CN106908499A - Immunosensor based on composite and its method for detecting HSP90 - Google Patents
Immunosensor based on composite and its method for detecting HSP90 Download PDFInfo
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- CN106908499A CN106908499A CN201710052768.7A CN201710052768A CN106908499A CN 106908499 A CN106908499 A CN 106908499A CN 201710052768 A CN201710052768 A CN 201710052768A CN 106908499 A CN106908499 A CN 106908499A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/308—Electrodes, e.g. test electrodes; Half-cells at least partially made of carbon
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Abstract
G C are based on the invention discloses one kind3N4The immunosensor of F127 Au composites, it includes：Working electrode, its basal electrode is glass-carbon electrode, and its surface is modified with g C successively from inside to outside3N4F127 Au and antiHSP90；g‑C3N4F127 Au are prepared by following methods：100 200 weight portion F127 add 38 weight portion water dissolves to stand overnight；5 20 weight portion g C are added afterwards3N4Ultrasonic 6h；0.1 0.3 weight portion HAuCl are added under dark condition4, stir 1 3h；It is rapidly added 0.3 0.8 weight portion NaBH4Stir 0.5 1.5h；Dialysed overnight.Immunosensor preparation cost of the present invention is low, and biocompatibility is good, and detection HSP90 sensitivity is high, strong interference immunity.The present invention also provides the method that detection HSP90 is carried out using the immunosensor.
Description
Technical field
The present invention relates to Protein Detection field.It is more particularly related to a kind of be based on g-C3N4- F127-Au is combined
The immunosensor of material and its method for being used to detect HSP90.
Background technology
Heat shock protein (heat shock proteins, HSPs) is also referred to as stress protein, is body under stress situation
The class protein molecule of synthesis.Wherein HSP90 includes the different binding sites of ATP, and many reactive compounds can be with
HSP90 reacts.Its rich content in the cell, accounts for the 1%~2% of normal cell total protein.It can protect cell
Function and structure, suppresses the polymerization and denaturation of other protein.Except it has obtained good science in medical science anticancer effect
Research, it is also of increased attention in the effect of Food Chemistry.Report explanation in recent years, HSP90 and meat quality
Closely related, its content influences the aspects such as the tenderness of meat, water-holding capacity, mouthfeel, therefore very necessary to its quantitative determination.It is existing
The method of inspection have the methods such as liquid chromatogram, fluorescent quantitation, but these method testing costs are larger, time of measuring is long.For
This, in the urgent need to developing a kind of HSP90 quantitative detecting methods that testing cost is low, simple to operate, sensitivity is high.
Electrochemical immunosensor can carry out specific quantitatively or semi-quantitatively analyzing based on antigen-antibody reaction
Self contained integrated device, it has biology sensor selectively good, and species is more, the low advantage of testing expense.But traditional electricity
When detection HSP90 is applied to, its biocompatibility and sensitivity can not all reach requirement to chemo-immunity sensor.
The content of the invention
It is an object of the invention to solve at least the above, and provide the advantage that at least will be described later.
It is a still further object of the present invention to provide a kind of immunosensor based on composite, its preparation cost is low, green
Color is harmless；Construction method is quick, and biocompatibility is good, has relatively good sensitivity to detection HSP90, to other interfering materials
Such as HSP70 etc. has good anti-interference.
In order to realize these purposes of the invention and further advantage, there is provided a kind of immune biography based on composite
Sensor, including：Working electrode, its basal electrode is glass-carbon electrode, and its surface is modified with g-C successively from inside to outside3N4-F127-Au
And antiHSP90；
Wherein, the g-C3N4- F127-Au is prepared from by following methods：100-200 weight portions F127 adds 3-8 weight
The water dissolves of part simultaneously stand overnight；5-20 weight portions g-C is added afterwards3N4Ultrasonic 6h；0.1-0.3 is added under dark condition
Weight portion HAuCl4, stir 1-3h；Then it is rapidly added the NaBH of 0.3-0.8 weight portion brand-news4Stirring 0.5-1.5h；By saturating
Analysis overnight, obtains g-C3N4-F127-Au。
Preferably, the preparation method of the working electrode is comprised the following steps：
Step one：Glass-carbon electrode GCE is polished in minute surface, then supersound washing, dry at room temperature；
Step 2, takes 3-7 μ Lg-C3N4- F127-Au drips to electrode surface, dries；
Step 3, g-C is dropped in by 3-7 μ LantiHSP90 solution3N4On the electrode surface of-F127-Au modifications, do at room temperature
It is dry；
The concentration of wherein described antiHSP90 solution is 1 μm of ol/L.
