CN108827948A - Acid phosphatase electrogenerated chemiluminescence measuring method based on gold nano cluster probe - Google Patents
Acid phosphatase electrogenerated chemiluminescence measuring method based on gold nano cluster probe Download PDFInfo
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- G—PHYSICS
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- 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
- G01N21/76—Chemiluminescence; Bioluminescence
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Abstract
The present invention discloses a kind of acid phosphatase electrogenerated chemiluminescence measuring method based on gold nano cluster probe.The present invention prepares high quantum production rate gold nano 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 acid phosphatase selective hydrolysis 2- phosphoric acid-L-AA trisodium salt restores electrochemiluminescence signal, realizes the measurement to acid phosphatase content.2.0 × 10‑4~2.0×102 The logarithm of the concentration range Acid Phosphatase concentration of U/L and opposite electrogenerated chemiluminescence Strength Changes value are in a linear relationship, and detection is limited to 6.5 × 10‑5 U/L.Operation of the present invention is simple, at low cost, high sensitivity, favorable reproducibility, can be used for the measurement of acid phosphatase in actual sample, has preferable potential applicability in clinical practice.
Description
Technical field
The present invention relates to a kind of acid phosphatase electrogenerated chemiluminescence measuring methods of gold nano cluster probe, belong to analysis
Chemistry and field of nanometer technology.
Background technique
Acid phosphatase（Acid phosphatase, ACP）It is one of mammalian body fluid and tissue hydrolase, it
Can effectively hydrolyse phosphate esters in acid condition, phosphate group therein is sloughed, in gene transfer, cell metabolism, nerve
It is played a key role in the bioprocess such as system.Acid phosphatase in normal human serum be mainly derived from the bone of human body, liver,
In the tissue such as kidney, spleen, pancreas.Clinically, abnormal acid phosphatase enzyme concentration implys that the pathologic process of some diseases, for example,
Prostate cancer, hemolytic anemia, hyperparathyroidism and Huppert's disease etc..Acid phosphatase has been considered as
The biomarker of disease has great importance in terms of medical diagnosis on disease, postoperative evaluation and drug screening.Currently, for examining
Survey the method such as high performance liquid chromatography of acid phosphatase detection, colorimetric method, fluorescence analysis, chemoluminescence method, electrochemistry side
Method etc. exists such as the deficiencies of sensitivity and accuracy are low, operating process is cumbersome, costly, detection time is long, greatly limits
Its application in clinical diagnosis.Therefore, establishing simple, quick, highly sensitive acid phosphatase content detection new method has
Important practical significance.
Electrogenerated chemiluminescence（Electrochemiluminescence, abbreviation ECL）Refer to electroactive material in electrode table
The phenomenon that face or electrode nearby occur redox reaction, form excitation state, then return to ground state by excitation state and generate light radiation.
In a sense, ECL is the desirable combination of electrochemistry and spectrographic technique.ECL not only shines with traditional chemical（CL）'s
Sensitivity and wide dynamic response range, but also several advantages of electrochemical method are shown, including equipment is simple, controllability
Strong and favorable reproducibility.So far, ECL has become a kind of powerful analytical technology, is widely used in life analysis, environment
The fields such as monitoring, clinical detection.
With the continuous development of science and technology, it is nano luminous body that nano luminous body is gradually developed by molecule emitter, these
Material includes semiconductor-quantum-point, carbon nanomaterial, Transition-metal dichalcogenide, metal cluster etc..Gold nano cluster（Au
Nanocluster, AuNCs）To have recently been developed a kind of novel ECL illuminator, because its unique property show it is huge
Application potential, arouse widespread concern.AuNCs diameter close to electronics Fermi's wavelength, therefore may occur in which size according to
Bad HOMO-LUMO energy level transition and fluorescence phenomenon.Furthermore the advantages that hypotoxicity, catalytic performance and good biocompatibility, make it
It has a wide range of applications in ECL analysis.Although having been achieved with phase currently based on AuNCs fluorescence Quality Research and application
When achievement abundant, but the research in terms of the ECL based on AuNCs is also relatively fewer, it may be possible to small since there is also ECL intensity,
The problems such as luminous mechanism not yet illustrates.Therefore, constructing a new and effective ECL sensor is particularly important.This seminar
Regulate and control successfully to realize the preparation of high quantum production rate gold nano cluster electrogenerated chemiluminescence probe by nanoscale, and basic herein
On establish the efficient electrogenerated chemiluminescence life analysis platform of a series of new.
