CN108872209A - Alkaline phosphatase assay method based on nanogold cluster electrogenerated chemiluminescence probe - Google Patents
Alkaline phosphatase assay method based on nanogold cluster electrogenerated chemiluminescence probe Download PDFInfo
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Abstract
The present invention discloses a kind of alkaline phosphatase assay method based on nanogold cluster electrogenerated chemiluminescence probe.The alkaline phosphatase assay method of the nanogold cluster electrogenerated chemiluminescence probe is to prepare high quantum production rate nanogold cluster electrogenerated chemiluminescence probe using reduction method, using nano material of manganese dioxide as electrogenerated chemiluminescence quencher, the redox reaction of the ascorbic acid and manganese dioxide that are generated using alkaline phosphatase selective hydrolysis 2- phosphoric acid-L-AA trisodium salt restores electrochemiluminescence signal, realizes the measurement to content of alkaline phosphatase.1.0 × 10‑4~2.0×102 The logarithm of alkaline phosphatase concentration is worth in a linear relationship with opposite electrogenerated chemiluminescence Strength Changes in the concentration range of U/L, and detection is limited to 1.5 × 10‑5 U/L.It with easy to operate, at low cost, high sensitivity, high repeatability and other advantages, can be used for the measurement of actual sample alkaline phosphatase, there is preferable potential applicability in clinical practice.
Description
Technical field
The present invention relates to a kind of alkaline phosphatase assay methods of nanogold cluster electrogenerated chemiluminescence probe, belong to analysisization
And field of nanometer technology.
Background technique
Alkaline phosphatase（Alkaline phosphates）It is a kind of plasma membrane combination glycoprotein, it being capable of catalytic nucleic acid, egg
Phosphate group therein is sloughed in the hydrolysis of the molecules such as white matter, phosphatide, in bioprocess such as gene transfer, cell metabolism, nervous systems
In play a key role.Alkaline phosphatase is widely distributed in nature（Other than the higher plant of part）, and be widely present
In a variety of organs of human body, especially in liver and bone, content is higher, and content is relatively fewer in placenta, kidney.
The study found that the content height of alkaline phosphatase and the occurrence and development of a variety of diseases have close relationship, such as in osteomalacia, liver
In the serum of the Diseases such as dysfunction, obstructive jaundice, alkaline phosphatase is in high expression status.Therefore, in clinical detection
In, alkaline phosphatase is by the biomarker as many diseases.As the key organism marker for diagnosing a variety of diseases, alkalinity
Phosphatase is one of most common measurement enzyme in clinical practice.Currently, the method such as colorimetric for detection of alkaline phosphatase detection
There is such as sensitivity and accuracy in method, fluorescence analysis, chemoluminescence method, Surface enhanced Raman spectroscopy method, electrochemical method etc.
Low, the deficiencies of operating process is cumbersome, costly, detection time is long, greatly limit its application in clinical diagnosis.Cause
This, establishes simple, quick, highly sensitive content of alkaline phosphatase new detecting method with important practical significance.
Electrogenerated chemiluminescence（Electrochemiluminescence, abbreviation ECL）Bioanalysis is to develop in recent years
A kind of novel analysis method come, is that chemiluminescence, electrochemistry, bioanalysis, microelectric technique and sensing technology combine
Newest product.The analysis method has easy to operate, quick, the good, range of linearity of selectivity wide and easy to control etc. many excellent
Performance characteristics, and ECL technology be not necessarily to excitation light source, background signal bottom, not only it is possible to prevente effectively from conventional fluorescent analysis in
Photobleaching and background interference problem, and it is more sensitiveer than conventional fluorescent analytic approach, be widely used in clinical diagnosis, medicine
The measurement of object, immune, food quality and other materials.In recent years, the electrogenerated chemiluminescence point based on quantum dot or nanocluster
Analysis method, it is wide etc. excellent because having that background signal is low, high sensitivity, controllability are good, reagent can be recycled and analyze test scope
Point is widely used in fields such as biology, medicine and environment.Nanogold cluster has as novel nano illuminator
Nontoxic, good water solubility, large specific surface area, preparation condition are mild, surface is easily modified, and have the characteristics that special photoelectric property, oneself
It is widely applied in fields such as biological medicine, clinical analysis, sensing detection and catalysis.This seminar is regulated and controled successfully by nanoscale
Realize the preparation of high quantum production rate nanogold cluster electrogenerated chemiluminescence probe, and it is efficient to establish a series of new on this basis
Electrogenerated chemiluminescence life analysis platform.
