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

The new method that a kind of hypersensitive alkaline phosphatase detects Download PDF

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CN103969436B
CN103969436B CN201410194039.1A CN201410194039A CN103969436B CN 103969436 B CN103969436 B CN 103969436B CN 201410194039 A CN201410194039 A CN 201410194039A CN 103969436 B CN103969436 B CN 103969436B
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alkaline phosphatase
solution
sers
bromo
chloro
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CN103969436A (en
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董健
李媛
张明月
郝振宇
朱珠
张里
钱卫平
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Southeast University
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Southeast 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
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)

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  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses the new method that a kind of hypersensitive alkaline phosphatase detects, the chloro-3-indoylphosphate of the bromo-4-of alkaline phosphatase enzyme hydrolysis 5-in testing sample, generate the chloro-3-indoles of the bromo-4-of 5-and inorganic phosphate, the bromo-4-of 5-chloro-3-indoles reduction Nile blue A, reduce the SERS intensity of Nile blue A, indirect detection alkaline phosphatase.The present invention compared with the ALP activity test method that optical density detects or light intensity detects, the present invention is based on SERS super sensitivity detection can in the level of lower concentration detection of alkaline phosphatase; Compared with existing alkaline phosphatase SERS detection method, what the present invention detected is the SERS signal of Nile blue A molecule (NBA) molecule, the inherent Raman active of Nile blue A molecule, than the height of BCIP product in the alkaline phosphatase SERS detection method had, is conducive to improving sensitivity for analysis further.

