoxal phosphate by taking nanogold as a chromogenic probe is characterized in that the concentration of pyridoxal phosphate is determined by utilizing the action of pyridoxal phosphate and nanogold to cause nanogold to aggregate to show the change of solution color and ultraviolet-visible absorption spectrum characteristics; using the absorbance ratio (A) of nano-gold at 700 nm and 525 nm700/A525) (ii) varying to determine the concentration of pyridoxal phosphate; the used nano-gold is prepared by the following steps: first, 400. mu.L of mercaptoethylamine hydrochloride having a concentration of 213 mmol/L and 2.23 mL of HAuCl having a concentration of 10mg/mL were taken4Adding into a beaker containing 37.5 mL of water, and stirring at room temperature for reaction for 20 min; after the reaction, 10. mu.L of NaBH of 10 mmol/L concentration was added to the reaction solution4Then, continuously stirring and reacting at room temperature for 30 min to obtain a mercaptoethylamine-nanogold crude product; purifying the prepared mercaptoethylamine-nanogold crude product by using a dialysis bag with the molecular weight cutoff of 7000 to obtain a mercaptoethylamine-nanogold pure product; sealing the obtained pure mercaptoethylamine-nanogold product in a dark place, and storing the product in a refrigerator at 4 ℃ for later use.
The method for rapidly determining pyridoxal phosphate by using nanogold as a chromogenic probe is characterized in that pyridoxal phosphate and mercaptoethylamine-nanogold with different concentrations are sequentially added into an acetate buffer solution, are uniformly mixed, react at room temperature for 0.25 ~ 10 minutes, and after the reaction is finished, color characteristics are visually observed or the detection is carried outDetermining the absorbance ratio A700/A525When the color characteristics are observed visually, the color of the mercaptoethylamine-nanogold changes from wine red → pink → purple → blue with the gradual increase of the pyridoxal phosphate concentration in the interval of 0 ~ 0.5.5 mu mol/L, and when the absorbance ratio A is determined700/A525When the concentration of pyridoxal phosphate is changed within the range of 0.25 ~ 0.5.5 mu mol/L, the absorbance ratio A of mercaptoethylamine to nanogold700/A525Increases with increasing pyridoxal phosphate concentration.
The volume ratio of the pyridoxal phosphate to the mercaptoethylamine-nanogold to the acetate buffer solution is 1:1:3, and the total reaction volume is 1 mL.
The pH and ionic strength of the acetate buffer solution used were 6.0 and 10 mmol/L, respectively, the reaction temperature was room temperature and the reaction time was 1 minute.
Specifically, the invention adopts the following technical scheme:
preparing mercaptoethylamine protected nanogold:
all glassware used in the following process is soaked in aqua regia, thoroughly washed with double distilled water and dried. Preparing mercaptoethylamine-nanogold: first, 400. mu.L of mercaptoethylamine hydrochloride having a concentration of 213 mmol/L and 2.23 mL of HAuCl having a concentration of 10mg/mL were taken4The mixture was added to a beaker containing 37.5 mL of water, and the reaction was stirred at room temperature for 20 min. After the reaction, 10. mu.L of NaBH of 10 mmol/L concentration was added to the reaction solution4And then continuously stirring and reacting for 30 min at room temperature to obtain the mercaptoethylamine-nanogold crude product. Purifying the prepared crude mercaptoethylamine-nanogold product by using a dialysis bag with the molecular weight cutoff of 7000 to obtain a pure mercaptoethylamine-nanogold product. Sealing the obtained pure mercaptoethylamine-nanogold product in a dark place, and storing the product in a refrigerator at 4 ℃ for later use.
Determination of pyridoxal (di) phosphate
0.2 mL of the sample solution and 0.2 mL of the mercaptoethylamine-nanogold prepared in the step (one) were sequentially added to 0.6 mL of acetate buffer solution (pH = 6.0, 10 mmol/L), mixed well, and reacted at room temperature for 1 minute. After completion, the color is visually observed to change orMeasuring the absorbance ratio (A) at 700 nm and 525 nm700/A525). And (4) carrying out quantification according to the comparison of the solution color and a colorimetric standard series or through an absorbance ratio.
The invention has the advantages that:
(1) the mercaptoethylamine-nanogold used in the method can be directly prepared by a one-step reduction method without further modification, and the preparation process is simple and rapid.
(2) The invention has high detection speed and can complete the detection of the sample within 1 minute.
(3) The detection sensitivity is high, and the mercaptoethylamine-nanogold can be completely aggregated by 0.5 mu mol/L pyridoxal phosphate.
Drawings
FIG. 1 is an appearance diagram of a solution before and after the action of mercaptoethylamine-nanogold and 0.5 mu mol/L pyridoxal phosphate.
