CN105928914A - Hydrogen sulfide detection sensor, preparation method thereof, quantitative detection method of hydrogen sulfide, and qualitative detection method of hydrogen sulfide in cells - Google Patents
Hydrogen sulfide detection sensor, preparation method thereof, quantitative detection method of hydrogen sulfide, and qualitative detection method of hydrogen sulfide in cells Download PDFInfo
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- CN105928914A CN105928914A CN201610235329.5A CN201610235329A CN105928914A CN 105928914 A CN105928914 A CN 105928914A CN 201610235329 A CN201610235329 A CN 201610235329A CN 105928914 A CN105928914 A CN 105928914A
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- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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Abstract
The invention discloses a hydrogen sulfide detection sensor, a preparation method thereof, a quantitative detection method of hydrogen sulfide, and a qualitative detection method of hydrogen sulfide in cells. The preparation method comprises the following steps: (1) preparing a water solution of copper complex by mixing disodium EDTA, soluble copper salts, and water; (2) subjecting the water solution of copper complex to hydrothermal reactions, filtration, centrifugation, and dialysis to obtain a water solution of copper doped carbon quantum dots in the dialysis bag; (3) mixing the water solution of copper doped carbon quantum dots with a buffer solution to obtain the hydrogen sulfide detection sensor. The hydrogen sulfide detection sensor can carry out quantitative determination on a hydrogen sulfide solution and qualitative detection on hydrogen sulfide in cells.
Description
Technical field
The present invention relates to the detection of hydrogen sulfide, in particular it relates to sulfurated hydrogen detection sensor and preparation side thereof
Method, the quantitative detecting method of hydrogen sulfide and the qualitative checking method of intracellular hydrogen sulfide.
Background technology
Hydrogen sulfide is found as novel signal projector a kind of in organism, thus to cause recently
The extensive concern of people.In addition to nitric oxide and carbon monoxide both gaseous signal molecules, it
It is acknowledged as the 3rd important gaseous signal molecule.Endogenous Hydrogen Sulfide is mainly enzymatic reaction to be passed through
Biological method synthesis obtains, and can participate in as various lifes such as antioxidation, antiinflammatory and apoptosis
Electrophysiologic procedure.It addition, the hydrogen sulfide of variable concentrations is probed into for pathological process also close contact.
In order to more accurately probe in these physiological process the change caused by the concentration of hydrogen sulfide, detection in real time is lived thin
The amount of the hydrogen sulfide in born of the same parents and organism is particularly important.But, owing to the high activity of hydrogen sulfide is with strong
Dispersibility, traditional detection technique is as gas chromatography, colorimetry and electrochemical methods, not
It is applicable to detect in real time.They need the last handling process of complexity, even can destroy tissue and competent cell.
It is noted that the Imaging-PAM constructed with fluorescent probe advantageously, it has been found that and its fluorescence visit
It is directed to the high selectivity of various biological sample, high sensitivity and good biocompatibility so that set
Meter and synthesis fluorescent probe have great importance for the detection of hydrogen sulfide.
In the numerous optical technologies for detecting hydrogen sulfide, fluorescence sense and imaging are considered as the most general
With most widely used technology in laboratory research and clinical practice, because it has high sensitivity, reality
Time the advantage such as monitoring, low ambient interferences and high s/n ratio.In 10 years of past, the method for various fluorescence
The detection of the hydrogen sulfide in biological fluid and living cells, generally can be divided into the chemistry of three types
Reaction is used for fluoroscopic examination hydrogen sulfide, is to utilize hydrogen sulfide that azide is reduced into amine, hydrogen sulfide respectively
The nucleophilic addition related to and the precipitation of copper sulfide.Due to copper ion paramagnetism significantly, it is easily caused
Fluorogen quencher, it is furthermore interesting that the complex that copper ion and fluorogen are formed can utilize again copper ion
It is used for detecting hydrogen sulfide, thus completes the process of fluorescence sense and imaging.But, so far, used
To fluorescent probe be organic fluorescent dye and quantum dot mostly, they itself have the biggest toxicity, and
Can not anti-light bleach, under the irradiation of daylight lamp, fluorescence intensity there will be and reduces significantly, not
It is applicable to intracellular sulfurated hydrogen detection and fluorescence imaging, and they need copper first for fluorescent probe
Quencher, then by add hydrogen sulfide play fluorescence go up thus detect the ternary system of hydrogen sulfide, its process
Loaded down with trivial details, error is bigger.
