CN109975260A - A kind of method and its application based on nanogold fluorescence detection lysozyme - Google Patents
A kind of method and its application based on nanogold fluorescence detection lysozyme Download PDFInfo
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Abstract
The present invention provides a kind of method and its application based on nanogold fluorescence detection lysozyme, it comprises the following steps that the preparation of (1) lysozyme standard solution: lysozyme standard product is dissolved in phosphate buffer solution, the lysozyme standard solution of various concentration is made；(2) nano-Au solution the measurement of standard curve: is added into the lysozyme standard solution of various concentration, 5min is reacted at 37 DEG C, rhodamine 6G solution is added, obtain mixed solution, it measures the fluorescence spectrum of mixed solution and records fluorescence intensity, fluorescence intensity at every curve 552nm is mapped with the logarithm of corresponding lysozyme standard solution concentration, obtains standard curve；(3) measurement of sample to be tested lysozyme: measuring the fluorescence intensity of sample to be tested, and reference standard curve calculates its lysozyme content.The advantages that quantitative detection of the invention is limited to 0.1 μ g/mL, has detection limit low, and operating process is simple, and reagent dosage is few, and detection method is quick, sensitive.
Description
Technical field
The present invention relates to a kind of method and its application based on nanogold fluorescence detection lysozyme, belong to biological medicine detection
Technical field.
Background technique
Lysozyme is protein important in organism, can promote the removing of oral cavity bacterium, is a kind of usually presence
In animal vegetable tissue liquid and the intracorporal albumen of certain micro-organisms.Abnormal lysozyme content in serum and urine be considered with
Disease is related, such as leukaemia, kidney diaseases, immune system disorder and meningitis, thus lysozyme can be used as system, organ,
The structure or function of tissue etc. change or may change biochemical indicator (referring to Selby D M, Valdez R,
Schnitzer B,et al.Blood, 2003,101(7):2895-2895；Filippatos T D,Milionis H J,
Elisaf M S.European journal of haematology, 2005,75(6):449-460.)。
Method currently used for detecting lysozyme mainly has high performance liquid chromatography, enzyme linked immunosorbent assay (ELISA), hair
Cons electrophoresis method and molecular imprinting method etc. (referring to: Jing T, Xia H, Guan Q, et al.Analytica chimica
acta,2011, 692(1-2):73-79；Zhang Z,Wang H,Wang H,et al.Analyst,2018,143(23):
5849-5856.).Chinese patent document CN107064504A discloses a kind of method for detecting lysozyme from egg white, this method packet
It includes following steps: being stayed overnight with monoclonal antibody coating elisa plate, PBST is washed and the closing of skimmed milk power solution is added, then is used
PBST, which is washed and the lysozyme standard solution being serially diluted is added etc., to be incubated for, and tmb substrate solution is added, it is whole that 3mol/L sulfuric acid is added
Only react and calculate the content of lysozyme in egg white in sample to be tested.But these methods need at prolonged sample
The detecting instrument of reason, complicated experimental implementation or valuableness, is not suitable for detecting lysozyme quick, sensitive, microly.
Currently, the method based on nanogold fluorescence detection content of material, since it is with high sensitivity, quick response, behaviour
Make simply and obtains extensive concern (referring to Chen J, Zheng A F, Chen A H, et the advantages that at low cost
al.Analytica chimica acta, 2007,599(1):134-142.).Therefore, develop it is a kind of simple, economical, quickly,
Method sensitively based on nanogold fluorescence detection lysozyme, diagnosis and detection to disease have important reference value and pole
Big meaning.
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides a kind of method based on nanogold fluorescence detection lysozyme and its
Using.