The present invention is using nanogold cluster as electrogenerated chemiluminescence probe, using manganese dioxide nano-plates as quencher, based on alkalinity
The redox reaction of ascorbic acid and manganese dioxide that phosphatase selective hydrolysis 2- phosphoric acid-L-AA trisodium salt generates
Restore electrochemiluminescence signal, establishes a kind of highly sensitive, high selection, quick alkaline phosphatase assay new method.
Summary of the invention
The purpose of the present invention is to provide a kind of using nanogold cluster as the alkaline phosphatase assay of electrogenerated chemiluminescence probe
Method.
To achieve the goals above, the present invention uses following technical scheme：One kind being based on nanogold cluster electrogenerated chemiluminescence
The alkaline phosphatase assay method of probe, it is characterized in that：First in electrode face finish nanogold cluster, prepared by reduction method high
Quantum yield nanogold cluster electrogenerated chemiluminescence probe, and further in electrode face finish nano material of manganese dioxide, by it
As electrogenerated chemiluminescence quencher；Alkaline phosphatase is added in 2- phosphoric acid-L-AA trisodium-salt solution, alkaline phosphatase
Enzyme selectivity hydrolyzes 2- phosphoric acid-L-AA trisodium salt and generates ascorbic acid, and then modified electrode is immersed above-mentioned reaction solution
In, redox reaction occurs for the ascorbic acid and manganese dioxide of generation；Acquire the electrochemiluminescence signal of modified electrode, root
The measurement of alkaline phosphatase is realized according to the change of electrochemiluminescence signal.
The reduction method prepares high quantum production rate nanogold cluster electrogenerated chemiluminescence probe using following electrochemical reductions
Method or chemical reduction method preparation：
（1）Electrochemical reducing：Using three-electrode system, using nanogold cluster modified glassy carbon electrode as working electrode, platinum electrode
For to electrode, Ag/AgCl is reference electrode, 0.1 mol/L, pH 7.4 of above-mentioned electrode insertion in phosphate buffer solution, is applied
Add negative potential voltage within the scope of -1.4 V of V ~ -2, carries out constant potential reduction treatment 5 min ~ 1 h, obtain electrochemical luminescence nanometer
Golden aggregate probe；
（2）Chemical reduction method：Nanogold cluster modified glassy carbon electrode is immersed in 0.1 ~ 1 mol/L sodium borohydride solution reaction 5
The min of min ~ 30 obtains the nanogold aggregate probe modified electrode of chemical reduction method preparation.
The nanogold cluster is functional modification nanogold cluster, using N- acetylation-L-cysteine-nanogold cluster or ox
Seralbumin-nanogold cluster.
The method in electrode face finish nano material of manganese dioxide is electrochemical deposition method：Using three electrode bodies
System, using reduction method processing nanogold cluster modified electrode as working electrode, platinum electrode is to electrode, and Ag/AgCl is reference electrode,
Using I-t method, 1:The KMnO of 1 V/V4-H2SO4In solution, electrochemical deposition manganese dioxide, current potential is -0.1 V ~ -0.5
V, sedimentation time are the s of 100 s ~ 600, and cleaning is dried with nitrogen.
The electrochemiluminescence signal method of the acquisition modified electrode is as follows：It is tested using three-electrode system, with
Modified electrode is working electrode, and platinum electrode is to electrode, and Ag/AgCl is reference electrode, and buffer solution is that phosphate-buffered is molten
Liquid or Tris-HCl buffer solution, electrolyte used are KCl or KNO3；The insertion of above-mentioned electrode is anti-altogether containing over cure acid ion
It answers in the buffer solution of agent, electrochemical luminescence test is carried out using step pulse method, initial potential is 0 V, burst length 10
S, termination current potential are -2 V, and the burst length is 1 s, and photomultiplier tube high pressure is set as 600 ~ 800 V, detect electrogenerated chemiluminescence
Radiation signal.
The pH that redox reaction occurs for the ascorbic acid and manganese dioxide of the generation is 8.0, the reaction time 4
min。
Acquire the phosphate buffer solution of the electrochemiluminescence signal of modified electrode or the pH of Tris-HCl buffer solution
Value is 7.4, and persulfuric acid ion concentration is 0.1 mol/L.