Summary of the invention
It is an object of the invention to provide a kind of sulfurated hydrogen detection sensor and preparation method thereof, the determining of hydrogen sulfide
Quantity measuring method and the qualitative checking method of intracellular hydrogen sulfide, hydrogen sulfide solution can be carried out by the method
Detection by quantitative, can carry out qualitative detection to intracellular hydrogen sulfide again.
To achieve these goals, the invention provides the preparation method of a kind of sulfurated hydrogen detection sensor,
Including:
1) disodium EDTA, soluble copper salt and water are mixed to form the water-soluble of copper complex
Liquid；
2) aqueous solution of copper complex is carried out hydro-thermal reaction, filter, be centrifuged, dialysing takes in bag filter
Liquid with the aqueous solution of the carbon quantum dot of prepared Copper-cladding Aluminum Bar；
3) aqueous solution of the carbon quantum dot of Copper-cladding Aluminum Bar is mixed with buffer solution pass with prepared sulfurated hydrogen detection
Sensor.
Present invention provides a kind of sulfurated hydrogen detection sensor, this sulfurated hydrogen detection sensor is by above-mentioned
Method be prepared.
Present invention also offers the quantitative detecting method of a kind of hydrogen sulfide, including:
1) the hydrogen sulfide standard solution of variable concentrations is respectively placed in above-mentioned sulfurated hydrogen detection sensor
And add water and be settled to solution to be measured, detect the fluorescence intensity of solution to be measured；
2) with fluorescence intensity as vertical coordinate, the concentration of hydrogen sulfide standard solution is abscissa, sets up fluorescence
The equation of the curve of spectrum；
3) hydrogen sulfide solution of unknown concentration it is placed in sulfurated hydrogen detection sensor and adds water and be settled to treat
Surveying solution, detect the fluorescence intensity of solution to be measured, then the hydrogen sulfide according to Equation for Calculating unknown concentration is molten
The concentration of liquid.
Invention further provides the qualitative checking method of a kind of intracellular hydrogen sulfide, it is characterised in that
Including:
1) human cancer cell is placed in the buffer solution that pH is 7.2-7.6 and carries out constant temperature culture；
2) the hydrogen sulfide standard solution addition of variable concentrations is carried out second incubation to human cancer cell；
3) human cancer cell is taken out, under Laser Scanning Confocal Microscope, then obtain human cancer cell at light field
Fluorescence imaging under exciting with blue light.
By technique scheme, Copper-cladding Aluminum Bar is entered carbon quantum dot by the way of fabricated in situ by the present invention
In, a length of 370nm of optimum excitation wave of this carbon quantum dot carbon quantum dot, quantum yield is up to 56%, with
(diammonium Sequestrene AA is the carbon point that carbon source prepares to simple carbon quantum dot, with quinoline sulfate for ginseng
Ratio, obtaining quantum yield is 25%, and undope copper) compare, have a surplus for high one times, thus the amount of overcoming
The defect that sub-productivity is low.Meanwhile, this quantum dot belongs to carbon nanomaterial, has the environmental protection characteristic of excellence,
And then efficiently solve organic dyestuff and quantum dot strong toxicity in prior art, and anti-light Bleachability weak etc.
Problem.Additionally, the synthetic method phase of the preparation method of this carbon quantum dot and traditional tube furnace high temperature carbon point
Ratio, has that temperature is low, preparation time is short, simple to operate, energy consumption is relatively low and eco-friendly advantage.
On the basis of above-mentioned carbon quantum dot, the present invention is by the aqueous solution of carbon quantum dot and buffer solution group
The sulfurated hydrogen detection sensor become carries out spectrophotomelric assay to hydrogen sulfide solution, and then can obtain linear
Excellent working curve such that it is able to effectively the concentration of hydrogen sulfide solution is carried out detection by quantitative.Meanwhile,
The present invention also utilizes hydrogen sulfide solution to cultivate human cancer cell with buffer solution, and then to human body cancer
Cell carries out fluorescence imaging, thus effectively detects the hydrogen sulfide of human body cell qualitatively.
Other features and advantages of the present invention will be described in detail in detailed description of the invention part subsequently.
Accompanying drawing explanation
Accompanying drawing is used to provide a further understanding of the present invention, and constitutes the part of description, with
Detailed description below is used for explaining the present invention together, but is not intended that limitation of the present invention.?
