CN103969436B - The new method that a kind of hypersensitive alkaline phosphatase detects - Google Patents

Abstract

The invention discloses a new method for ultrasensitive alkaline phosphatase detection. The alkaline phosphatase in the sample to be tested hydrolyzes 5-bromo-4-chloro-3-indole phosphate to generate 5-bromo-4-chloro- 3-indole and inorganic phosphate, 5-bromo-4-chloro-3-indole reduced Nile blue A, decreased the SERS intensity of Nile blue A, and indirectly detected alkaline phosphatase. Compared with the ALP activity detection method of optical density detection or light intensity detection of the present invention, the ultrasensitive detection based on SERS of the present invention can detect alkaline phosphatase on the level of lower concentration; Compared with the existing alkaline phosphatase SERS detection Compared with the method, what the present invention detects is the SERS signal of the Nile Blue A molecule (NBA) molecule, and the intrinsic Raman activity of the Nile Blue A molecule is higher than that of the BCIP product in the alkaline phosphatase SERS detection method that has, is conducive to Further improve analytical sensitivity.

Description

A new method for ultrasensitive alkaline phosphatase detection

technical field

The invention relates to the technical field of enzyme activity detection, more specifically, to a new method for ultrasensitive alkaline phosphatase detection based on surface-enhanced Raman detection technology.

Background technique

Alkaline phosphatase (ALP) is a hydrolytic enzyme that removes phosphate groups from biological molecules to exert biological effects and participate in life activities such as energy metabolism and signal transduction. Changes in its activity or content will indicate biochemical changes such as physiological and pathological changes reaction. Therefore, detection of ALP in body fluids can diagnose various diseases. In addition, ALP is also used as a labeling reagent for biological research. When it is used as an enzyme immunolabeling reagent, compared with horseradish peroxidase, ALP has the advantages of high stability and high sensitivity. It is also used as a quantitative indicator for gene expression studies. It is very important to develop an ultrasensitive detection method of ALP activity for the above research. Currently, the activity of alkaline phosphatase (ALP) is detected by measuring the optical density of the colored product or the intensity of fluorescence or chemiluminescence emitted by the product. The detection limits of the method were 500μU/L and 20μU/L, respectively.

Surface-enhanced Raman scattering (SERS) technology is a powerful tool for ultrasensitive detection. In practical applications, strong SERS signals are always obtained from molecules with high affinity for SERS materials and high intrinsic Raman activity. Many strategies have been developed for those molecules with low affinity for SERS materials or low Raman activity. In the study of SERS detection of enzyme activity, when the substrate and product of the enzyme cannot provide strong SERS signals, the synthesis of alternative substrates with dyes is an effective method.

5-Bromo-4-chloro-3-indole phosphate (BCIP), a commercially available chromogenic substrate commonly used in the detection of ALP activity, is converted to 5-bromo-4-chloro-3-indole (BCI) and inorganic phosphates. The resulting BCI can reduce nitroblue tetrazolium (NBT) to form an insoluble colored precipitate indicating the presence of ALP in immunoblotting, in situ hybridization, and immunohistochemical studies. Although the SERS detection method of ALP has been developed by collecting the SERS signal of BCI dimer, in the present invention, Nile Blue A (NBA) is a molecule with strong intrinsic Raman activity, which can establish the ultrasensitive ALP SERS detection method.

Contents of the invention

Purpose of the invention: The purpose of the present invention is to provide a new method for ultrasensitive alkaline phosphatase detection based on surface-enhanced Raman detection technology, which has the characteristics of easy operation.

Technical solution: In order to achieve the above object, the present invention is realized through the following technical solution: a new method for ultrasensitive alkaline phosphatase detection, the alkaline phosphatase in the sample to be tested hydrolyzes 5-bromo-4-chloro-3- Indole phosphoric acid, generate 5-bromo-4-chloro-3-indole and inorganic phosphate, 5-bromo-4-chloro-3-indole reduces Nile blue A, reduces the SERS intensity of Nile blue A, indirectly Detection of alkaline phosphatase.

The present invention uses Nile blue A molecule (NBA) and 5-bromo-4-chloro-3-indole phosphoric acid as the substrate of ALP, and detects a kind of SERS spectrum of Nile blue A molecule (NBA) in the reaction system A new method for ultrasensitive alkaline phosphatase detection.

Specifically include the following steps:

1) The sample to be tested containing alkaline phosphatase is diluted 1-10000 times with a tris-HCl buffer solution containing MgCl and _NaCl at pH 9.5 to obtain a solution;

2) Add 5-bromo-4-chloro-3-indole phosphoric acid aqueous solution and Nile blue A molecule aqueous solution to step 1) solution, and then incubate at 37°C for 30 minutes, the 5-bromo-4-chloro- The initial concentration of the aqueous solution of 3-indole phosphoric acid in the solution is 1×10 ^-3 -1×10 ^-6 mol/L, and the initial concentration of the aqueous solution of Nile Blue A molecule in the solution is 1×10 ^-4 - 1×10 ^-9 mol/L;

3) adding the SERS active material into the solution obtained in step 2), and measuring the SERS signal after 5 minutes.