The logarithm of electrogenerated chemiluminescence Strength Changes value and ascorbic acid concentrations is 1.0 × 10-4~2.0×102 U/L's
It is in good linear relationship in range, detection is limited to 1.5 × 10-5 U/L。
Specifically, to achieve the goals above, the present invention is using technical solution in detail below：The modification of nanogold cluster is existed
On glass-carbon electrode, high quantum production rate nanogold cluster electrogenerated chemiluminescence probe is prepared by reduction method, and then in electrode surface electricity
Deposit nano material of manganese dioxide；Alkaline phosphatase is added in 2- phosphoric acid-L-AA trisodium-salt solution, alkaline phosphatase
Selective hydrolysis 2- phosphoric acid-L-AA trisodium salt generates ascorbic acid, and manganese dioxide/nanogold cluster is further modified electricity
Pole is immersed in above-mentioned reaction solution；The electrochemiluminescence signal for acquiring modified electrode, utilizes the ascorbic acid generated in reaction solution
Redox reaction occurs with the manganese dioxide of electrode surface and restores electrochemiluminescence signal, according to electrochemiluminescence signal
Variation realize alkaline phosphatase measurement.
Above-mentioned nanogold aggregate probe is prepared by following methods：（1）Electrochemical reducing：It is gone back using three-electrode system
Original, using nanogold cluster modified glassy carbon electrode as working electrode, platinum electrode is to electrode, and Ag/AgCl is reference electrode, will be above-mentioned
Electrode is inserted into buffer solution, applies different negative potential voltages, is carried out constant potential reduction treatment, is obtained electrochemical luminescence nanogold
Aggregate probe；（2）Chemical reduction method：Nanogold cluster modified glassy carbon electrode is immersed in certain density sodium borohydride solution reaction one
It fixes time, obtains the nanogold aggregate probe modified electrode of chemical reduction method preparation.
The nanogold clustered materials are functional modification nanogold cluster, such as：N- acetylation-L-cysteine-nanogold
Cluster, bovine serum albumin(BSA)-nanogold cluster etc..
The method in electrode surface electro-deposition nano material of manganese dioxide is：Using three-electrode system, with reduction
It is working electrode that method, which handles nanogold cluster modified electrode, and platinum electrode is to electrode, and Ag/AgCl is reference electrode, using I-T
Method, in KMnO4-H2SO4（1:1 V/V）In solution, nano material of manganese dioxide modified electrode is made in potentiostatic electrodeposition.
The electrochemiluminescence signal is acquired by following methods：It is tested using three-electrode system, to modify glass
Carbon electrode is working electrode, and platinum electrode is to electrode, and Ag/AgCl is reference electrode, and the insertion of above-mentioned electrode is contained coreaction
In the buffer solution of agent.Using step pulse method, photomultiplier tube high pressure is set as the V of 600 V ~ 800, detects working electrode
The electrochemiluminescence signal that surface generates.
It is of the present invention it is a kind of based on nanogold cluster be electrogenerated chemiluminescence probe Methods For Quantitative Estimation of Ascorbic Acid, including
Following steps：1）By glass-carbon electrode Al2O3Powder successively polishes, polishes, until smooth mirror surface, then it is sequentially placed into HNO3Solution, nothing
It is cleaned by ultrasonic in water-ethanol and deionized water, N2Drying；2）Nanogold aggregate probe solution is added dropwise in the glass-carbon electrode handled well
Surface, drying at room temperature prepare high quantum production rate nanogold cluster electricity to get nanogold aggregate probe modified glassy carbon electrode, by reduction method
Chemiluminescence probe is caused, and nano material of manganese dioxide/nanogold cluster modified electrode is further made；3）Take the alkali of various concentration
Acid phosphatase solution is added in 2- phosphoric acid-L-AA trisodium-salt solution, is uniformly mixed, then by manganese dioxide/nanometer
Golden aggregate probe modified glassy carbon electrode, which is put into above-mentioned solution, to be impregnated.It is rinsed well after taking-up with distilled water, N2It is spare after drying；4）
It is tested using three-electrode system, using manganese dioxide after above-mentioned reaction/nanogold aggregate probe modified glassy carbon electrode as work electricity
Pole, platinum electrode are to electrode, and Ag/AgCl is reference electrode, and above-mentioned electrode is inserted into the buffer solution containing coreagent,
Detect its electrochemiluminescence signal value；Using step pulse method, photomultiplier tube high pressure is set as the V of 600 V ~ 800, inspection
Survey the electrochemiluminescence signal that working electrode surface generates；4）It is dense to alkaline phosphatase with electrochemiluminescence signal changing value
The logarithm of degree maps to obtain standard curve, is 1.0 × 10 in alkaline phosphatase concentration-4~2.0×102 Resist in the range of U/L
The logarithm and electrogenerated chemiluminescence intensity value of bad hematic acid concentration are in good linear relationship, and detection is limited to 1.5 × 10-5 U/L。
Above-mentioned coreagent is over cure acid ion, and concentration range is 0.01 ~ 1 mol/L, preferably 0.1 mol/L.