Technical scheme is as follows:
A method of based on nanogold fluorescence detection lysozyme, comprise the following steps that
(1) preparation of lysozyme standard solution: lysozyme standard product are dissolved in phosphate buffer solution, are made different dense
The lysozyme standard solution of degree；
(2) it the measurement of standard curve: is added receives into the lysozyme standard solution of various concentration made from step (1) respectively
Rice gold solution, reacts 5min at 37 DEG C, and rhodamine 6G solution is then added, obtains mixed solution, takes mixed solution respectively, passes through
Excitation light source excitation, exciting slit 2nm, transmite slit 2nm scan its fluorescence spectrum and record fluorescence intensity, by every
Fluorescence intensity at curve 552nm is mapped with the logarithm of corresponding lysozyme standard solution concentration, obtains standard curve；
(3) measurement of sample to be tested lysozyme: sample to be tested is dissolved in phosphate buffer solution, according to step (2)
Lysozyme in sample to be tested is calculated by the fluorescence intensity reference standard curve measured in the fluorescence intensity of method test system
Content.
Preferred according to the present invention, phosphate buffer solution described in step (1) includes: phosphatase 11 0mmol/L, NaCl
138 mmol/L, KCl 2.7mmol/L, the pH=7.4 of solution.
Preferred according to the present invention, the concentration of lysozyme standard Lysozyme in Solution described in step (1) is 0.1~10
μg/mL。
Preferred according to the present invention, the concentration of nano-Au solution described in step (2) is 0.35nmol/L, and described receives
The ratio between the addition volume of rice gold solution and lysozyme standard liquor capacity are 1:1.
Preferred according to the present invention, the preparation method of nano-Au solution described in step (2) is the prior art, can be pressed
State method preparation:
Beta-cyclodextrin solution, tetra chlorauric acid solution are added separately in deionized water, lye regulation system pH value is added
It is 10.5, then stirs 3h at 60 DEG C, be reduced tetra chlorauric acid sufficiently, obtains the mixed solution of stable homogeneous, it will be resulting
Mixed solution is centrifuged 20min in the case where revolving speed is 9000r/min, obtains nanogold particle, gained nanogold particle is dispersed in three times
In distilled water, nano-Au solution is obtained；The tetrachloro in beta-cyclodextrin and tetra chlorauric acid solution in the beta-cyclodextrin solution
The molar ratio of auric acid is 10:1；The concentration of the beta-cyclodextrin solution and tetra chlorauric acid solution is 0.01mol/L；Described
The ratio between volume of tetra chlorauric acid solution and deionized water is 1:38-40；The alkaline solution is that the sodium hydroxide of 1mol/L is molten
Liquid.
Preferred according to the present invention, the concentration of rhodamine 6G solution described in step (2) is 0.1 μm of ol/L, described
The ratio between the addition volume of rhodamine 6G solution and lysozyme standard liquor capacity are 1:1.
According to the present invention, the volume of the lysozyme standard solution of various concentration described in step (2) is identical, each
Group test is parallel three times, be averaged and be compared, test temperature is room temperature.
Preferred according to the present invention, the excitation wavelength of excitation light source described in step (2) is 520nm.
According to the present invention, the above-mentioned method based on nanogold fluorescence detection lysozyme, the detection for lysozyme in urine.
The principle of the present invention is as follows:
When method based on nanogold fluorescence detection lysozyme of the invention detects lysozyme, in wavelength 520nm exciting light
Under source excitation, when not having lysozyme in sample to be tested, the fluorescent emission peak intensity at 552nm is very weak, works as sample to be tested
In when containing lysozyme, the fluorescence intensity at the place 552nm enhances, and lysozyme content is more, and the fluorescence intensity at 552nm is got over
By force.
The present invention by fluorescence resonance energy transfer (FRET) effect between nanogold and rhodamine 6G reach detection it is molten
The purpose of bacterium enzyme, nanogold can make rhodamine 6G fluorescent quenching by FRET effect, but under conditions of pH=7.4, band
The lysozyme of positive electricity is added in electronegative nano-Au solution, and lysozyme and nanogold lead to nanometer by electrostatic interaction
Gold aggregation, is then added rhodamine 6G, and the nanogold after aggregation cannot generate FRET effect with rhodamine 6G again, and lysozyme can be with
Achieve the purpose that quantization detection by the fluorescence that detection rhodamine 6G generates, and the content of lysozyme is more, rhodamine 6G
Fluorescence intensity is stronger.
Beneficial effects of the present invention are as follows:
1, the present invention is based on the method for nanogold fluorescence detection lysozyme, operating process is simple, at low cost, detection is accurate,
High sensitivity, quantitative detection are limited to 0.1 μ g/mL.