Description

The new method that a kind of hypersensitive alkaline phosphatase detects
Technical field
The present invention relates to a kind of technical field of Enzyme assay, more specifically, relate to the new method that a kind of hypersensitive alkaline phosphatase set up based on surface-enhanced Raman detection technique detects.
Background technology
Alkaline phosphatase (ALP) is a kind of hydrolytic enzyme, plays biological effect, participate in the vital movement such as energetic supersession, signal transduction from biomolecule removing phosphate group, and change that is active at it or content will indicate the biochemical reaction such as physiology and Pathologic.Therefore, detect ALP in body fluid and can diagnose various diseases.In addition, ALP is also used as labelled reagent is biological study.When it is that compared to horseradish peroxidase, ALP has high stability as the immune labeled reagent of enzyme, highly sensitive advantage.It is also used as gene expression research quantizating index.The detection method developing a kind of overdelicate ALP activity is very important for above-mentioned research.At present, the activity of alkaline phosphatase (ALP) detects by measuring the optical density of color products or the fluorescence sent of product or chemiluminescence intensity.The detection limit of method is respectively 500 μ U/L and 20 μ U/L.
Surface enhanced raman spectroscopy (SERS) technology is the powerful of overdelicate detection.In actual applications, strong SERS signal always obtains from molecule SERS material to high-affinity and high inherent Raman active.Many strategies have been developed for those the molecule that SERS material affinity is low or Raman active is low.Detect in the activity research of enzyme at SERS, when the substrate of enzyme and product can not provide strong SERS signal, anamorphic zone has the alternative substrate of dyestuff to be a kind of effective method.
The chloro-3-indoylphosphate (BCIP) of the bromo-4-of 5-detects commercialization chromogenic substrate conventional in ALP activity, and be translated into the chloro-3-indoles (BCI) of the bromo-4-of 5-and inorganic phosphate.In Western blot, immunofluorescent double labeling research, the BCI of generation can reduce nitroblue tetrazolium (NBT), to form insoluble colored precipitate thing, to indicate the existence of ALP.Although the SERS detection method of ALP has been developed by collecting the dimeric SERS signal of BCI, in the present invention, Nile blue A (NBA) has been a kind of molecule with stronger inherent Raman active, can set up the hypersensitive SERS detection method of ALP.
Summary of the invention
Goal of the invention: the object of this invention is to provide the new method that a kind of hypersensitive alkaline phosphatase set up based on surface-enhanced Raman detection technique detects, there is feature easy and simple to handle.
Technical scheme: for achieving the above object, the present invention is achieved through the following technical solutions: the new method that a kind of hypersensitive alkaline phosphatase detects, the chloro-3-indoylphosphate of the bromo-4-of alkaline phosphatase enzyme hydrolysis 5-in testing sample, generate the chloro-3-indoles of the bromo-4-of 5-and inorganic phosphate, the bromo-4-of 5-chloro-3-indoles reduction Nile blue A, reduce the SERS intensity of Nile blue A, indirect detection alkaline phosphatase.
The present invention using Nile blue A molecule (NBA) and the chloro-3-indoylphosphate of the bromo-4-of the 5-substrate as ALP, the new method detected by a kind of hypersensitive alkaline phosphatase of the SERS spectrum of Nile blue A molecule (NBA) in detection reaction system.
Specifically comprise the following steps:
1) testing sample containing alkaline phosphatase is used containing MgCl 2doubly solution is obtained with the tris-HCl buffer solution dilution 1-10000 of the pH9.5 of NaCl;
2) bromo-for 5-4-chloro-3-indoylphosphate aqueous solution and Nile blue A molecular water solution are added step 1) in solution, then 37 DEG C of incubations 30 minutes, the bromo-4-of described 5-chloro-3-indoylphosphate aqueous solution initial concentration was in the solution 1 × 10 -3-1 × 10 -6mol/L, described Nile blue A molecular water solution initial concentration is in the solution 1 × 10 -4-1 × 10 -9mol/L;
3) SERS active material is added step 2) in the solution that obtains, after 5 minutes, measure SERS signal.
Wherein, above-mentioned MgCl 2concentration in tris-HCl buffer solution is 0.1-50mmol/L, and the concentration of described NaCl in tris-HCl buffer solution is 10-1000mmol/L.
Wherein, the concentration of above-mentioned tris-HCl buffer solution is 1-1000mmol/L.
Wherein, above-mentioned SERS active material be gold nanoparticle suspension, one in the nanoparticle suspension of the nanoparticle suspension of silver nanoparticle suspension, gold and silver compound, gold and silver and inorganic or organic material compound or be integrated in nano material that solid phase substrate is formed.
Wherein, the nanoparticle suspension of above-mentioned gold and silver and inorganic material compound is silicon or silicon dioxide is the golden shell of core or silver-colored shell suspending liquid; The nanoparticle suspension of described gold and silver and organic material compound is polystyrene is the golden shell of core or silver-colored shell suspending liquid; The described solid phase substrate nano material that is integrated in is that any one or a few nanoparticle suspension above-mentioned is adsorbed onto the solid phase substrate nano material that microslide or acupuncture needle are formed.
Beneficial effect: compared with prior art, tool of the present invention has the following advantages:
(1) the present invention is compared with the ALP activity test method that optical density detects or light intensity detects, the present invention is based on SERS super sensitivity detection can in the level of lower concentration detection of alkaline phosphatase.