The present invention is using gold nano cluster as electrogenerated chemiluminescence probe, using manganese dioxide nano-plates as quencher, based on acid
The redox of ascorbic acid and manganese dioxide that acid phosphatase selective hydrolysis 2- phosphoric acid-L-AA trisodium salt generates is anti-
It should restore electrochemiluminescence signal, establish a kind of highly sensitive, high selection, quick Assay of acid phosphatase content new method.
Summary of the invention
The purpose of the present invention is to provide a kind of using gold nano cluster as the survey of the acid phosphatase of electrogenerated chemiluminescence probe
Determine method.
To achieve the goals above, the present invention uses following technical scheme：A kind of acidity based on gold nano cluster probe
Phosphatase electrogenerated chemiluminescence measuring method, it is characterized in that：First in electrode face finish gold nano cluster, by restoring legal system
Standby high quantum production rate gold nano cluster electrogenerated chemiluminescence probe, and further in electrode face finish manganese dioxide nano material
Material, as electrogenerated chemiluminescence quencher；Acid phosphatase is added in 2- phosphoric acid-L-AA trisodium-salt solution,
Acid phosphatase selective hydrolysis 2- phosphoric acid-L-AA trisodium salt generates ascorbic acid, and then modified electrode is immersed
It states in reaction solution, redox reaction occurs for the ascorbic acid and manganese dioxide of generation；Acquire the electroluminescent chemistry hair of modified electrode
Optical signal realizes the measurement of acid phosphatase according to the change of electrochemiluminescence signal.
2, a kind of acid phosphatase electrogenerated chemiluminescence based on gold nano cluster probe according to claim 1 is surveyed
Method is determined, it is characterized in that the reduction method prepares high quantum production rate gold nano cluster electrogenerated chemiluminescence probe using following electricity
Chemical reduction method or chemical reduction method preparation：
（1）Electrochemical reducing：Using three-electrode system, using gold nano cluster modified glassy carbon electrode as working electrode, platinum filament electricity
Extremely to electrode, Ag/AgCl is reference electrode, and above-mentioned electrode is inserted into 0.1 mol/L pH, 7.4 phosphate buffer solution,
Apply negative potential voltage within the scope of -1.4 V of V ~ -2, carry out constant potential reduction treatment 5 min ~ 1 h, obtains electrochemical luminescence gold
Nanocluster probe；
（2）Chemical reduction method：Gold nano 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 gold nano cluster probe modification electrode of chemical reduction method preparation.
The gold nano cluster is functional modification gold nano cluster, using N- acetylation-L-cysteine-gold nano group
Cluster or glutathione-gold nano 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 gold nano 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 is -0.1 ~ -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 2.0 × 10-4~2.0×102 U/L's
In a linear relationship in range, detection is limited to 6.5 × 10-5 U/L。
Specifically, to achieve the goals above, the present invention is using technical solution in detail below：Gold nano cluster is modified
On glass-carbon electrode, high quantum production rate gold nano cluster electrogenerated chemiluminescence probe is prepared by reduction method, and then in electrode table
Face electro-deposition nano material of manganese dioxide；Acid phosphatase is added in 2- phosphoric acid-L-AA trisodium-salt solution, acid phosphorus
Sour enzyme selectivity hydrolysis 2- phosphoric acid-L-AA trisodium salt generates ascorbic acid, further by manganese dioxide/gold nano cluster
Modified electrode immerses in above-mentioned reaction solution；The electrochemiluminescence signal for acquiring modified electrode, it is anti-using what is generated in reaction solution
The manganese dioxide of bad hematic acid and electrode surface occurs redox reaction and restores electrochemiluminescence signal, is sent out according to electroluminescent chemistry
The measurement of acid phosphatase is realized in the variation of optical signal.
