CN108680566B - Novel method for detecting hydrogen peroxide based on MoS2 nano enzyme luminescence system - Google Patents

Abstract

The invention discloses a new method for detecting hydrogen peroxide based on a MoS2 nano enzyme luminescence system, which comprises the following steps of S1, preparing MoS2 two-dimensional nano enzyme, S2, preparing 0.1 mol/L luminol mother liquor and Tris-HCl buffer solution, S3, diluting the prepared luminol mother liquor to 5 millimole/liter luminol solution by using Tris-HCl buffer solution, then uniformly mixing 40 microliter of the obtained solution and 40 microliter of hydrogen peroxide solution with different concentrations in a measuring cup, adding 40 microliter of 1 millimole/liter MoS2 nano enzyme into the measuring cup, reacting for 30 seconds, putting the whole reaction system into a chemiluminescence determinator, and collecting and recording optical signals generated by the reaction through the chemiluminometer.

Description

Novel method for detecting hydrogen peroxide based on MoS2 nano enzyme luminescence system

Technical Field

The invention relates to a hydrogen peroxide detection method, in particular to a new method for detecting hydrogen peroxide based on a MoS2 nano enzyme luminescence system.

Background

Hydrogen peroxide is an oxidizing agent and is a common metabolite in a living body, whether the living metabolism is normal can be known by detecting and monitoring the hydrogen peroxide, and meanwhile, one of disease symptoms is abnormal hydrogen peroxide content, so that the hydrogen peroxide detection can provide scientific and objective evidence for monitoring the living body metabolism and diagnosing diseases, and has very important research and development values.

At present, the specific and sensitive means for detecting hydrogen peroxide is known to be catalase of organisms, however, the biological nature of catalase is harsh in purification, storage and use conditions and high in cost, so the requirement of enzyme is extremely urgent. The MoS2 two-dimensional nano material is a nano enzyme with better hydrogen peroxide enzyme activity, and the nano enzyme and a tetramethyl benzidine substrate (TMB) share the same effect and show very good enzyme-like characteristics in a hydrogen peroxide colorimetric detection method. However, the hydrogen peroxide detection system composed of the MoS2 nano-class enzyme and TMB is based on color change caused by oxidation of TMB substrate (colorimetric detection), and has weak signal, slow reaction and high detection limit, so that further research is needed to develop a detection method with better detection performance.

Disclosure of Invention

In order to solve the problems, the invention provides a novel method for detecting hydrogen peroxide based on a MoS2 nano enzyme luminescent system, which enhances the intensity of a luminescent signal during hydrogen peroxide detection and shortens the reaction time.

In order to achieve the purpose, the invention adopts the technical scheme that:

the novel method for detecting hydrogen peroxide based on the MoS2 nano enzyme luminescent system comprises the following steps:

s1, preparation of MoS2 two-dimensional nano enzyme

Adding 50mgMoS2 into 10ml water solution containing 10mg Bovine Serum Albumin (BSA), carrying out ultrasonic treatment for 6h under the condition of power of 240W by using a gun type ultrasonic probe, placing the mixture into a high-speed centrifuge, centrifuging for 45min at the speed of 5000r/min, carrying out ultrasonic redispersion for 10min in a general ultrasonic cleaning bath, centrifuging for 45min at the speed of 15000r/min, collecting supernatant, and storing for later use at 4 ℃;

s2, preparing luminol solution and Tris-HCl buffer solution

Preparing luminol solution

Weighing 7.08mg of luminol reagent, dissolving the reagent in 4ml of sodium hydroxide solution with the concentration of 0.1 mol/L to prepare 0.1 mol/L luminol mother solution, and storing the solution at 4 ℃ in a dark place;

preparing Tris-HCl buffer solution

Weighing 1.211mg of tris (hydroxymethyl) aminomethane, dissolving the tris (hydroxymethyl) aminomethane in 75ml of ultrapure water, respectively adjusting the pH to 11 by using 1 mol/L hydrochloric acid solution, adding 25mg of ethylenediamine tetraacetic acid, and fixing the volume of the ultrapure water to 1000ml for later use;

s3 measurement of Hydrogen peroxide

Diluting the prepared luminol mother solution to 5 millimole liter of luminol solution by using Tris-HCl buffer solution, then uniformly mixing 40 microliter of the obtained solution and 40 microliter of hydrogen peroxide solution with different concentrations in a measuring cup, adding 40 microliter of MoS2 nano enzyme with 1 millimole liter of the solution, reacting for 30 seconds, putting into a chemiluminescence determinator, collecting and recording an optical signal generated by the reaction through the chemiluminiscence instrument, and quantifying the hydrogen peroxide according to a characteristic signal frequency peak and a peak intensity on an optical signal spectrum.

In the scheme, the luminol/MoS 2 nano enzyme is used as a hydrogen peroxide detection system, so that the intensity of the generated optical signal can be obviously improved, the detection limit of the hydrogen peroxide can be reduced, the concentration of the detectable hydrogen peroxide can be further reduced, and a new method is provided for detecting the high-sensitivity micro-trace hydrogen peroxide.

Drawings

FIG. 1 is a UV-vis spectroscopy of the supernatant after centrifugation of ultrasonically stripped MoS2 in an example of the invention.

FIG. 2 is a two-dimensional MoS with exfoliation in an embodiment of the invention₂TEM images of the nanoplates.