Wherein, the concentration of the above MgCl ₂ in the tris-HCl buffer solution is 0.1-50mmol/L, and the concentration of the NaCl in the tris-HCl buffer solution is 10-1000mmol/L.

Wherein, the concentration of the tris-HCl buffer solution is 1-1000mmol/L.

Wherein, the above-mentioned SERS active material is a gold nanoparticle suspension, a silver nanoparticle suspension, a gold-silver composite nanoparticle suspension, a gold-silver composite nanoparticle suspension or an inorganic or organic material, or a composite formed on a solid substrate. One of the nanomaterials.

Wherein, the above-mentioned nanoparticle suspension compounded with gold and silver and inorganic materials is a gold shell or silver shell suspension with silicon or silicon dioxide as the core; the nanoparticle suspension compounded with gold and silver with organic materials is polystyrene Gold shell or silver shell suspension as the core; the nanomaterial integrated on the solid phase substrate is the solid phase substrate nanomaterial formed by absorbing any one or several kinds of nanoparticle suspensions mentioned above onto glass slides or acupuncture needles.

Beneficial effect: compared with the prior art, the present invention has the following advantages:

(1) Compared with the ALP activity detection method of optical density detection or light intensity detection, the ultrasensitive detection based on SERS of the present invention can detect alkaline phosphatase at a lower concentration level.

(2) Compared with the existing alkaline phosphatase SERS detection method, what the present invention detects is the SERS signal of Nile Blue A molecule (NBA) molecule, and the inherent Raman activity of Nile Blue A molecule is more basic than some The high BCIP product in the phosphatase SERS detection method is conducive to further improving the analytical sensitivity.

Description of drawings

Fig. 1 is a schematic diagram of the detection principle of the present invention.

Figure 2 is a standard curve for detecting alkaline phosphatase based on a solid-phase SERS substrate.

Detailed ways

The following non-limiting examples can enable those skilled in the art to understand the present invention more fully, but do not limit the present invention in any way.

The SERS detection of embodiment 1 pure alkaline phosphatase

Pure enzyme is diluted to 1,10,100,1000,10000mU/L with the tris-HCl buffer solution (pH9.5) containing the MgCl of 5mmol/L ₂ and the NaCl of 100mmol/L; BCIP aqueous solution and Nile blue A molecule ( NBA) aqueous solution was added to the above solution, and after incubation at 37°C for 30 minutes, the concentration of tris-HCl buffer solution (pH9.5) was 1 mmol/L; the initial concentration of BCIP aqueous solution in the solution was 4.5×10 ^-4 mol/L, the initial concentration of the Nile blue A molecule (NBA) aqueous solution in the solution is 4×10 ^-6 mol/L; the glass slide integrated with the SERS active material is immersed in the above solution, and the SERS signal is measured after 5 minutes, according to The SERS intensity at 592cm ^-1 and the corresponding amount of alkaline phosphatase were used to draw a standard curve (see Figure 2). The linear equation is obtained: Y=11.6397X+33413.429. The SERS active material is a gold-silver composite nanoparticle suspension.

Embodiment 2: SERS detection of alkaline phosphatase in blood

The blood is diluted to ₁ , 10, 100, 1000, 10000 times with the tris-HCl buffer solution (pH9.5) containing the MgCl of 5mmol/L and 100mmol/L NaCl; wherein the tris-HCl buffer solution (pH9. 5) the concentration is 10mmol/L; BCIP aqueous solution and Nile blue A molecule (NBA) aqueous solution are added in the above-mentioned solution, and after incubating at 37 ℃ for 30 minutes, wherein, the initial concentration of described BCIP aqueous solution in the solution is 4.5×10 ^-4 mol/L, the initial concentration of the Nile blue A molecule (NBA) aqueous solution in the solution is 4×10 ^-6 mol/L; the glass slide integrated with the SERS active material is immersed in the above solution, The SERS signal was measured after 5 minutes, and the SERS active material was a nanoparticle suspension composed of gold, silver and silicon dioxide, with silicon dioxide as the core. The SERS intensity diluted 10 times is 1.03×10 ⁵ . The specific enzyme content can be calculated according to the standard curve in Example 1 to be about 119.98 U/L.

Example 3: SERS detection of phosphatase in blood

The blood is diluted to ₁ , 10, 100, 1000, 10000 times with the tris-HCl buffer solution containing 0.1mmol/L MgCl and 10mmol/L NaCl-containing pH9.5 to obtain a solution; wherein the tris-HCl buffer solution (pH9 .5) the concentration is 20mmol/L; 5-bromo-4-chloro-3-indole phosphoric acid aqueous solution and nile blue A molecular aqueous solution are added to the above solution, and then incubated at 37°C for 30 minutes, the 5- The initial concentration of bromo-4-chloro-3-indole phosphoric acid aqueous solution in the solution is 1×10 ^-3 mol/L, and the initial concentration of the aqueous solution of Nile Blue A molecule in the solution is 1×10 ^-4 mol /L; the SERS active material was added to the resulting solution, and the SERS signal was measured after 5 minutes. The SERS active material is a suspension of gold-shell nanoparticles with polystyrene as the core. Among them, the SERS intensity diluted 10 times is 8.2×10 ⁴ . The specific enzyme content can be calculated according to the standard curve in Example 1 to be about 493.45 U/L.