Above-mentioned gold nano cluster probe is prepared by following methods：（1）Electrochemical reducing：It is gone back using three-electrode system
Original, using gold nano cluster modified glassy carbon electrode as working electrode, platinum electrode is to electrode, and Ag/AgCl is reference electrode, will be upper
It states in electrode insertion buffer solution, applies different negative potential voltages, carry out constant potential reduction treatment, obtain electrochemical luminescence Jenner
Rice cluster probe；（2）Chemical reduction method：Gold nano cluster modified glassy carbon electrode is immersed in certain density sodium borohydride solution
Certain time is reacted, the gold nano cluster probe modification electrode of chemical reduction method preparation is obtained.
The gold nano cluster material is functional modification gold nano cluster, such as：N- acetylation-L-cysteine-gold
Nanocluster, bovine serum albumin(BSA)-gold nano 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 gold nano 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 gold nano cluster be electrogenerated chemiluminescence probe Methods For Quantitative Estimation of Ascorbic Acid, packet
Include following steps：1）By glass-carbon electrode Al2O3Powder successively polishes, polishes, until smooth mirror surface, then it is sequentially placed into HNO3Solution,
It is cleaned by ultrasonic in dehydrated alcohol and deionized water, N2Drying；2）Gold nano cluster probe solution is added dropwise in the glass carbon handled well
Electrode surface, drying at room temperature prepare high quantum production rate Jenner to get gold nano cluster probe modification glass-carbon electrode, by reduction method
Rice cluster electrogenerated chemiluminescence probe, and nano material of manganese dioxide/gold nano cluster modified electrode is further made；3）It takes not
It with the acid phosphatase enzyme solutions of concentration, is added in 2- phosphoric acid-L-AA trisodium-salt solution, is uniformly mixed, then by two
Manganese oxide/gold nano cluster probe modification glass-carbon electrode is put into above-mentioned solution and impregnates.It is rinsed well after taking-up with distilled water, N2
It is spare after drying；4）It is tested using three-electrode system, with manganese dioxide after above-mentioned reaction/gold nano cluster probe modification 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, its electrochemiluminescence signal value is detected；Using step pulse method, photomultiplier tube high pressure is set as
The V of 600 V ~ 800, the electrochemiluminescence signal that detection working electrode surface generates；4）Changed with electrochemiluminescence signal
Value maps to obtain standard curve to the logarithm of acid phosphatase enzyme concentration, is 2.0 × 10 in acid phosphatase enzyme concentration-4~2.0×
102 The logarithm of ascorbic acid concentrations and electrogenerated chemiluminescence intensity value are in good linear relationship, detection limit in the range of U/L
It is 6.5 × 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-gold nano cluster
N-acetyl-L-cysteine-gold nano cluster synthesis step is as follows：The sodium hydroxide for being 0.1 ~ 0.8 mol/L by concentration
It is the N-acetyl-L-cysteine that 0.01 ~ 0.1 g/L chlorauric acid solution is added to that concentration is 0.02 ~ 0.18 mol/L with concentration
In solution, mixing is placed on 20 ~ 70 DEG C of water bath with thermostatic control isothermal reactions 0 ~ 3.5 hour.It is handled to dialysis purification after reaction,
N-acetyl-L-cysteine-gold nano cluster aqueous solution is obtained, N-acetyl-L-cysteine-Jenner can be obtained after freeze-drying
Rice cluster powder.