FIG. 2 is a diagram showing UV-VIS absorption spectra before and after the action of mercaptoethylamine-nanogold with 0.5. mu. mol/L pyridoxal phosphate.
FIG. 3 is a transmission electron microscope image before and after the action of mercaptoethylamine-nanogold and pyridoxal phosphate of 0.5 mu mol/L.
FIG. 4 is a dynamic light scattering particle size distribution diagram before and after the action of mercaptoethylamine-nanogold and 0.5 mu mol/L pyridoxal phosphate.
FIG. 5 is Zeta potential diagram before and after the action of mercaptoethylamine-nanogold and pyridoxal phosphate of 0.5. mu. mol/L.
FIG. 6 is the X-ray photoelectron spectrum before and after the reaction of mercaptoethylamine-nanogold with 0.5. mu. mol/L pyridoxal phosphate.
FIG. 7 is a graph showing the effect of system pH on the interaction between mercaptoethylamine-nanogold and pyridoxal phosphate.
FIG. 8 is a graph showing the effect of temperature on the interaction between mercaptoethylamine-nanogold and pyridoxal phosphate.
FIG. 9 is a graph showing the effect of reaction time on the interaction between mercaptoethylamine-nanogold and pyridoxal phosphate.
FIG. 10 is a color change diagram of the interaction of mercaptoethylamine-nanogold with pyridoxal phosphate at different concentrations.
FIG. 11 is a graph showing the change of absorbance ratio of mercaptoethylamine-nanogold to pyridoxal phosphate at different concentrations.
Detailed Description
Example 1:
preparing mercaptoethylamine-nanogold: first, 400. mu.L of mercaptoethylamine hydrochloride having a concentration of 213 mmol/L and 2.23 mL of HAuCl having a concentration of 10mg/mL were taken4The mixture was added to a beaker containing 37.5 mL of water, and the reaction was stirred at room temperature for 20 min. After the reaction, 10. mu.L of NaBH of 10 mmol/L concentration was added to the reaction solution4And then continuously stirring and reacting for 30 min at room temperature to obtain the mercaptoethylamine-nanogold crude product. Purifying the prepared crude mercaptoethylamine-nanogold product by using a dialysis bag with the molecular weight cutoff of 7000 to obtain a pure mercaptoethylamine-nanogold product. Sealing the obtained pure mercaptoethylamine-nanogold product in a dark place, and storing the product in a refrigerator at 4 ℃ for later use. All glassware used in the above process is soaked in aqua regia, thoroughly washed with double distilled water, and air dried.
Example 2:
0.2 mL of pyridoxal phosphate at a final concentration of 0.5. mu. mol/L and 0.2 mL of the mercaptoethylamine-nanogold prepared in example 1 were sequentially added to 0.6 mL of acetate buffer solution (pH = 6.0, 10 mmol/L), mixed well and reacted at room temperature for 1 minute. The color of the mercaptoethylamine-nanogold changes from wine red to blue (shown in figure 1, A: mercaptoethylamine-nanogold, B: mercaptoethylamine-nanogold + pyridoxal phosphate), and a new absorption peak is generated at the wavelength of 700 nm (shown in figure 2, A: mercaptoethylamine-nanogold, B: mercaptoethylamine-nanogold + pyridoxal phosphate).
Example 3:
0.2 mL of pyridoxal phosphate at a final concentration of 0.5. mu. mol/L and 0.2 mL of the mercaptoethylamine-nanogold prepared in example 1 were sequentially added to 0.6 mL of acetate buffer (pH = 6.0, 10 mmol/L), mixed well, reacted at room temperature for 1 minute, and then subjected to transmission electron microscopy. As can be seen from fig. 3, the mercaptoethylamine-nanogold is in a sphere-like shape and has good dispersibility without reacting with pyridoxal phosphate (a in the figure); and after the reaction with pyridoxal phosphate, mercaptoethylamine-nanogold is obviously aggregated (B in the figure).
Example 4:
0.2 mL of pyridoxal phosphate at a final concentration of 0.5. mu. mol/L and 0.2 mL of the mercaptoethylamine-nanogold prepared in example 1 were sequentially added to 0.6 mL of acetate buffer solution (pH = 6.0, 10 mmol/L), mixed well, reacted at room temperature for 1 minute, and then subjected to dynamic light scattering particle size distribution test. As shown in FIG. 4, the dispersibility of mercaptoethylamine/nanogold was good without reacting with pyridoxal phosphate, and the average particle size was 28 nm (A in the figure); after the reaction with pyridoxal phosphate, the mercaptoethylamine-nanogold is obviously aggregated, and the average particle size is increased to 813 nm (B in the figure).