Preferably, the immunosensor based on composite also includes：Reference electrode, it is saturation calomel electricity
Pole；
And to electrode, it is platinum electrode.
Preferably, the HAuCl4Weight percent concentration be 2%；The NaBH4Weight percent concentration be
0.5wt%.
Preferably, the molecular cut off MW of the dialysis is 14000.
The present invention also provides a kind of method that HSP90 is detected using the immunosensor, and it is comprised the following steps：
Step one, using antiHSP90/g-C3N4- F127-Au electrodes are working electrode, saturated calomel electrode as reference
Electrode, Pt an electrode are used as to electrode, are correctly connected on electrochemical workstation；
Step 2, sets up the standard curve of detection HSP90 concentration：The HSP90 solution of different concentration is prepared, is dropped in
antiHSP90/g-C3N4On-F127-Au working electrodes, differential pulse voltammetry scanning is carried out to it, record peak current Ipa, will
Described peak current Ipa and HSP90 concentration of standard solution c draws Ipa-c working curves, sets up the standard of detection HSP90 concentration
Curve.
Preferably, the HSP90 solution of various concentrations is configured, wherein the concentration of the HSP90 is respectively 0.049,
0.097,0.24,0.49,0.97,1.70,2.43mM；The linear equation of the standard curve is：Ipa=8.73c+6.29；Its
In, R=0.99.
The present invention at least includes following beneficial effect：Immunosensor based on composite of the present invention, from g-
C3N4- F127-Au builds antiHSP90/g-C3N4- F127-Au/GCE electrodes are used as working electrode, and construction method is quick；Wherein
g-C3N4The preparation of-F127-Au.Preparation cost is low for the traditional chromatogram measurement of contrast, and green is harmless；It is of the present invention to be based on
The immunosensor of composite has good biocompatibility；In terms of antijamming capability, to other interfering materials for example
HSP70 etc. has good anti-interference.Improve the accuracy in detection of HSP90.Using exempting from based on composite of the present invention
The method that epidemic disease sensor detects HSP90, foundation detects that the linear equation of HSP90 is：Ipa (μ A)=8.73c (mg/mL)+6.29
(R=0.99) calculate simple.Test limit is low；
Further advantage of the invention, target and feature embody part by following explanation, and part will also be by this
The research and practice of invention and be understood by the person skilled in the art.
Brief description of the drawings
Fig. 1 is the g-C of the immunosensor based on composite of the present invention3N4The TEM figures of-F127-Au；
Fig. 2 is g-C in the immunosensor based on composite of the present invention3N4(a) and g-C3N4-F127-Au(b)
FTIR figure；
Fig. 3 is the ultraviolet spectrogram of the immunosensor main body based on composite of the present invention；
Fig. 4 is the CD curve maps of the immunosensor based on composite of the present invention；
Fig. 5 is the LSV curve maps of the immunosensor based on composite of the present invention；
Fig. 6 be HSP90 of the present invention detection method in various concentrations HSP90 DPV curve maps；
Fig. 7 be HSP90 of the present invention detection method in various concentrations HSP90 canonical plotting；
Fig. 8 be HSP90 of the present invention detection method in different material testing result comparison diagram.
Specific embodiment
The present invention is described in further detail below in conjunction with the accompanying drawings, to make those skilled in the art with reference to specification text
Word can be implemented according to this.
It should be appreciated that it is used herein such as " have ", "comprising" and " including " term is not precluded from one or many
The presence or addition of individual other elements or its combination.