（Two）The preparation of gold nano 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 gold nano cluster water
Solution is added dropwise in the glassy carbon electrode surface handled well, and drying at room temperature is to get gold nano cluster modified glassy carbon electrode.
（Three）The preparation of gold nano cluster probe
（1）Electrochemical reducing：It is restored using three-electrode system, using gold nano cluster 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 buffer solution, apply 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 gold nano cluster probe is obtained.
（2）Chemical reduction method：It is anti-that gold nano cluster modified glassy carbon electrode is immersed in 0.1 ~ 1 mol/L sodium borohydride solution
Answer 5 ~ 30 min（It is preferred that 0.1 mol/L sodium borohydride solution reacts 5 min）, obtain the gold nano cluster of chemical reduction method preparation
Probe modification 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 gold nano 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 -0.2 V, and sedimentation time is
The s of 100 s ~ 600, preferably 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 acid phosphatase
50 μ L, 40 ~ 100 mM 2- phosphoric acid-L-AA trisodium-salt solution is added in the acid phosphatase of various concentration, is mixed
It closes uniformly, adds 400 μ L 50mM HAc buffers（pH 5.0）After reaction 30 minutes, 0.1 M sodium hydroxide is then added
70 ~ 80 μ L, regulation system pH value to 8.0.And then manganese dioxide/gold nano cluster modified electrode is immersed anti-in above-mentioned reaction solution
4 ~ 10 min are answered, are rinsed well after taking-up with distilled water, N2Drying.The electrogenerated chemiluminescence letter that collecting work electrode surface generates
Number, it 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 gold nano cluster probe as electrogenerated chemiluminescence material, with nano-manganese dioxide for electroluminescentization
Learn luminescence quenchers, the ascorbic acid generated based on acid phosphatase selective hydrolysis 2- phosphoric acid-L-AA trisodium salt with
The redox reaction of manganese dioxide restores electrochemiluminescence signal, realizes the detection to acid phosphatase content.The present invention
, at low cost, high sensitivity easy to operate to the detection of acid phosphatase, stability are good, accuracy is good, the range of linearity is wide（2.0×
10-4~2.0×102 U/L）, and it is low to detect limit（6.5×10-5U/L）, thus there is good market value.
Detailed description of the invention
Fig. 1 is electrogenerated chemiluminescence-time plot of gold nano cluster probe modification glass-carbon electrode.
Fig. 2 is manganese dioxide/gold nano cluster probe modification glass-carbon electrode electrogenerated chemiluminescence-time plot.
Fig. 3 is that manganese dioxide/gold nano cluster probe modification electrode immerses acid phosphatase and 2- phosphoric acid-L-AA
Electrogenerated chemiluminescence-time plot after trisodium 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 acid phosphatase log concentration value.
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-gold nano cluster aqueous solution obtains N-acetyl-L-cysteine-gold nano 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-gold nano cluster solution of 1 preparation is added dropwise in the glassy carbon electrode surface handled well, drying at room temperature,
Obtain N-acetyl-L-cysteine-gold nano cluster modified glassy carbon electrode.By N-acetyl-L-cysteine-gold nano cluster modification
Glass-carbon electrode, which is immersed in 0.1 mol/L sodium borohydride solution, reacts 5 min, obtains gold nano cluster probe modification electrode.It will
Above-mentioned gold nano cluster probe modification electrode is working electrode, and platinum filament is to electrode, and Ag/AgCl electrode is reference electrode, insertion
In 0.1 mol/L pH, 7.4 phosphate buffer solution containing 0.1 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 transit
Pipe high pressure is set as 750 V, and 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 gold nano cluster solution of the N-acetyl-L-cysteine protection of 1 preparation is added dropwise in the glassy carbon electrode surface handled well, room temperature
It is dry, and further the electrode is immersed in 0.1 mol/L sodium borohydride solution and is reacted 5 minutes, obtain gold nano cluster spy
Needle modified glassy carbon electrode.Using three-electrode system, using gold nano cluster probe modification glass-carbon electrode as working electrode, platinum electrode
For to electrode, Ag/AgCl is reference electrode, using chronoamperometry, 1:1 V/V KMnO4-H2SO4In solution, electro-deposition two
Manganese oxide, sedimentation potential are -0.2 V, and sedimentation time is 300 s, and obtained electrode is manganese dioxide/gold nano cluster modification
Glass-carbon electrode 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 mistake
In 0.1 mol/L pH, 7.4 phosphate buffer solution of potassium sulfate and 0.1 mol/L KCl.Using step pulse method, initially
Current potential is 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 as 750
V, the electrochemiluminescence signal that detection working electrode surface generates, obtains electrochemical luminescence signals（See Fig. 2）.