The SERS detection of phosphatase in embodiment 4 blood

1) The blood is diluted to 1, 10, 100, 1000, 10000 times with the tris-HCl buffer solution containing the MgCl of 25mmol/L ₂ and the pH9.5 of 500mmol/L NaCl to obtain the solution; wherein the tris-HCl buffer solution ( The concentration of pH9.5) is 500mmol/L; 2) Add 5-bromo-4-chloro-3-indole phosphoric acid aqueous solution and Nile blue A molecular aqueous solution to step 1) solution, and then incubate at 37°C for 30 minutes , the initial concentration of 5-bromo-4-chloro-3-indole phosphoric acid aqueous solution in the solution is 0.5×10 ^-3 mol/L, the initial concentration of Nile blue A molecular aqueous solution in the solution is 0.5×10 ^-4 mol /L; 3) Add the SERS active material to the solution obtained in step 2), and measure the SERS signal after 5 minutes. The SERS active material is a solid-phase substrate nanomaterial formed by absorbing a silver nanoparticle suspension onto a glass slide. The SERS intensity diluted 10 times is 4.5×10 ⁴ . The specific enzyme content can be calculated according to the standard curve in Example 1 to be about 31.65 U/L.

The SERS detection of phosphatase in embodiment 5 blood

1) The blood is diluted to 1, 10, 100, 1000, 10000 times with the tris-HCl buffer solution containing the MgCl of 50mmol/L ₂ and the pH9.5 of 1000mmol/L NaCl to obtain the solution; wherein the tris-HCl buffer solution ( The concentration of pH9.5) is 1000mmol/L; 2) Add 5-bromo-4-chloro-3-indole phosphoric acid aqueous solution and Nile blue A molecular aqueous solution to step 1) solution, and then incubate at 37°C for 30 minutes , the initial concentration of 5-bromo-4-chloro-3-indole phosphoric acid aqueous solution in the solution is 1×10 ^-6 mol/L, the initial concentration of Nile blue A molecular aqueous solution in the solution is 1×10 ^-9 mol /L; 3) Add the SERS active material to the solution obtained in step 2), and measure the SERS signal after 5 minutes. The SERS active material is a composite of gold, silver and silicon dioxide, and a solid-phase substrate nanomaterial formed by adsorbing a suspension of nanoparticles with silicon dioxide as the core on acupuncture needles. The SERS intensity diluted 10 times is 8.6×10 ⁴ . The specific enzyme content can be calculated according to the standard curve in Example 1 to be about 498.8 U/L.

Claims (5)

1. A method for ultrasensitive alkaline phosphatase detection, characterized in that the alkaline phosphatase in the sample to be tested hydrolyzes 5-bromo-4-chloro-3-indole phosphate to generate 5-bromo-4-chloro -3-indole and inorganic phosphate, 5-bromo-4-chloro-3-indole reduce Nile blue A, reduce the surface-enhanced Raman scattering intensity of Nile blue A, and indirectly detect alkaline phosphatase. 2. the method for a kind of ultrasensitive alkaline phosphatase detection according to claim 1, is characterized in that, specifically comprises the following steps: 1) Dilute the test sample containing alkaline phosphatase by 1-10000 times with Ttris-HCl buffer solution containing MgCl ₂ and NaCl at pH 9.5 to obtain a solution; 2) Add 5-bromo-4-chloro-3-indole phosphoric acid aqueous solution and Nile blue A molecular aqueous solution to the solution in step 1), and incubate at 37°C for 30 minutes, the 5-bromo-4-chloro- The initial concentration of the aqueous solution of 3-indolephosphoric acid in the solution is 1×10 ^-3 -1×10 ^-6 mol/ L, and the initial concentration of the aqueous solution of Nile Blue A molecule in the solution is 1×10 ^-4 - 1×10 ^-9 mol/L; 3) Add the surface-enhanced Raman scattering active material to the solution obtained in step 2), and measure the SERS signal after 5 minutes. 3. the method for a kind of ultrasensitive alkaline phosphatase detection according to claim ₂ , is characterized in that, described MgCl Concentration in Tris-HCl buffer solution is 0.1-50 mmol/ L, and described NaCl is in The concentration in Tris-HCl buffer solution is 10-1000 mmol/L. 4. the method for a kind of ultrasensitive alkaline phosphatase detection according to claim 2, is characterized in that, the concentration of described Tris-HCl buffer solution is 1-1000 mmol/L. 5. the method for a kind of ultrasensitive alkaline phosphatase detection according to claim 2, is characterized in that, described surface enhanced Raman scattering active material is gold nanoparticle suspension, silver nanoparticle suspension, gold-silver composite One of nanoparticle suspensions, composite nanoparticle suspensions of gold and silver and inorganic or organic materials, or nanomaterials integrated on solid substrates.