The present invention provides a kind of immunosensor based on composite, including：Working electrode, its basal electrode is glass carbon
Electrode, its surface is modified with g-C successively from inside to outside3N4- F127-Au and antiHSP90；
Wherein, the g-C3N4- F127-Au is prepared from by following methods：100-200 weight portions F127 adds 3-8 weight
The water dissolves of part simultaneously stand overnight；5-20 weight portions g-C is added afterwards3N4Ultrasonic 6h；0.1-0.3 is added under dark condition
Weight portion HAuCl4, stir 1-3h；Then it is rapidly added the NaBH of 0.3-0.8 weight portion brand-news4Stirring 0.5-1.5h；By saturating
Analysis overnight, obtains g-C3N4-F127-Au。
Wherein in one embodiment, the preparation method of the working electrode is comprised the following steps：
Step one：Glass-carbon electrode GCE is polished in minute surface, then supersound washing, dry at room temperature；
Step 2, takes 3-7 μ L g-C3N4- F127-Au drips to electrode surface, dries；
Step 3, g-C is dropped in by 3-7 μ LantiHSP90 solution3N4On the electrode surface of-F127-Au modifications, do at room temperature
It is dry；
The concentration of wherein described antiHSP90 solution is 1 μm of ol/L.
Wherein in one embodiment, the immunosensor based on composite also includes：Reference electrode, it is full
And calomel electrode；
And to electrode, it is platinum electrode.
Wherein in one embodiment, the HAuCl4Weight percent concentration be 2%；The NaBH4Weight percent
Specific concentration is 0.5wt%.
Wherein in one embodiment, the molecular cut off MW of the dialysis is 14000.
Embodiment 1
Immunosensor based on composite of the present invention includes：
Working electrode, its basal electrode is glass-carbon electrode, and its surface is modified with g-C successively from inside to outside3N4- F127-Au and
antiHSP90；Reference electrode, it is saturated calomel electrode；And to electrode, it is platinum electrode.
The working electrode is prepared using following methods：
Step one, g-C3N4The preparation of-F127-Au：
0.15g F127 are taken to be dissolved in 5mL water overnight.10mg g-C are added in the solution3N4Ultrasonic 6h.In dark bar
200 μ L HAuCl are added under part4(2%) in above-mentioned suspension, 2h is stirred.In order to reduce HAuCl4, it is fast in above-mentioned solution
Speed adds the NaBH of 0.6mL brand-news4(0.5wt%) is stirred 1 hour.Finally, by dialysed overnight (MW=14000), g- is obtained
C3N4-F127-Au.The composite for preparing is stored in the refrigerator of 4 DEG C of normal temperature.
Step 2, antiHSP90/g-C3N4The preparation of-F127-Au electrodes：
Glass-carbon electrode (GCE) is polished in minute surface, then supersound washing, dry at room temperature.5μL g-C3N4-F127-Au
Electrode surface is dripped to, after drying, 5 μ LantiHSP90 (1 μm of ol/L) solution g-C is dropped in into3N4The electrode table of-F127-Au modifications
On face, dry at room temperature.
G-C is determined using HITACHI H-7650 (HITACHI, Japan)3N4The shape characteristic of-F127-Au, wherein voltage
It is 80kV.Result is shown in Fig. 1, as shown in Figure 1, g-C3N4- F127-Au particles are evenly distributed, and particle is in regular spherical.Prove that this is answered
Condensation material is successfully synthesized.
G-C is determined using the infrared transform spectrophotometer (VARIAN, the U.S.) of Cary 50003N4And g-C3N4-F127-
Au simultaneously obtains its infrared spectrogram, and measurement range is 3500cm-1To 500cm-1.As shown in Fig. 2 in curve a and curve b, in ripple
Number 2850cm-1Neighbouring absworption peak is the characteristic absorption peak of C-H.In 1400cm-1Neighbouring peak is the characteristic peak of C-N, illustrates g-
C3N4- F127-Au composites are successfully prepared.