Example 5:
0.2 mL of pyridoxal phosphate having a final concentration of 0.5. mu. mol/L and 0.2 mL of mercaptoethylamine-nanogold prepared in example 1 were sequentially added to 0.6 mL of acetate buffer solution (pH = 6.0, 10 mmol/L), mixed well, reacted at room temperature for 1 minute, and then subjected to a Zeta potential test. As can be seen from FIG. 5, the average surface potential of mercaptoethylamine-nanogold was + 20.5. + -. 1.8 mV (A in the figure) when it was not reacted with pyridoxal phosphate; and the surface potential of mercaptoethylamine-nanogold is reduced to-4.6 mV. + -. 1.0 mV (B in the figure) after the reaction with pyridoxal phosphate.
Example 6:
0.2 mL of pyridoxal phosphate at a final concentration of 0.5. mu. mol/L and 0.2 mL of the mercaptoethylamine-nanogold prepared in example 1 were sequentially added to 0.6 mL of acetate buffer solution (pH = 6.0, 10 mmol/L), mixed well and reacted at room temperature for 1 minute. The obtained solution is freeze-dried to obtain powder, and the obtained powder is taken for X-ray photoelectron spectroscopy measurement, a characteristic peak of-C = N-appears at 399.0 eV, and a 2p peak of phosphorus appears at 134.5 eV (shown in a figure of 1s of nitrogen and a figure of 2p of phosphorus in figure 6), which shows that pyridoxal phosphate is combined to the surface of mercaptoethylamine-nanogold through Schiff base reaction.
Example 7:
0.2 mL of pyridoxal phosphate with a final concentration of 0.5. mu. mol/L and 0.2 mL of the mercaptoethylamine-nanogold prepared in example 1 were added sequentially to 0.6 mL of acetate buffer solutions (10 mmol/L) with different pH, mixed well and reacted at room temperatureFor 1 minute. Measuring the absorbance ratio A after the reaction700/A525And a blank control group (containing no pyridoxal phosphate) was set. As can be seen from FIG. 7, when the pH value of the system is 6.0, the effect of pyridoxal phosphate on inducing aggregation of mercaptoethylamine-nanogold is the most significant (in the figure, A: mercaptoethylamine-nanogold, B: mercaptoethylamine-nanogold + pyridoxal phosphate).
Example 8:
0.2 mL of pyridoxal phosphate at a final concentration of 0.5. mu. mol/L and 0.2 mL of the mercaptoethylamine-nanogold prepared in example 1 were added sequentially to 0.6 mL of acetate buffer solution (pH = 6.0, 10 mmol/L), mixed well, and left to react at different temperatures for 1 minute. Measuring the absorbance ratio A after the reaction700/A525And a blank control group (without pyridoxal phosphate) is set, as can be seen from fig. 8, when the reaction temperature is changed at 20 ~ 50 ℃, the aggregation-inducing effect of pyridoxal phosphate on mercaptoethylamine-nanogold does not change significantly (in the figure, a: mercaptoethylamine-nanogold, B: mercaptoethylamine-nanogold + pyridoxal phosphate).
Example 9:
0.2 mL of pyridoxal phosphate (final concentration: 0.5. mu. mol/L) and 0.2 mL of the mercaptoethylamine-nanogold prepared in example 1 were sequentially added to 0.6 mL of acetate buffer (pH = 6.0, 10 mmol/L), mixed well, and reacted at room temperature for 0.25 ~ 10 min, and the absorbance ratio A was measured after the reaction was completed700/A525And a blank control group (containing no pyridoxal phosphate) was set. As can be seen from FIG. 9, when the reaction time is 1 minute, the aggregation-inducing effect of pyridoxal phosphate on mercaptoethylamine-nanogold tends to be stable (in the figure, A: mercaptoethylamine-nanogold, B: mercaptoethylamine-nanogold + pyridoxal phosphate).
Example 10:
measurement of pyridoxal phosphate 0.2 mL of pyridoxal phosphate at various concentrations and 0.2 mL of the mercaptoethylamine-nanogold prepared in example 1 were sequentially added to 0.6 mL of acetate buffer solution (pH = 6.0, 10 mmol/L), mixed well, and reacted at room temperature for 1 minute after completion of the reaction, the change in color was visually observed, and the result is shown in FIG. 10. the color of mercaptoethylamine-nanogold was changed from wine red → pink → purple → blue with the gradual increase in the concentration of pyridoxal phosphate (0 ~ 0.5.5. mu. mol/L).