The buffer solution is phosphate buffer or Tris-HCl buffer solution, the added electrolyte in buffer solution
For KCl or KNO3, concentration is 0.01 ~ 1 mol/L.
The specific technical solution that the present invention uses is as follows：
（One）The preparation of N-acetyl-L-cysteine-nanogold cluster
N-acetyl-L-cysteine-nanogold cluster synthesis step is as follows：By concentration be 0.1 ~ 0.8 mol/L sodium hydroxide with
It is molten that concentration is that 0.01 ~ 0.1 g/L chlorauric acid solution is added to the N-acetyl-L-cysteine that concentration is 0.02 ~ 0.18 mol/L
In liquid, mixing is placed on 20 ~ 70 DEG C of water bath with thermostatic control isothermal reactions 0 ~ 3.5 hour.To the processing of dialysis purification after reaction, obtain
To N-acetyl-L-cysteine-nanogold cluster aqueous solution, N-acetyl-L-cysteine-nanogold cluster can be obtained after freeze-drying
Material powder.
（Two）The preparation of nanogold cluster modified electrode
By glass-carbon electrode with 1.0 μm, 0.3 μm and 0.05 μm of Al2O3Powder successively polishes, polishes, until smooth mirror surface, then
It is sequentially placed into HNO3It is cleaned by ultrasonic 3 minutes in solution, dehydrated alcohol and deionized water, N2Drying.Take 5 μ L nanogold clusters water-soluble
Drop is added in the glassy carbon electrode surface handled well, and drying at room temperature is to get nanogold cluster modified glassy carbon electrode.
（Three）The preparation of nanogold aggregate probe
（1）Electrochemical reducing：It is restored using three-electrode system, using nanogold cluster modified glassy carbon electrode as working electrode,
Platinum electrode is to electrode, and Ag/AgCl is reference electrode, and above-mentioned electrode is inserted into buffer solution, applies negative potential voltage（-
Within the scope of 1.4 V of V ~ -2）, constant potential reduction treatment 5 min ~ 1 h is carried out, electrochemical luminescence nanogold aggregate probe is obtained.
（2）Chemical reduction method：Nanogold cluster modified glassy carbon electrode is immersed in the reaction of 0.1 ~ 1 mol/L sodium borohydride solution
5~30 min（It is preferred that 0.1 mol/L sodium borohydride solution reacts 5 min）, obtain the nanogold aggregate probe of chemical reduction method preparation
Modified electrode.
（Four）Nano material of manganese dioxide method of modifying
Nano material of manganese dioxide is modified using electrochemical deposition method, specific step is as follows：Using three-electrode system, with reduction method
Processing nanogold cluster modified electrode is working electrode, and platinum electrode is to electrode, and Ag/AgCl is reference electrode, using I-t method,
In KMnO4-H2SO4（1:1 V/V）In solution, electrochemical deposition manganese dioxide, sedimentation potential is -0.1 V of V ~ -0.5, when deposition
Between be the s of 100 s ~ 600, preferably -0.2 V, 300 s are rinsed well with distilled water later, are dried with nitrogen spare.
（Five）Electrochemiluminescence signal acquisition method
By polishing electrode, then it is sequentially placed into HNO3It is cleaned by ultrasonic in solution, dehydrated alcohol and deionized water, N2Drying.Using three
Electrode system is tested, and using modified glassy carbon electrode as working electrode, platinum electrode is to electrode, and Ag/AgCl is reference electrode,
Above-mentioned electrode is inserted into the buffer solution containing coreagent.Using step pulse method, initial potential is 0 V, burst length
For 10 s, termination current potential is -2 V, and the burst length is 1 s.Photomultiplier tube high pressure is set as the V of 600 V ~ 800, detects work
Make the electrochemiluminescence signal of electrode surface generation.