2, method of the invention has many advantages, such as that reagent consumption is few, and sample processing time is short, solves existing detection method
Problem complicated, at high cost.
3, the method Successful utilization of detection lysozyme of the invention to the lysozyme content in detection urine and detects knot
Fruit is good, provides prospect for later clinical diagnosis and application development.
Detailed description of the invention
Fig. 1 is transmission electron microscope (TEM) photo of nano-Au solution used in embodiment.
Fig. 2 be embodiment 1 in various concentration lysozyme standard solution fluorescence spectra, at 552nm fluorescence intensity from
Under to the lysozyme standard solution concentration being above corresponding in turn to be respectively 0.1,0.5,1,5,10 μ g/mL.
Fig. 3 is the standard curve of relationship between the logarithm and fluorescence intensity of lysozyme standard solution concentration in embodiment 1.
Specific embodiment
Technical solution of the present invention is further elaborated combined with specific embodiments below, but the scope of the present invention is unlimited
In this.
Experimental method described in embodiments following simultaneously is unless otherwise specified conventional method；The reagent and material
Material, unless otherwise specified, commercially obtains.
Wherein tetra chlorauric acid is bought from Sa En Chemical Engineering Technology (Shanghai) Co., Ltd., China.
Nano-Au solution used is prepared using following methods in embodiment:
The tetra chlorauric acid solution 2mL of beta-cyclodextrin solution 20mL, 0.01mol/L of 0.01mol/L are added separately to
In 78mL deionized water, the sodium hydroxide solution 0.2mL of 1mol/L is added, the pH value of regulation system is 10.5, then in 60 DEG C
Lower stirring 3h, is reduced tetra chlorauric acid sufficiently, obtains the mixed solution of stable homogeneous, is in revolving speed by resulting mixed solution
It is centrifuged 20min under the conditions of 9000 r/min, obtains nanogold particle, gained nanogold particle is dispersed in distilled water three times, obtains
The nano-Au solution for being 0.35nmol/L to concentration.
The nano-Au solution of preparation is added drop-wise on copper mesh, is dried with infrared lamp and is observed under transmission electron microscope,
Transmission electron microscope (TEM) photo is as shown in Figure 1, as can be seen from Figure 1 nanogold particle partial size about 17nm, and size
Uniformly, favorable dispersibility.
Embodiment 1
A method of based on nanogold fluorescence detection lysozyme, comprise the following steps that
(1) preparation of lysozyme standard solution: lysozyme standard product are dissolved in phosphate buffer solution, and concentration point is made
Not Wei 0.1,0.5,1,5,10 μ g/mL lysozyme standard solution；
The phosphate buffer solution includes: phosphatase 11 0mmol/L, NaCl 138mmol/L, KCl 2.7mmol/L, molten
The pH=7.4 of liquid.
(2) measurement of standard curve: respectively into the lysozyme standard solution of various concentration made from 350 μ L steps (1)
350 μ L of 0.35nmol/L nano-Au solution is added, 5min is reacted at 37 DEG C, 0.1 μm of ol/L rhodamine 6G solution is then added
350 μ L, place it in Fluorescence Spectrometer, set excitation wavelength as 520nm, exciting slit 2nm, transmite slit 2nm,
At room temperature scan system fluorescence spectrum and record its fluorescence intensity, each group of test carry out in parallel three times, be averaged into
Row compares, and the fluorescence intensity at every curve 552nm is mapped with the logarithm of the concentration of corresponding lysozyme standard solution, is obtained
To standard curve；The fluorescence spectra of various concentration lysozyme standard solution is as shown in Fig. 2, standard curve is as shown in Figure 3；
(3) measurement of sample to be tested lysozyme: sample to be tested is dissolved in phosphate buffer solution, according to step (2)
Bacteriolyze in sample to be tested is calculated in fluorescence intensity reference standard curve at 552nm by the fluorescence intensity of method test system
The content of enzyme.