Example 11:
determination of pyridoxal phosphate: 0.2 mL of pyridoxal phosphate at various concentrations and 0.2 mL of mercaptoethylamine-nanogold prepared in example 1 were sequentially added to 0.6 mL of acetate buffer solution (pH = 6.0, 10 mmol/L), mixed well, and reacted at room temperature for 1 minute. After the reaction is finished, measuring the absorbance ratio A of the mixture after the reaction is finished700/A525The results are shown in FIG. 11, where the concentration of pyridoxal phosphate was varied within the range of 0 ~ 0.2.2. mu. mol/L, the absorbance ratio A of mercaptoethylamine to nanogold was found to be700/A525The change is not obvious, when the concentration of the pyridoxal phosphate is changed within the range of 0.25 ~ 0.5.5 mu mol/L, the absorbance ratio A of the mercaptoethylamine to the nanogold700/A525(ii) increases with increasing pyridoxal phosphate concentration; when the concentration of pyridoxal phosphate is more than 0.5 mu mol/L, the absorbance ratio A of mercaptoethylamine to nanogold700/A525The variation tends to be smooth.

Claims (5)

1. A method for rapidly determining pyridoxal phosphate by taking nanogold as a chromogenic probe is characterized in that the concentration of pyridoxal phosphate is determined by utilizing the action of pyridoxal phosphate and nanogold to cause the nanogold to aggregate to show the change of solution color and ultraviolet-visible absorption spectrum characteristics; judging the concentration of pyridoxal phosphate by using the color change of the nanogold solution; the used nano-gold is prepared by the following steps: first, 400. mu.L of mercaptoethylamine hydrochloride having a concentration of 213 mmol/L and 2.23 mL of HAuCl having a concentration of 10mg/mL were taken4Adding into a beaker containing 37.5 mL of water, and stirring at room temperature for reaction for 20 min; after the reaction, 10. mu.L of NaBH of 10 mmol/L concentration was added to the reaction solution4Then, continuously stirring and reacting at room temperature for 30 min to obtain a mercaptoethylamine-nanogold crude product; purifying the prepared mercaptoethylamine-nanogold crude product by using a dialysis bag with the molecular weight cutoff of 7000 to obtain a mercaptoethylamine-nanogold pure product; the obtained mercaptoethylamine-nano-scaleSealing the pure gold product in a dark place, and storing the pure gold product in a refrigerator at 4 ℃ for later use.
2. A method for rapidly determining pyridoxal phosphate by taking nanogold as a chromogenic probe is characterized in that the concentration of pyridoxal phosphate is determined by utilizing the action of pyridoxal phosphate and nanogold to cause nanogold to aggregate to show the change of solution color and ultraviolet-visible absorption spectrum characteristics; using the absorbance ratio (A) of nano-gold at 700 nm and 525 nm700/A525) (ii) varying to determine the concentration of pyridoxal phosphate; the used nano-gold is prepared by the following steps: first, 400. mu.L of mercaptoethylamine hydrochloride having a concentration of 213 mmol/L and 2.23 mL of HAuCl having a concentration of 10mg/mL were taken4Adding into a beaker containing 37.5 mL of water, and stirring at room temperature for reaction for 20 min; after the reaction, 10. mu.L of NaBH of 10 mmol/L concentration was added to the reaction solution4Then, continuously stirring and reacting at room temperature for 30 min to obtain a mercaptoethylamine-nanogold crude product; purifying the prepared mercaptoethylamine-nanogold crude product by using a dialysis bag with the molecular weight cutoff of 7000 to obtain a mercaptoethylamine-nanogold pure product; sealing the obtained pure mercaptoethylamine-nanogold product in a dark place, and storing the product in a refrigerator at 4 ℃ for later use.
3. The method for rapid determination of pyridoxal phosphate using nanogold as a chromogenic probe according to claim 1 or 2, wherein pyridoxal phosphate and mercaptoethylamine-nanogold at different concentrations are sequentially added to an acetate buffer solution, mixed uniformly, reacted at room temperature for 0.25 ~ 10 minutes, and after completion of the reaction, the color characteristics are visually observed or the absorbance ratio A is determined700/A525When the color characteristics are observed visually, the color of the mercaptoethylamine-nanogold changes from wine red → pink → purple → blue with the gradual increase of the pyridoxal phosphate concentration in the interval of 0 ~ 0.5.5 mu mol/L, and when the absorbance ratio A is determined700/A525When the concentration of pyridoxal phosphate is changed within the range of 0.25 ~ 0.5.5 mu mol/L, the absorbance ratio A of mercaptoethylamine to nanogold700/A525Increases with increasing pyridoxal phosphate concentration.
4. The method for rapidly measuring pyridoxal phosphate by using nanogold as a chromogenic probe according to claim 3, wherein the volume ratio of pyridoxal phosphate to mercaptoethylamine-nanogold to an acetate buffer solution is 1:1:3, and the total reaction volume is 1 mL.
5. The method for rapid determination of pyridoxal phosphate according to claim 3, wherein acetate buffer solution is used having pH and ionic strength of 6.0 and 10 mmol/L, respectively, the reaction temperature is room temperature, and the reaction time is 1 minute.
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