The electrode is glass-carbon electrode, screen printing electrode or ITO electrode etc..Above-mentioned coreagent be over cure acid group from
Son, concentration range are 0.01 ~ 1 mol/L, preferably 0.1 mol/L.The buffer solution is phosphate buffer or Tris-HCl
Buffer solution, added electrolyte is KCl or KNO in buffer solution3, concentration is 0.01 ~ 1 mol/L, preferably 0. 1 mol/
L。
（Six）The measurement of alkaline phosphatase
37.5 μ L, 4 ~ 10 mM 2- phosphoric acid-L-AA trisodium-salt solution is added in the alkaline phosphatase of various concentration, is mixed
It closes uniformly, adds 300 μ L Tris-HCl buffer solutions, and then manganese dioxide/nanogold cluster modified electrode immersion is above-mentioned
4 ~ 10 min are reacted in reaction solution, are rinsed well after taking-up with distilled water, N2Drying.Collecting work electrode surface generates electroluminescent
Chemiluminescence signal is mapped with electrochemiluminescence signal Ascorbic Acid concentration and draws standard curve.
Compared with prior art, beneficial effects of the present invention are：
The present invention is using high quantum production rate nanogold aggregate probe as electrogenerated chemiluminescence material, with nano-manganese dioxide for electroluminescent chemistry
Luminescence quenchers, the ascorbic acid and two generated based on alkaline phosphatase selective hydrolysis 2- phosphoric acid-L-AA trisodium salt
The redox reaction of manganese oxide restores electrochemiluminescence signal, realizes the detection to content of alkaline phosphatase.The present invention couple
The detection of alkaline phosphatase is easy to operate, at low cost, high sensitivity, stability is good, accuracy is good, the range of linearity is wide（1.0×
10-9~1.0×10-2 mol/L）, and it is low to detect limit（2.56×10-10mol/L）, thus there is good market value.
Detailed description of the invention
Fig. 1 is electrogenerated chemiluminescence-time plot of nanogold aggregate probe modified glassy carbon electrode.
Fig. 2 is manganese dioxide/nanogold aggregate probe modified glassy carbon electrode electrogenerated chemiluminescence-time plot.
Fig. 3 is that manganese dioxide/nanogold aggregate probe modified electrode immerses alkaline phosphatase and 2- phosphoric acid-L-AA three
Electrogenerated chemiluminescence-time plot after sodium salt reaction solution.
Fig. 4 is 2- phosphoric acid-L-AA trisodium salinity optimization figure.
Linear relationship chart of the Fig. 5 between electrogenerated chemiluminescence Strength Changes and alkaline phosphatase concentration logarithm.
Specific embodiment
The present invention is further elaborated in the following with reference to the drawings and specific embodiments, and the present invention is not limited thereto.
Embodiment 1
It is 0.5 mol/L that 0.6 mL concentration is added into the N-acetyl-L-cysteine solution that 4 mL concentration are 0.08 mol/L
Sodium hydroxide and 0.4 mL concentration be 20 mg/mL chlorauric acid solutions, mixing is placed in 37 DEG C of thermostatic water baths and is incubated for 3 h.
Reaction solution after reaction carries out dialysis purification processing with the bag filter that retention molecule is 3500, obtains half Guang of N- acetyl-L-
Propylhomoserin-nanogold cluster aqueous solution obtains N-acetyl-L-cysteine-nanogold cluster powder after freeze-drying.
Embodiment 2
By the glass-carbon electrode of diameter 3mm with 1.0 μm, 0.3 μm and 0.05 μm of Al2O3Powder successively polishes, polishing, until light
Sliding mirror surface, then it is sequentially placed into HNO3Solution, dehydrated alcohol are cleaned by ultrasonic 3 minutes in deionized water, N2Drying.Take 5 μ L embodiments
N-acetyl-L-cysteine-nanogold cluster solution of 1 preparation is added dropwise in the glassy carbon electrode surface handled well, and drying at room temperature obtains
N-acetyl-L-cysteine-nanogold cluster modified glassy carbon electrode.By N-acetyl-L-cysteine-nanogold cluster modification glass carbon electricity
Pole, which is immersed in 0.1 mol/L sodium borohydride solution, reacts 5 min, obtains nanogold aggregate probe modified electrode.By above-mentioned nanometer
Golden aggregate probe modified electrode is working electrode, and platinum filament is to electrode, and Ag/AgCl electrode is reference electrode, and insertion contains 0.1
In 0.1 mol/L pH, 7.4 phosphate buffer solution of mol/L potassium peroxydisulfate and 0.1 mol/L KCl.Using step pulse
Method, initial potential are 0 V, and the burst length is 10 s, and termination current potential is -2 V, and the burst length is 1 s.Photomultiplier tube high pressure is set
750 V are set to, the electrochemiluminescence signal that detection working electrode surface generates obtains electrochemical luminescence signals（See Fig. 1）.