(2) compared with existing alkaline phosphatase SERS detection method, what the present invention detected is the SERS signal of Nile blue A molecule (NBA) molecule, the inherent Raman active of Nile blue A molecule, than the height of BCIP product in the alkaline phosphatase SERS detection method had, is conducive to improving sensitivity for analysis further.
Accompanying drawing explanation
Fig. 1 is the principle schematic that the present invention detects.
Fig. 2 is the typical curve based on a kind of solid phase SERS substrate detection of alkaline phosphatase.
Embodiment
Following non-limiting example can make the present invention of those of ordinary skill in the art's comprehend, but does not limit the present invention in any way.
The SERS of embodiment 1 soda ash acid phosphatase detects
The MgCl of pure enzyme containing 5mmol/L 2be diluted to 1 with the tris-HCl buffer solution (pH9.5) of the NaCl of 100mmol/L, 10,100,1000,10000mU/L; BCIP aqueous solution and Nile blue A molecule (NBA) aqueous solution add in above-mentioned solution, then at 37 DEG C of incubations after 30 minutes, wherein the concentration of tris-HCl buffer solution (pH9.5) is 1mmol/L; BCIP aqueous solution initial concentration is in the solution 4.5 × 10 -4mol/L, Nile blue A molecule (NBA) aqueous solution initial concentration is in the solution 4 × 10 -6mol/L; The microslide of integrated SERS active material is immersed above-mentioned solution, measures SERS signal after 5 minutes, according to 592cm -1the SERS intensity at place and the amount drawing standard curve (see Fig. 2) of corresponding alkaline phosphatase.Obtain linear equation: Y=11.6397X+33413.429.SERS active material is the nanoparticle suspension of gold and silver compound.
Embodiment 2: the SERS of blood alkaline phosphatase detects
By the MgCl of blood containing 5mmol/L 21,10,100,1000,10000 times is diluted to the tris-HCl buffer solution (pH9.5) containing NaCl of 100mmol/L; Wherein the concentration of tris-HCl buffer solution (pH9.5) is 10mmol/L; BCIP aqueous solution and Nile blue A molecule (NBA) aqueous solution add in above-mentioned solution, then at 37 DEG C of incubations after 30 minutes, wherein, described BCIP aqueous solution initial concentration is in the solution 4.5 × 10 -4mol/L, described Nile blue A molecule (NBA) aqueous solution initial concentration is in the solution 4 × 10 -6mol/L; The microslide of integrated SERS active material is immersed above-mentioned solution, measures SERS signal after 5 minutes, SERS active material is gold and silver and silicon dioxide compound, take silicon dioxide as the nanoparticle suspension of core.The SERS intensity of wherein diluting 10 times is 1.03 × 10 5.Concrete enzyme content can be calculated according to the typical curve of embodiment 1 and be about 119.98U/L.
Embodiment 3: in blood, the SERS of phosphatase detects
By the MgCl of blood containing 0.1mmol/L 2be diluted to 1,10,100,1000,10000 times with 10mmol/L containing the tris-HCl buffer solution of pH9.5 of NaCl and obtain solution; Wherein the concentration of tris-HCl buffer solution (pH9.5) is 20mmol/L; Bromo-for 5-4-chloro-3-indoylphosphate aqueous solution and Nile blue A molecular water solution are added in above-mentioned solution, then 37 DEG C of incubations 30 minutes, the bromo-4-of described 5-chloro-3-indoylphosphate aqueous solution initial concentration was in the solution 1 × 10 -3mol/L, described Nile blue A molecular water solution initial concentration is in the solution 1 × 10 -4mol/L; SERS active material is added in the solution obtained, after 5 minutes, measure SERS signal.The golden core/shell nanoparticles suspending liquid of SERS active material to be polystyrene be core.Wherein dilute the SERS intensity 8.2 × 10 of 10 times 4.Concrete enzyme content can be calculated according to the typical curve of embodiment 1 and be about 493.45U/L.
In embodiment 4 blood, the SERS of phosphatase detects
1) by the MgCl of blood containing 25mmol/L 2be diluted to 1,10,100,1000,10000 times with the tris-HCl buffer solution of the pH9.5 of the NaCl of 500mmol/L and obtain solution; Wherein the concentration of tris-HCl buffer solution (pH9.5) is 500mmol/L; 2) bromo-for 5-4-chloro-3-indoylphosphate aqueous solution and Nile blue A molecular water solution are added step 1) in solution, then 37 DEG C of incubations 30 minutes, the bromo-4-of 5-chloro-3-indoylphosphate aqueous solution initial concentration was in the solution 0.5 × 10 -3mol/L, Nile blue A molecular water solution initial concentration is in the solution 0.5 × 10 -4mol/L; 3) SERS active material is added step 2) in the solution that obtains, after 5 minutes, measure SERS signal.SERS active material is that silver nanoparticle suspension is adsorbed onto the solid phase substrate nano material that microslide is formed.Wherein dilute the SERS intensity 4.5 × 10 of 10 times 4.Concrete enzyme content can be calculated according to the typical curve of embodiment 1 and be about 31.65U/L.
In embodiment 5 blood, the SERS of phosphatase detects
1) by the MgCl of blood containing 50mmol/L 2be diluted to 1,10,100,1000,10000 times with the tris-HCl buffer solution of the pH9.5 of the NaCl of 1000mmol/L and obtain solution; Wherein the concentration of tris-HCl buffer solution (pH9.5) is 1000mmol/L; 2) bromo-for 5-4-chloro-3-indoylphosphate aqueous solution and Nile blue A molecular water solution are added step 1) in solution, then 37 DEG C of incubations 30 minutes, the bromo-4-of 5-chloro-3-indoylphosphate aqueous solution initial concentration was in the solution 1 × 10 -6mol/L, Nile blue A molecular water solution initial concentration is in the solution 1 × 10 -9mol/L; 3) SERS active material is added step 2) in the solution that obtains, after 5 minutes, measure SERS signal.SERS active material is gold and silver and silicon dioxide compound, is that the nanoparticle suspension of core is adsorbed onto the solid phase substrate nano material that acupuncture needle is formed with silicon dioxide.The SERS intensity of wherein diluting 10 times is 8.6 × 10 4.Concrete enzyme content can be calculated according to the typical curve of embodiment 1 and be about 498.8U/L.