Alternatively, it is also possible to determine g-C using Cary 5000UV-vis-NIR spectrophotometers (Varian, the U.S.)3N4–
F127-Au simultaneously obtains its ultraviolet spectrogram.From the figure 3, it may be seen that F127 does not have absworption peak in the wavelength of 200~800nm.
At 320nm, g-C3N4And g-C3N4- F127-Au has similar absworption peak, it is known from literature that this is the characteristic absorption of nitrogen carbide
Peak.Additionally, g-C3N4- F127-Au has a strong absworption peak at 520nm, it is known from literature that this is the characteristic absorption peak of Au.
Therefore, g-C is further proved3N4- F127-Au is successfully synthesized.
Determined by Chirascan (Applied photophysics, England) and obtain two spectrograms of circle, thus proved
g-C3N4- F127-Au good biocompatibility.It is g-C in Fig. 43N4-F127-Au(a)、HSP90/g-C3N4-F127-Au/
The CD curves of GCE (b).As can be known from Fig. 4, g-C is being added3N4Before and after-F127-Au, the peak height of HSP90 and peak position all do not have
Obvious change.Thus g-C is illustrated3N4- F127-Au has good biocompatibility.
Using linear voltammetry (LSV), to GCE, g-C3N4-F127-Au/GCE、antiHSP90/g-C3N4-F127-Au/
GCE、HSP90/antiHSP90/g-C3N4- F127-Au/GCE carries out chemical property detection respectively.(contained using PBS solution
0.1mol/L KCl) pH=7, the sweep speed of detection is 100mV/s, and sweep limits is -0.2~0.7V.
Using linear voltammetry (LSV) to g-C3N4-F127-Au、antiHSP90/g-C3N4-F127-Au/GCE、
HSP90/antiHSP90/g-C3N4The chemical property of-F127-Au/GCE is further detected：The PBS solution for preparing pH=7 (contains
Have 0.1mol/L KCl), wherein the sweep speed for detecting is 100mV/s, sweep limits is 0.2~0.8V.Curve a is in Fig. 5
Glassy carbon electrode surface has modified g-C3N4The LSV figures of-F127-Au, due to g-C3N4- F127-Au has good electric conductivity, because
This electric current is larger, and curve b is antiHSP90/g-C3N4The LSV figures of-F127-Au/GCE, because the conductive capability of antibody is poor,
Current signal reduction, curve c is HSP90/HSP90 α/g-C3N4The LSV figures of-F127-Au/GCE, compared to curve a and curve b
It is lower, illustrate that HSP90 can be by specifically binding to HSP90 α/g-C3N4- F127-Au/GCE, substantially increases space bit
Resistance, it is suppressed that [Fe (CN)6]3-/4-Electron exchange.The sensor chemical property that we build is good, to detection HSP90
There is relatively good sensitivity.
Hb, Mb, HSP70 and HSP90 are detected respectively using the immunosensor based on composite of the present invention, as a result
See Fig. 8.As can be seen from Figure 8 during detection method detection HSP90 concentration of the present invention, the sensitivity to HSP90 is far above
Other three materials, illustrate that the sensor has good anti-interference for Hb, Mb and HSP70.Due to HSP70 and HSP90
Belong to HSP families and molecular weight is close, illustrate the detection method of HSP90 of the present invention, the accuracy of detection is very high.
The method that immunosensor based on composite of the present invention detects HSP90, comprises the following steps：
(1) antiHSP90/g-C is used3N4- F127-Au/GCE electrodes are working electrode, saturated calomel electrode as reference
Electrode, Pt an electrode are used as to electrode, are correctly connected on electrochemical workstation；Electrochemical workstation be CHI660D (on
Sea, China).
(2) standard curve of detection HSP90 concentration is set up：The HSP90 of the different concentration of preparation, respectively 0.049,
0.097,0.24,0.49,0.97,1.70,2.43mM, drop in HSP90 α/g-C3N4On-F127-Au/GCE, it is carried out to show difference
Pulse Voltammetry is scanned, and records peak current Ipa, and described peak current Ipa and HSP90 concentration of standard solution c is drawn into Ipa-c work
Curve, sets up the standard curve of detection HSP90 concentration.Calculating linear equation is：Ipa (μ A)=8.73c (mg/mL)+
6.29 (R=0.99).Fig. 6 and Fig. 7 is seen respectively.