Embodiment 3
By the glass-carbon electrode of 3 mm of diameter with 1.0 μm, 0.3 μm and 0.05 μm of Al2O3Powder successively polishes, polishing, until light
Sliding mirror surface, then it is sequentially placed into HNO3Solution, dehydrated alcohol are cleaned by ultrasonic 3 minutes in deionized water, N2Drying.Take 5 μ L embodiments
The nanogold cluster solution of the N-acetyl-L-cysteine protection of 1 preparation is added dropwise in the glassy carbon electrode surface handled well, and room temperature is dry
It is dry, and further the electrode is immersed in 0.1 mol/L sodium borohydride solution and is reacted 5 minutes, it obtains nanogold aggregate probe and repairs
Adorn glass-carbon electrode.Using three-electrode system, using nanogold aggregate probe modified glassy carbon electrode as working electrode, platinum electrode is to electricity
Pole, Ag/AgCl are reference electrode, using chronoamperometry, in KMnO4-H2SO4（1:1 V/V）In solution, electro-deposition titanium dioxide
Manganese, sedimentation potential are -0.2 V, and sedimentation time is 300 s, and obtained electrode is that manganese dioxide/nanogold cluster modifies glass carbon electricity
Pole is rinsed well with distilled water later, N2Gas gently dries up spare.The insertion of above-mentioned electrode is contained into 0.1 mol/L potassium peroxydisulfate
In 0.1 mol/L pH, 7.4 phosphate buffer solution of 0.1 mol/L KCl.Using step pulse method, initial potential 0
V, burst length are 10 s, and termination current potential is -2 V, and the burst length is 1 s.Photomultiplier tube high pressure is set as 750 V, detection
The electrochemiluminescence signal that working electrode surface generates, obtains electrochemical luminescence signals（See Fig. 2）.
Embodiment 4
37.5 μ L, 4 mM 2- phosphoric acid-L-AA trisodium-salt solution is added in 400 U/L alkaline phosphatases（2-
Phospho-L-ascorbic acid trisodium salt is purchased from Sigma-Aldrich company）, it is uniformly mixed, then plus
Enter 300 μ L Tris-HCl buffer solutions, and then manganese dioxide/nanogold cluster modified electrode is immersed in above-mentioned reaction solution and is reacted
4 min.Using above-mentioned manganese dioxide/nanogold aggregate probe modified electrode as working electrode, platinum filament is to electrode, Ag/AgCl electrode
For reference electrode, insertion contains 0.1 mol/L potassium peroxydisulfate and 0.1 8.0 phosphate of mol/L, pH of 0.1 mol/L KCl are slow
It rushes in solution.Using step pulse method, initial potential is 0 V, and the burst length is 10 s, and termination current potential is -2 V, burst length
For 1 s.Photomultiplier tube high pressure is set as 750 V, after reacting 4 min, rinsed well after taking-up with distilled water, N2Drying.Inspection
The electrochemiluminescence signal that working electrode surface generates is surveyed, obtained electrochemiluminescence signal ratio is not inserted into alkaline phosphatase
It is significantly increased with the electroluminescent chemical signal in 2- phosphoric acid-L-AA trisodium-salt solution reaction solution（See Fig. 3）.
Embodiment 5
37.5 μ L various concentration 2- phosphoric acid-L-AA trisodium-salt solution, mixing is added in 400 U/L alkaline phosphatases
Uniformly, 300 μ L Tris-HCl buffer solutions are added, and then manganese dioxide prepared by embodiment 3/nanogold cluster is modified
Electrode immerses in above-mentioned reaction solution and reacts 4 min, is rinsed well after taking-up with distilled water, N2Drying.With the dioxy of above-mentioned drying
Change manganese/nanogold aggregate probe modified electrode is working electrode, and platinum filament is to electrode, and Ag/AgCl electrode is reference electrode, and insertion contains
0.1 mol/L, pH 8.0 of 0.1 mol/L potassium peroxydisulfate and 0.1 mol/L KCl are in phosphate buffer solution.Using step
Impulse method, initial potential are 0 V, and the burst length is 10 s, and termination current potential is -2 V, and the burst length is 1 s.Photomultiplier tube is high
Pressure is set as 750 V, and after reacting 4 min, detecting 2- phosphoric acid-L-AA trisodium-salt solution concentration respectively is 0,1 mM, and 2
The electrochemiluminescence signal that working electrode surface generates when mM, 3 mM, 4 mM, 6 mM, 8 mM and 10 mM, obtains best 2-
Phosphoric acid-L-AA trisodium-salt solution concentration is 4 mM（See Fig. 4）.