In accompanying drawing:
Fig. 1 is the fluorescent emission collection of illustrative plates of the carbon quantum dot of Copper-cladding Aluminum Bar in detection example 1；
Fig. 2 is the fluoroscopic examination cartogram of the carbon quantum dot of Copper-cladding Aluminum Bar in detection example 3；
Fig. 3 is the transmission electron microscope picture of the carbon quantum dot of Copper-cladding Aluminum Bar in detection example 4；
Fig. 4 is the result cartogram of Fig. 3；
Fig. 5 is the infrared spectrogram of the carbon quantum dot of Copper-cladding Aluminum Bar in detection example 5；
Fig. 6 is the XPS spectrum figure of the carbon quantum dot of Copper-cladding Aluminum Bar in detection example 6；
Fig. 7 is the fluoroscopic examination cartogram of the carbon quantum dot of Copper-cladding Aluminum Bar in detection example 7；
Fig. 8 is fluoroscopic examination cartogram in application examples 1；
Fig. 9 is fluorescence imaging result figure in application examples 2.
Detailed description of the invention
Hereinafter the detailed description of the invention of the present invention is described in detail.It should be appreciated that this place is retouched
The detailed description of the invention stated is merely to illustrate and explains the present invention, is not limited to the present invention.
The invention provides the preparation method of a kind of sulfurated hydrogen detection sensor, including:
1) disodium EDTA, soluble copper salt and water are mixed to form the water-soluble of copper complex
Liquid；
2) aqueous solution of copper complex is carried out hydro-thermal reaction, filter, be centrifuged, dialysing takes in bag filter
Liquid with the aqueous solution of the carbon quantum dot of prepared Copper-cladding Aluminum Bar；
3) aqueous solution of the carbon quantum dot of Copper-cladding Aluminum Bar is mixed with buffer solution pass with prepared sulfurated hydrogen detection
Sensor.
Step 1 in the present invention) in, the consumption of each material can select in wide scope, but is
Improve the productivity of the copper complex prepared, it is preferable that in step 1) in, second two relative to 3.7g
Amine Sequestrene AA, the consumption of soluble copper salt is 1.5-2.5g, and the consumption of water is 20-40ml；
Step 1 in the present invention) in, the concrete kind of soluble copper salt can select in wide scope,
But in order to improve the productivity and taking cost into account of prepared copper complex, it is preferable that soluble copper
One or more in copper chloride, copper sulfate and copper nitrate of salt；
Step 1 in the present invention) in, the condition of mixing can select in wide scope, but in order to
Improve the productivity of the copper complex prepared, it is preferable that mixing at least meets following condition: mixing temperature is
20-30 DEG C, incorporation time is 5-10min.
Step 2 in the present invention) in, the condition of hydro-thermal reaction can select in wide scope, but
In order to improve the productivity of the carbon quantum dot of prepared Copper-cladding Aluminum Bar, it is preferable that in step 2) in, hydro-thermal is anti-
Should at least meet following condition: reaction temperature is 180-220 DEG C, the response time is 2-6h.
Step 3 in the present invention) in, the consumption of raw material can select in wide scope, but in order to
Improve the sensitivity for hydrogen sulfide of the sulfurated hydrogen detection sensor, it is preferable that the copper relative to 1mL is mixed
The aqueous solution of miscellaneous carbon quantum dot, the consumption of buffer solution is 1-3mL；
Step 3 in the present invention) in, the pH of buffer solution can select in wide scope, but
In order to improve the sensitivity for hydrogen sulfide of the sulfurated hydrogen detection sensor, it is preferable that the pH of buffer solution
For 7.2-7.6.
Step 3 in the present invention) in, the kind of buffer solution can select in wide scope, but
In order to improve the sensitivity for hydrogen sulfide of the sulfurated hydrogen detection sensor, it is preferable that buffer solution is selected from phosphorus
Hydrochlorate buffer solution and/or carbon acid solution.
Step 2 in the present invention) in, the molecular cut off of bag filter can select in wide scope,
But in order to improve the sensitivity for hydrogen sulfide of the sulfurated hydrogen detection sensor, it is preferable that cutting of bag filter
Staying molecular weight is 500-1000.
Present invention provides a kind of sulfurated hydrogen detection sensor, this sulfurated hydrogen detection sensor is by above-mentioned
Method be prepared.