Embodiment 2
A method of based on lysozyme in nanogold fluorescence detection urine, comprise the following steps that
(1) normal human urine phosphate buffer solution is diluted 10 times, lysozyme standard product is added thereto, are made molten
The concentration of bacterium enzyme is respectively the urine sample of 1,5,10 μ g/mL；
The phosphate buffer solution includes: phosphatase 11 0mmol/L, NaCl 138mmol/L, KCl 2.7mmol/L, molten
The pH=7.4 of liquid；
(2) the 0.35 of 350 μ L are added in the urine sample of the different lysozyme concentrations obtained respectively to 350 μ L steps (1)
The nano-Au solution of nmol/L, the water-bath 5min at 37 DEG C；The rhodamine 6G that 0.1 μm of ol/L of 350 μ L is added later is molten
Liquid places it in Fluorescence Spectrometer, sets excitation wavelength as 520nm, exciting slit 2nm, transmite slit is 2 nm, detection
Fluorescence intensity at 552nm, each group of test carry out three times, being averaged and being compared, test temperature is room temperature in parallel.Root
According to the standard curve in embodiment 1, the content of lysozyme in urine sample is calculated, and calculates the rate of recovery (Recovery) and phase
To standard deviation (RSD).Calculated result is as shown in table 1.
Table 1
As it can be seen from table 1 method of the invention enzyme rapidly and sensitively can effectively examine the lysozyme in urine
It surveys.
The above description is merely a specific embodiment, and scope of protection of the present invention is not limited thereto, any ripe
Know those skilled in the art in the technical scope disclosed by the present invention, any changes or substitutions that can be easily thought of, should all contain
Lid is within protection scope of the present invention.Therefore, the protection scope that protection scope of the present invention should be defined with claim
Subject to.
Claims (9)
1. a kind of method based on nanogold fluorescence detection lysozyme, comprises the following steps that
(1) preparation of lysozyme standard solution: lysozyme standard product are dissolved in phosphate buffer solution, and various concentration is made
Lysozyme standard solution；
(2) nanogold the measurement of standard curve: is added into the lysozyme standard solution of various concentration made from step (1) respectively
Solution reacts 5min at 37 DEG C, and rhodamine 6G solution is then added, obtains mixed solution, takes mixed solution respectively, through exciting
Light source activation, exciting slit 2nm, transmite slit 2nm scan its fluorescence spectrum and record fluorescence intensity, by every curve
Fluorescence intensity at 552nm is mapped with the logarithm of corresponding lysozyme standard solution concentration, obtains standard curve；
(3) measurement of sample to be tested lysozyme: sample to be tested is dissolved in phosphate buffer solution, according to the method for step (2)
Containing for lysozyme in sample to be tested is calculated by the fluorescence intensity reference standard curve measured in the fluorescence intensity of test system
Amount.
2. the method according to claim 1 based on nanogold fluorescence detection lysozyme, which is characterized in that in step (1)
The phosphate buffer solution includes: phosphatase 11 0mmol/L, NaCl 138mmol/L, KCl 2.7mmol/L, the pH=of solution
7.4。
3. the method according to claim 1 based on nanogold fluorescence detection lysozyme, which is characterized in that in step (1)
The concentration of the lysozyme standard Lysozyme in Solution is 0.1~10 μ g/mL.
4. the method according to claim 1 based on nanogold fluorescence detection lysozyme, which is characterized in that in step (2)
The concentration of the nano-Au solution is 0.35nmol/L.
5. the method according to claim 1 based on nanogold fluorescence detection lysozyme, which is characterized in that in step (2)
The ratio between the addition volume of the nano-Au solution and lysozyme standard liquor capacity are 1:1.
6. the method according to claim 1 based on nanogold fluorescence detection lysozyme, which is characterized in that in step (2)
The concentration of the rhodamine 6G solution is 0.1 μm of ol/L.
7. the method according to claim 1 based on nanogold fluorescence detection lysozyme, which is characterized in that in step (2)
The ratio between the addition volume of the rhodamine 6G solution and lysozyme standard liquor capacity are 1:1.
8. the method according to claim 1 based on nanogold fluorescence detection lysozyme, which is characterized in that in step (2)
The excitation wavelength of the excitation light source is 520nm.
9. the described in any item methods based on nanogold fluorescence detection lysozyme of claim 1-8, for lysozyme in urine
Detection.
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