Embodiment 6
The alkaline phosphatase of various concentration is added to 2- phosphoric acid-L-AA trisodium-salt solution of 37.5 μ L, 4 mM, mixing
Uniformly, 300 μ L Tris-HCl buffer solutions are added, and then manganese dioxide prepared by embodiment 3/nanogold cluster is modified
Electrode immerses in above-mentioned reaction solution and reacts 4 min, is rinsed well after taking-up with distilled water, N2Drying.With the dioxy of above-mentioned drying
Change manganese/nanogold aggregate probe modified electrode is working electrode, and platinum filament is to electrode, and Ag/AgCl electrode is reference electrode, and insertion contains
0.1 mol/L, pH 8.0 of 0.1 mol/L potassium peroxydisulfate and 0.1 mol/L KCl are in phosphate buffer solution.Using step
Impulse method, initial potential are 0 V, and the burst length is 10 s, and termination current potential is -2 V, and the burst length is 1 s.Photomultiplier tube is high
Pressure is set as 750 V, and after reacting 4 min, working electrode surface is generated under the conditions of detecting the alkaline phosphatase of various concentration respectively
Electrochemiluminescence signal, alkaline phosphatase concentration be 1.0 × 10-4~2.0×102 Glutathione is dense in the range of U/L
The logarithm and electrogenerated chemiluminescence intensity value of degree are in good linear relationship, and detection is limited to 1.5 × 10-5 U/L（See Fig. 5）.
Embodiment 7
Fresh 2 μ L of blood serum sample is taken, 20 mM Tris-HCl buffers are added and are diluted to 20 mL, by serum sample after dilution
Product are added to containing two in 2- phosphoric acid-L-AA trisodium salt Tris-HCl buffer, then prepared embodiment 3
Manganese oxide/nanogold cluster modified electrode, which is put into above-mentioned solution, impregnates 4 min of reaction, is rinsed well after taking-up with distilled water, N2
Drying.Using the manganese dioxide of above-mentioned drying/nanogold aggregate probe modified electrode as working electrode, platinum filament is to electrode, Ag/AgCl
Electrode is reference electrode, 0.1 mol/L, pH 8.0 phosphoric acid of the insertion containing 0.1 mol/L potassium peroxydisulfate and 0.1 mol/L KCl
In salt buffer solution.Using step pulse method, initial potential is 0 V, and the burst length is 10 s, and termination current potential is -2 V, pulse
Time is 1 s.Photomultiplier tube high pressure is set as 750 V, after reacting 4 min, detects respectively work in different sample solutions respectively
The electrochemiluminescence signal that electrode surface generates, is quantified by standard curve, obtains containing for sample alkaline phosphatase
Amount, and being detected using international alkaline phosphatase examination criteria method PNPP azo method, then with the detection of this experimental method
As a result statistics comparison is carried out（Table 1）.There was no significant difference with PNPP method for this method, and therefore, this experimental method is suitable for practical
The detection of sample.
Table 1 is the testing result of actual sample alkaline phosphatase.
The foregoing is merely exemplary embodiments of the invention, are not intended to limit the invention, all in essence of the invention
Made any modification within mind and principle, equivalent replacement and improvement etc., should all be included in the protection scope of the present invention.
Claims (8)
1. a kind of alkaline phosphatase assay method based on nanogold cluster electrogenerated chemiluminescence probe, it is characterized in that：First in electricity
Pole surface modified nano gold cluster prepares high quantum production rate nanogold cluster electrogenerated chemiluminescence probe by reduction method, and further
In electrode face finish nano material of manganese dioxide, as electrogenerated chemiluminescence quencher；2- is added in alkaline phosphatase
In phosphoric acid-L-AA trisodium-salt solution, alkaline phosphatase selective hydrolysis 2- phosphoric acid-L-AA trisodium salt generates anti-
Bad hematic acid, and then modified electrode is immersed in above-mentioned reaction solution, it is anti-that redox occurs for ascorbic acid and the manganese dioxide of generation
It answers；The electrochemiluminescence signal for acquiring modified electrode realizes alkaline phosphatase according to the change of electrochemiluminescence signal
Measurement.