Present invention also offers the quantitative detecting method of a kind of hydrogen sulfide, including:
1) the hydrogen sulfide standard solution of variable concentrations is respectively placed in above-mentioned sulfurated hydrogen detection sensor
And add water and be settled to solution to be measured, detect the fluorescence intensity of solution to be measured；
2) with fluorescence intensity as vertical coordinate, the concentration of hydrogen sulfide standard solution is abscissa, sets up fluorescence
The equation of the curve of spectrum；
3) hydrogen sulfide solution of unknown concentration it is placed in sulfurated hydrogen detection sensor and adds water and be settled to treat
Surveying solution, detect the fluorescence intensity of solution to be measured, then the hydrogen sulfide according to Equation for Calculating unknown concentration is molten
The concentration of liquid.
In the quantitative detecting method of above-mentioned hydrogen sulfide, for the ease of detecting the concentration of hydrogen sulfide solution,
Preferably, equation is y=-0.242x+2119.15, and wherein, y is fluorescence intensity, and x is hydrogen sulfide standard
The concentration of solution.
In the present invention, hydrogen sulfide standard solution can be to configure voluntarily and obtain, it is also possible to is obtained by purchase
, in order to avoid the impurity pollution to hydrogen sulfide solution, it is preferable that hydrogen sulfide standard solution will be for vulcanizing
Sodium solid is dissolved in water and is prepared.
Invention further provides the qualitative checking method of a kind of intracellular hydrogen sulfide, it is characterised in that
Including:
1) human cancer cell is placed in the buffer solution that pH is 7.2-7.6 and carries out constant temperature culture；
2) the hydrogen sulfide standard solution addition of variable concentrations is carried out second incubation to human cancer cell；
3) human cancer cell is taken out, under Laser Scanning Confocal Microscope, then obtain human cancer cell at light field
Fluorescence imaging under exciting with blue light.
In the qualitative checking method of above-mentioned intracellular hydrogen sulfide, the concrete kind of buffer solution can be at width
In the range of select, but in order to improve the effect of fluorescence imaging, it is preferable that in step 1) in, slow
Dissolved liquid is selected from phosphate buffered solution and/or carbon acid solution.
Wherein, in step 1) and 2) in, the actual conditions that cell is cultivated can select in wide scope
Select, but in order to improve the progress that cell is cultivated, it is preferable that constant temperature culture and second incubation are each independent
Ground at least meets following condition: cultivation temperature is 35-38 DEG C, and incubation time is 25-35min, CO2's
Content is 4-6 volume %.
Meanwhile, in step 3) in, the wavelength of blue light detection can select in wide scope, but is
Obtain maximum fluorescence intensity, it is preferable that in step 3) in, the wavelength of blue light is 365-375mm.
On the basis of the above, in order to shorten human cancer cell further in step 1) and 2) in
Incubation time, it is preferable that in step 1) before, qualitative checking method also includes: at 35-38 DEG C
And CO in air2Content be 4-6 volume % under conditions of, human cancer cell is placed in containing 8 weight
The culture dish of %-12 weight % hyclone is cultivated 22-26h.
Hereinafter will be described the present invention by embodiment.In following example, human cancer cell
Buy in Wuhan doctor spy's biology company limited (Wuhan, China)；And human cancer cell is before use:
CO in 37 DEG C and air2Content be 5 volume % under conditions of, human cancer cell is placed in and contains
The culture dish of 10 weight % hyclones is cultivated 24h；Hydrogen sulfide standard solution is by molten for sodium sulfide solid
It is prepared in water.
Embodiment 1
1) at 25 DEG C, by disodium EDTA 3.7224g, copper chloride 1.7048g and water 30ml
Mixing 8min forms the aqueous solution of copper complex；
2) aqueous solution of above-mentioned copper complex is carried out at 200 DEG C hydro-thermal reaction 4h, filter, be centrifuged,
After dialysis 76h, take the liquid in bag filter (molecular cut off of bag filter is 700) and mix with prepared copper
The aqueous solution of miscellaneous carbon quantum dot；
3) by phosphate buffered solution that the aqueous solution of the carbon quantum dot of above-mentioned Copper-cladding Aluminum Bar and pH are 7.4 by
Volume ratio according to 1:2 mixes to prepare described sulfurated hydrogen detection sensors A 1.