Claims (5)

1. the method for a hypersensitive alkaline phosphatase detection, it is characterized in that, the chloro-3-indoylphosphate of the bromo-4-of alkaline phosphatase enzyme hydrolysis 5-in testing sample, generate the chloro-3-indoles of the bromo-4-of 5-and inorganic phosphate, the bromo-4-of 5-chloro-3-indoles reduction Nile blue A, reduce the Surface enhanced raman spectroscopy intensity of Nile blue A, indirect detection alkaline phosphatase.
2. the method for a kind of hypersensitive alkaline phosphatase detection according to claim 1, is characterized in that, specifically comprise the following steps:
1) testing sample containing alkaline phosphatase is used containing MgCl 2doubly solution is obtained with the Ttris-HCl buffer solution dilution 1-10000 of the pH9.5 of NaCl;
2) bromo-for 5-4-chloro-3-indoylphosphate aqueous solution and Nile blue A molecular water solution are added in step 1) solution, then 37 DEG C of incubations 30 minutes, the bromo-4-of described 5-chloro-3-indoylphosphate aqueous solution initial concentration was in the solution 1 × 10 -3-1 × 10 -6mol/ L, described Nile blue A molecular water solution initial concentration is in the solution 1 × 10 -4-1 × 10 -9mol/L;
3) surface-enhanced Raman scattering activity material is added step 2) in the solution that obtains, after 5 minutes, measure SERS signal.
3. the method for a kind of hypersensitive alkaline phosphatase detection according to claim 2, is characterized in that, described MgCl 2concentration in Tris-HCl buffer solution is 0.1-50 mmol/ L, the described NaCl concentration in Tris-HCl buffer solution is 10-1000 mmol/L.
4. the method for a kind of hypersensitive alkaline phosphatase detection according to claim 2, it is characterized in that, the concentration of described Tris-HCl buffer solution is 1-1000 mmol/L.
5. the method for a kind of hypersensitive alkaline phosphatase detection according to claim 2, it is characterized in that, described surface-enhanced Raman scattering activity material is gold nanoparticle suspension, one in solid phase substrate is formed for the nanoparticle suspension of the nanoparticle suspension of silver nanoparticle suspension, gold and silver compound, gold and silver and inorganic or organic material compound or be integrated in nano material.
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