2. a kind of alkaline phosphatase assay side based on nanogold cluster electrogenerated chemiluminescence probe according to claim 1
Method, it is characterized in that the reduction method, which prepares high quantum production rate nanogold cluster electrogenerated chemiluminescence probe, uses following electrochemistry also
Former method or chemical reduction method preparation：
（1）Electrochemical reducing：Using three-electrode system, using nanogold cluster modified glassy carbon electrode as working electrode, platinum electrode
For to electrode, Ag/AgCl is reference electrode, 0.1 mol/L, pH 7.4 of above-mentioned electrode insertion in phosphate buffer solution, is applied
Add negative potential voltage within the scope of -1.4 V of V ~ -2, carries out constant potential reduction treatment 5 min ~ 1 h, obtain electrochemical luminescence nanometer
Golden aggregate probe；
（2）Chemical reduction method：Nanogold cluster modified glassy carbon electrode is immersed in 0.1 ~ 1 mol/L sodium borohydride solution reaction 5
The min of min ~ 30 obtains the nanogold aggregate probe modified electrode of chemical reduction method preparation.
3. a kind of according to claim 1, alkaline phosphatase assay side based on nanogold cluster electrogenerated chemiluminescence probe described in 2
Method, it is characterized in that the nanogold cluster is functional modification nanogold cluster, using N- acetylation-L-cysteine-nanogold cluster
Or bovine serum albumin(BSA)-nanogold cluster.
4. a kind of alkaline phosphatase assay side based on nanogold cluster electrogenerated chemiluminescence probe according to claim 1
Method, it is characterized in that the method in electrode face finish nano material of manganese dioxide is electrochemical deposition method：Using three electrodes
System, using reduction method processing nanogold cluster modified electrode as working electrode, platinum electrode is to electrode, and Ag/AgCl is reference electricity
Pole, using I-t method, 1:The KMnO of 1 V/V4-H2SO4In solution, electrochemical deposition manganese dioxide, current potential be -0.1 V ~ -
0.5 V, sedimentation time are the s of 100 s ~ 600, and cleaning is dried with nitrogen.
5. a kind of alkaline phosphatase assay side based on nanogold cluster electrogenerated chemiluminescence probe according to claim 1
Method, it is characterized in that the electrochemiluminescence signal method of the acquisition modified electrode is as follows：It is tested using three-electrode system,
Using modified electrode as working electrode, platinum electrode is to electrode, and Ag/AgCl is reference electrode, and buffer solution is phosphate-buffered
Solution or Tris-HCl buffer solution, electrolyte used are KCl or KNO3；The insertion of above-mentioned electrode is total to containing over cure acid ion
In the buffer solution of reactant, electrochemical luminescence test is carried out using step pulse method, initial potential is 0 V, and the burst length is
10 s, termination current potential are -2 V, and the burst length is 1 s, and photomultiplier tube high pressure is set as 600 ~ 800 V, detects electroluminescent chemistry
Luminous radiation signal.
6. a kind of alkaline phosphatase assay side based on nanogold cluster electrogenerated chemiluminescence probe according to claim 1
Method, it is characterized in that the pH that redox reaction occurs for the ascorbic acid of the generation and manganese dioxide is 8.0, the reaction time 4
min。
7. a kind of alkaline phosphatase assay side based on nanogold cluster electrogenerated chemiluminescence probe according to claim 5
Method, it is characterized in that the phosphate buffer solution or Tris-HCl buffer solution of the electrochemiluminescence signal of acquisition modified electrode
PH value is 7.4, and persulfuric acid ion concentration is 0.1 mol/L.
8. a kind of alkaline phosphatase assay side based on nanogold cluster electrogenerated chemiluminescence probe according to claim 1
Method, it is characterized in that the logarithm of electrogenerated chemiluminescence Strength Changes value and ascorbic acid concentrations is 1.0 × 10-4~2.0×102 U/
It is in good linear relationship in the range of L, detection is limited to 1.5 × 10-5 U/L。
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