Embodiment 4
50 μ L, 40 mM 2- phosphoric acid-L-AA trisodium salt is added in 50 μ L, 400 U/L acid phosphatase（2-
Phospho-L-ascorbic acid trisodium salt is purchased from Sigma-Aldrich company）Solution is uniformly mixed,
Add 400 μ L 50mM HAc buffers（pH 5.0）, 0.1 M sodium hydroxide, 79.3 μ L, regulation system pH value is then added
To 8.0 reaction 30 minutes.And then the manganese dioxide for preparing embodiment 3/gold nano cluster modified electrode immerses above-mentioned reaction
4 min are reacted in liquid, are rinsed well after taking-up with distilled water, N2Drying.With manganese dioxide/gold nano cluster after above-mentioned drying
Probe modification electrode is working electrode, and platinum filament is to electrode, and Ag/AgCl electrode is reference electrode, and insertion contains 0.1 mol/L over cure
0.1 mol/L, pH 8.0 of sour potassium and 0.1 mol/L KCl are in phosphate buffer solution.Using step pulse method, initial potential
For 0 V, 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 as 750 V,
It after reacting 4 min, is rinsed well after taking-up with distilled water, N2Drying.Detect the electrogenerated chemiluminescence that working electrode surface generates
Signal, obtained electrochemiluminescence signal are more anti-than acid phosphatase and 2- phosphoric acid-L-AA trisodium-salt solution are not added
The electroluminescent chemical signal in liquid is answered to significantly increase（See Fig. 3）.
Embodiment 5
50 μ L various concentration 2- phosphoric acid-L-AA trisodium-salt solution is added in 50 μ L, 400 U/L acid phosphatase, is mixed
It closes uniformly, adds 400 μ L 50mM HAc buffers（pH 5.0）After reaction 30 minutes, 0.1 M sodium hydroxide is then added
79.3 μ L, regulation system pH value to 8.0.And then the manganese dioxide for preparing embodiment 3/gold nano cluster modified electrode is soaked
Enter and react 4 min in above-mentioned reaction solution, is rinsed well after taking-up with distilled water, N2Drying.With the manganese dioxide after above-mentioned drying/
Gold nano cluster probe modification electrode is working electrode, and platinum filament is to electrode, and Ag/AgCl electrode is reference electrode, and insertion contains 0.1
0.1 mol/L, pH 8.0 of mol/L potassium peroxydisulfate and 0.1 mol/L KCl are in phosphate buffer solution.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, after reacting 4 min, detecting 2- phosphoric acid-L-AA trisodium-salt solution concentration respectively is 0,1 mM, 2 mM, 3
The electrochemiluminescence signal that working electrode surface generates when 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 acid phosphatase of 50 μ L various concentrations is added to 2- phosphoric acid-L-AA trisodium-salt solution of 50 μ L, 40 mM,
It is uniformly mixed, adds 400 μ L 50mM HAc buffers（pH 5.0）After reaction 30 minutes, 0.1 M hydroxide is then added
79.3 μ L of sodium, regulation system pH value to 8.0.And then the manganese dioxide for preparing embodiment 3/gold nano cluster modified electrode leaching
Enter and react 4 min in above-mentioned reaction solution, is rinsed well after taking-up with distilled water, N2Drying.With the manganese dioxide after above-mentioned drying/
Gold nano cluster probe modification electrode is working electrode, and platinum filament is to electrode, and Ag/AgCl electrode is reference electrode, and insertion contains 0.1
0.1 mol/L, pH 8.0 of mol/L potassium peroxydisulfate and 0.1 mol/L KCl are in phosphate buffer solution.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, after reacting 4 min, detect the electricity of working electrode surface generation under the conditions of the acid phosphatase of various concentration respectively
Chemiluminescence signal is caused, is 2.0 × 10 in acid phosphatase enzyme concentration-4~2.0×102 Glutathione concentrations in the range of U/L
Logarithm and electrogenerated chemiluminescence intensity value are in good linear relationship, and detection is limited to 6.5 × 10-5 U/L（See Fig. 5）.