Embodiment 2
1) at 20 DEG C, by disodium EDTA 3.7224g, copper chloride 1.5g and water 20-40ml
Mixing 5min forms the aqueous solution of copper complex；
2) aqueous solution of above-mentioned copper complex is carried out at 180 DEG C hydro-thermal reaction 2h, filter, be centrifuged,
After dialysis 76h, take the liquid in bag filter (molecular cut off of bag filter is 500) and mix with prepared copper
The aqueous solution of miscellaneous carbon quantum dot；
3) by phosphate buffered solution that the aqueous solution of the carbon quantum dot of above-mentioned Copper-cladding Aluminum Bar and pH are 7.2 by
Volume ratio according to 1:1 mixes to prepare described sulfurated hydrogen detection sensors A 2.
Embodiment 3
1) at 30 DEG C, by disodium EDTA 3.7224g, copper chloride 2.5g and water 40ml
Mixing 10min forms the aqueous solution of copper complex；
2) aqueous solution of above-mentioned copper complex is carried out at 220 DEG C hydro-thermal reaction 6h, filter, be centrifuged,
After dialysis 76h, take the liquid in bag filter (molecular cut off of bag filter is 1000) and mix with prepared copper
The aqueous solution of miscellaneous carbon quantum dot；
3) by phosphate buffered solution that the aqueous solution of the carbon quantum dot of above-mentioned Copper-cladding Aluminum Bar and pH are 7.6 by
Volume ratio according to 1:3 mixes to prepare described sulfurated hydrogen detection sensors A 3.
Detection example 1
By F-4500 spectrofluorophotometer (Hitachi, Ltd, Japan), the copper in embodiment 1 is mixed
The aqueous solution of miscellaneous carbon quantum dot carries out fluorescent emission detection under different excitation wavelengths, and testing result is shown in figure
1, as seen from the figure, a length of 370nm of optimum excitation wave of the carbon quantum dot of Copper-cladding Aluminum Bar.
In like manner, the carbon quantum dot of the Copper-cladding Aluminum Bar in embodiment 2 and 3 is detected, acetonideexample 1
In the carbon quantum dot of Copper-cladding Aluminum Bar keep consistent.
Detection example 2
The aqueous solution of the carbon quantum dot of the Copper-cladding Aluminum Bar in embodiment 1 is diluted, then passes through F-4500
Spectrofluorophotometer (Hitachi, Ltd, Japan) is water-soluble to the carbon quantum dot of the Copper-cladding Aluminum Bar of variable concentrations
Liquid carries out fluorescent emission detection under the wavelength of 370nm, can obtain the denseest of Copper-cladding Aluminum Bar carbon quantum dot
Degree is 0.5mg/ml.
In like manner, the carbon quantum dot of the Copper-cladding Aluminum Bar in embodiment 2 and 3 is detected, acetonideexample 1
In the carbon quantum dot of Copper-cladding Aluminum Bar keep consistent.
Detection example 3
By the carbon quantum dot solution of Copper-cladding Aluminum Bar that the optium concentration in embodiment 2 is 0.5mg/ml in ultraviolet
It is irradiated under lamp, then F-4500 spectrofluorophotometer (Hitachi, Ltd, Japan) detection fluorescence
The change of intensity, result is shown in Fig. 2, as shown in Figure 2, As time goes on, Copper-cladding Aluminum Bar carbon quantum dot
Fluorescence intensity do not change.Illustrate that carbon quantum dot has the strongest anti-light Bleachability, be not required to
Want lucifuge to process, easily preserve.
Detection example 4
By the TEM-1200EX transmission electron microscope carbon quantum dot to the Copper-cladding Aluminum Bar in embodiment 1
Carrying out transmission electron microscope detection, testing result is shown in Fig. 3 and Fig. 4, and as seen from the figure, Copper-cladding Aluminum Bar carbon quantum dot has
Having good dispersibility, particle diameter is little and is evenly distributed, and Average Particle Diameters is probably at about 4nm.From figure
The high-resolution transmission plot in the upper right corner of 4 is it can be seen that have lattice clearly inside carbon quantum dot.
In like manner, the carbon quantum dot of the Copper-cladding Aluminum Bar in embodiment 2 and 3 is detected, acetonideexample 1
In the carbon quantum dot of Copper-cladding Aluminum Bar keep consistent.
Detection example 5
Right by Spectrum GX fourier transform infrared spectroscopy instrument (Perkin Elmer company of the U.S.)