(3) duck, three kinds of samples of pork and goose are taken and is analyzed instrument detection and electrochemistry of the present invention respectively
Immunosensor detection detection, as a result see the table below：
Institute's value is that parallel five measure of each sample is obtained, and takes its average value.
As seen from the above table：The data that immunosensor of the present invention is measured are and the data result measured in analytical instrument
Match.These data illustrate that sensitivity, the degree of accuracy and the anti-interference of immunosensor of the present invention are higher,
It is used to detect HSP90 that testing result to be accurate.
Although embodiment of the present invention is disclosed as above, it is not restricted to listed in specification and implementation method
With, it can be applied to various suitable the field of the invention completely, for those skilled in the art, can be easily
Other modification is realized, therefore under the universal limited without departing substantially from claim and equivalency range, the present invention is not limited
In specific details and shown here as the legend with description.
Claims (7)
1. a kind of immunosensor based on composite, it is characterised in that including：
Working electrode, its basal electrode is glass-carbon electrode, and its surface is modified with g-C successively from inside to outside3N4- F127-Au and
antiHSP90；
Wherein, the g-C3N4- F127-Au is prepared from by following methods：100-200 weight portions F127 adds 3-8 weight portions
Water dissolves simultaneously stand overnight；5-20 weight portions g-C is added afterwards3N4Ultrasonic 6h；0.1-0.3 weight is added under dark condition
Part HAuCl4, stir 1-3h；Then it is rapidly added the NaBH of 0.3-0.8 weight portion brand-news4Stirring 0.5-1.5h；By dialysing
At night, obtain g-C3N4-F127-Au。
2. the immunosensor of composite is based on as claimed in claim 1, it is characterised in that the preparation of the working electrode
Method is comprised the following steps：
Step one：Glass-carbon electrode GCE is polished in minute surface, then supersound washing, dry at room temperature；
Step 2, takes 3-7 μ L g-C3N4- F127-Au drips to electrode surface, dries；
Step 3, g-C is dropped in by 3-7 μ LantiHSP90 solution3N4On the electrode surface of-F127-Au modifications, drying at room temperature is
Can；
The concentration of wherein described antiHSP90 solution is 1 μm of ol/L.
3. the immunosensor of composite is based on as claimed in claim 2, it is characterised in that also included：Reference electrode, its
It is saturated calomel electrode；
And to electrode, it is platinum electrode.
4. the immunosensor of composite is based on as claimed in claim 1, it is characterised in that the HAuCl4Weight hundred
Specific concentration is divided to be 2%；The NaBH4Weight percent concentration be 0.5wt%.
5. the immunosensor of composite is based on as claimed in claim 1, it is characterised in that the retention molecule of the dialysis
Amount MW is 14000.
6. a kind of method that immunosensor using as described in claim any one of 1-5 detects HSP90, it is characterised in that
Comprise the following steps：
Step one, using antiHSP90/g-C3N4It is electric that-F127-Au electrodes are working electrode, saturated calomel electrode is used as reference
Pole, Pt electrode are used as to electrode, are correctly connected on electrochemical workstation；
Step 2, sets up the standard curve of detection HSP90 concentration：The HSP90 of different concentration is prepared, antiHSP90/g- is dropped in
C3N4On-F127-Au working electrodes, differential pulse voltammetry scanning is carried out to it, peak current Ipa is recorded, by described peak current
Ipa and HSP90 concentration of standard solution c draws Ipa-c working curves, sets up the standard curve of detection HSP90 concentration.
7. the method for detecting HSP90 as claimed in claim 6, it is characterised in that the HSP90 solution of configuration various concentrations, its
Described in the concentration of HSP90 be respectively 0.049,0.097,0.24,0.49,0.97,1.70,2.43mM；The standard curve
Linear equation is：Ipa=8.73c+6.29；Wherein, R=0.99.
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