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 acid phosphatase electrogenerated chemiluminescence measuring method based on gold nano cluster probe, it is characterized in that：Exist first
Electrode face finish gold nano cluster prepares high quantum production rate gold nano cluster electrogenerated chemiluminescence probe by reduction method, and
Further in electrode face finish nano material of manganese dioxide, as electrogenerated chemiluminescence quencher；By acid phosphatase
It is added in 2- phosphoric acid-L-AA trisodium-salt solution, acid phosphatase selective hydrolysis 2- phosphoric acid-L-AA trisodium salt
Ascorbic acid is generated, and then modified electrode is immersed in above-mentioned reaction solution, ascorbic acid and the manganese dioxide of generation aoxidize
Reduction reaction；The electrochemiluminescence signal for acquiring modified electrode realizes acid phosphorus according to the change of electrochemiluminescence signal
The measurement of sour enzyme.
2. a kind of acid phosphatase electrogenerated chemiluminescence measurement side based on gold nano cluster probe according to claim 1
Method, it is characterized in that the reduction method prepares high quantum production rate gold nano cluster electrogenerated chemiluminescence probe using following electrochemistry
Reduction method or chemical reduction method preparation：
（1）Electrochemical reducing：Using three-electrode system, using gold nano cluster modified glassy carbon electrode as working electrode, platinum filament electricity
Extremely to electrode, Ag/AgCl is reference electrode, and above-mentioned electrode is inserted into 0.1 mol/L pH, 7.4 phosphate buffer solution,
Apply negative potential voltage within the scope of -1.4 V of V ~ -2, carry out constant potential reduction treatment 5 min ~ 1 h, obtains electrochemical luminescence gold
Nanocluster probe；
（2）Chemical reduction method：Gold nano 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 gold nano cluster probe modification electrode of chemical reduction method preparation.
3. a kind of according to claim 1, acid phosphatase electrogenerated chemiluminescence measurement based on gold nano cluster probe described in 2
Method, it is characterized in that the gold nano cluster is functional modification gold nano cluster, using N- acetylation-L-cysteine-gold
Nanocluster or glutathione-gold nano cluster.
4. a kind of acid phosphatase electrogenerated chemiluminescence measurement side based on gold nano cluster 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 gold nano 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 be -0.1 ~ -
0.5 V, sedimentation time are the s of 100 s ~ 600, and cleaning is dried with nitrogen.
5. a kind of acid phosphatase electrogenerated chemiluminescence measurement side based on gold nano cluster 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 acid phosphatase electrogenerated chemiluminescence measurement side based on gold nano cluster 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 acid phosphatase electrogenerated chemiluminescence measurement side based on gold nano cluster 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 acid phosphatase electrogenerated chemiluminescence measurement side based on gold nano cluster probe according to claim 1
Method, it is characterized in that the logarithm of electrogenerated chemiluminescence Strength Changes value and ascorbic acid concentrations is 2.0 × 10-4~2.0×102 U/
In a linear relationship in the range of L, detection is limited to 6.5 × 10-5 U/L。
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