Detailed Description

In order that the objects and advantages of the invention will be more clearly understood, the invention is further described in detail below with reference to examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Examples

S1, preparation of MoS2 two-dimensional nano enzyme

Adding 50mgMoS2 into 10ml water solution containing 10mg Bovine Serum Albumin (BSA), carrying out ultrasonic treatment for 6h under the condition of power of 240W by using a gun type ultrasonic probe, placing the mixture into a high-speed centrifuge, centrifuging for 45min at the speed of 5000r/min, carrying out ultrasonic redispersion for 10min, then centrifuging for 45min at the speed of 15000r/min, collecting supernatant, and storing at 4 ℃ for later use;

s2, preparing luminol solution and Tris-HCl buffer solution

Preparing luminol solution

Weighing 7.08mg of luminol reagent, dissolving in 4ml of 0.1 mol/L-concentration sodium hydroxide solution to prepare 0.1 mol/L luminol mother solution, storing at 4 ℃ in a dark place, and diluting with Tris-HCl buffer solution to the required concentration when in use.

Preparing Tris-HCl buffer solution

1.211mg of tris (hydroxymethyl) aminomethane 1 part was weighed using an electronic balance, and dissolved in 75ml of ultrapure water, respectively, the pH was adjusted to 11 with 1 mol/L of hydrochloric acid solution, respectively, 25mg of ethylenediaminetetraacetic acid was added to each buffer solution adjusted, and the volume was adjusted to 1000ml using ultrapure water for use.

Preparing hydrogen peroxide solution

Hydrogen peroxide solution with the concentration of 30 percent is taken as mother solution, is placed at 4 ℃ and is kept away from light, and is diluted into the required concentration by using ultrapure water when in use.

S3 measurement of Hydrogen peroxide

Diluting the prepared luminol mother solution to 5 millimole liter of luminol solution by using Tris-HCl buffer solution, then uniformly mixing 40 microliter of the 5 millimole liter of luminol solution and 40 microliter of hydrogen peroxide solution with different concentrations (the detection limit can be determined by the concentration of the hydrogen peroxide solution) in a measuring cup, adding 40 microliter of MoS2 nano enzyme with 1 millimole liter of the solution, reacting for 30 seconds, and then putting into a chemiluminescence determinator. The light signal generated by the reaction was collected and recorded by a chemiluminescence apparatus. The chemiluminescence apparatus measures the high voltage set to-900V, measures the detection set to 0.05s, and records the integrated signal of 10 s.

The results show that our detection limit is at 37 nanomoles per liter, 1 order of magnitude higher than that based on TMB (which is at 0.08 micromoles per liter in micromoles per liter-reference "guo xinrong, ni, renegon, detection of hydrogen peroxide based on molybdenum disulfide chromogenic).

FIG. 1 can help to prove the stripping of MoS2, and obviously, typical single-layer or few-layer MoS2 characteristic peaks appear near the wavelengths of 430nm, 610nm and 670nm, which proves the existence of MoS2 two-dimensional nano enzyme. FIG. 2 is a typical MoS2 two-dimensional nanoenzyme topography with few layers on the left and a monolayer on the right, which is very similar to the base color, indicating that the thinner the sheet, the closer the monolayer.

2) Fluorescence test result data

When the MoS2 two-dimensional nano enzyme is not added to detect the hydrogen peroxide and when the MoS2 two-dimensional nano enzyme is added to detect the hydrogen peroxide, the luminous peaks are displayed at the spectrum position with the wavelength of 450nm, which means that the MoS 2/luminol luminous system can actually detect the hydrogen peroxide, and meanwhile, when the MoS2 two-dimensional nano enzyme is added to detect the hydrogen peroxide, the luminous peak intensity is 2000 units, while when the MoS2 two-dimensional nano enzyme is not added to detect the hydrogen peroxide, the luminous peak intensity is only 500 units, and obviously, the intensity of the luminous signal can be greatly enhanced by adding the MoS2 two-dimensional nano enzyme.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be construed as the protection scope of the present invention.

Claims (1)

1. The new method for detecting hydrogen peroxide based on the MoS2 nano enzyme luminescence system is characterized by comprising the following steps: the method comprises the following steps:

s1, preparation of MoS2 two-dimensional nano enzyme

Adding 50mgMoS2 into 10ml water solution containing 10mg Bovine Serum Albumin (BSA), carrying out ultrasonic treatment for 6h under the condition of power of 240W by using a gun type ultrasonic probe, placing the mixture into a high-speed centrifuge, centrifuging for 45min at the speed of 5000r/min, carrying out ultrasonic redispersion for 10min in a general ultrasonic cleaning bath, centrifuging for 45min at the speed of 15000r/min, collecting supernatant, and storing for later use at 4 ℃;

s2, preparing luminol solution and Tris-HCl buffer solution

Preparing luminol solution

Weighing 7.08mg of luminol reagent, dissolving the reagent in 4ml of sodium hydroxide solution with the concentration of 0.1 mol/L to prepare 0.1 mol/L luminol mother solution, and storing the solution at 4 ℃ in a dark place;

preparing Tris-HCl buffer solution

Weighing 1.211mg of tris (hydroxymethyl) aminomethane, dissolving the tris (hydroxymethyl) aminomethane in 75ml of ultrapure water, respectively adjusting the pH to 11 by using 1 mol/L hydrochloric acid solution, adding 25mg of ethylenediamine tetraacetic acid, and fixing the volume of the ultrapure water to 1000ml for later use;

s3 measurement of Hydrogen peroxide

Diluting the prepared luminol mother solution to 5 millimole liter of luminol solution by using Tris-HCl buffer solution, then uniformly mixing 40 microliter of the obtained solution and 40 microliter of hydrogen peroxide solution with different concentrations in a measuring cup, adding 40 microliter of MoS2 nano enzyme with 1 millimole liter of the solution, reacting for 30 seconds, putting into a chemiluminescence determinator, collecting and recording an optical signal generated by the reaction through the chemiluminiscence instrument, and quantifying the hydrogen peroxide according to a characteristic signal frequency peak and a peak intensity on an optical signal spectrum.

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