The carbon quantum dot of the Copper-cladding Aluminum Bar in embodiment 1 carries out infrared spectrum detection, and testing result is shown in Fig. 5, by scheming
Understand, at 1634cm-1There is strong absworption peak at place, illustrates that there is carbon-carbon double bond (C=C) on carbon quantum dot surface
Stretching vibration, doping carbon quantum dot respectively 1343,1187,1090 and 1020cm-1Place has bright
Aobvious absworption peak, indicates and may have the stretching vibration of N-O key, the bending vibration of N-O-H, C-N
Stretching vibration and the bending vibration of N-O-H.From Copper-cladding Aluminum Bar carbon quantum dot 900 to 1100cm-1Red
Outer spectrogram can be seen that at 1020cm-1And 1050cm-1Place, Copper-cladding Aluminum Bar carbon quantum dot has obvious spy
Levy peak, thus it is speculated that the surface of Copper-cladding Aluminum Bar carbon quantum dot is likely to be of the stretching vibration of N-Cu-N.These are a series of
Characterization result show, raw material Na2[Cu (EDTA)] successful conversion becomes the carbon quantum dot that metallic copper adulterates,
And carbon quantum dot surface contains the site of substantial amounts of carboxyl, hydroxyl and amino and copper.
In like manner, the carbon quantum dot of the Copper-cladding Aluminum Bar in embodiment 2 and 3 is detected, acetonideexample 1
In the carbon quantum dot of Copper-cladding Aluminum Bar keep consistent.
Detection example 6
By X-ray photoelectricity energy disperse spectroscopy (ESCALAB250, thermoelectricity, the U.S.) in embodiment 1
The carbon quantum dot of Copper-cladding Aluminum Bar carry out XPS detection, testing result is shown in Fig. 6, and as seen from the figure, metallic copper is mixed
Miscellaneous carbon quantum dot contains four kinds of elements, is C, N, O, Cu respectively, and contained the hundred of four kinds of elements
Component is respectively 67.49%, 7.76%, 21.72% and 3.03%.
In like manner, the carbon quantum dot of the Copper-cladding Aluminum Bar in embodiment 2 and 3 is detected, acetonideexample 1
In the carbon quantum dot of Copper-cladding Aluminum Bar keep consistent.
Detection example 7
Respectively by F-, Cl-, Br-, I-, NO3 -, SO4 2-, CH3COO-, HCO3 -, HPO4 2-And HSO4
Add in the carbon quantum dot solution of the Copper-cladding Aluminum Bar to embodiment 1, then by F-4500 fluorescence spectrophotometer
Photometer (Hitachi, Ltd, Japan) fluorescence intensity, testing result is shown in Fig. 7, as seen from the figure, when
The when of adding sulfur hydrogen radical ion, significantly reducing occurs in the fluorescence intensity of copper carbon quantum dot, and sulfur hydrogen is described
Radical ion can cause the fluorescent quenching of copper carbon quantum dot, and when adding other anion when, copper carbon amounts
The fluorescence intensity of son point does not changes, and illustrates under conditions of other anion exists, copper carbon amounts
Son point has good selectivity to hydrogen sulfide.
In like manner, the carbon quantum dot of the Copper-cladding Aluminum Bar in embodiment 2 and 3 is detected, acetonideexample 1
In the carbon quantum dot of Copper-cladding Aluminum Bar keep consistent.
Application examples 1
1) the hydrogen sulfide standard solution of variable concentrations is respectively placed in above-mentioned sulfurated hydrogen detection sensors A 1
In and add water and be settled to solution to be measured, detect the fluorescence intensity of solution to be measured, concrete outcome is shown in Fig. 8；
2) with fluorescence intensity as vertical coordinate, the concentration of hydrogen sulfide standard solution is abscissa, sets up fluorescence
The equation of the curve of spectrum, equation is Y=-0.242X+2119.15；
In like manner, sensors A 2 is identical for the testing result of hydrogen sulfide solution with A3.
Application examples 2
1) human cancer cell is placed in the phosphate buffered solution that pH is 7.4 and carries out constant temperature culture 0.5h；
2) by the hydrogen sulfide standard solution (concentration is respectively 100 μMs, 200 μMs and 500 μMs) of variable concentrations
Add and carry out second incubation 0.5h to described human cancer cell；Wherein, above-mentioned constant temperature culture and secondary
Cultivation is satisfied by following condition: cultivation temperature is 37 DEG C, CO2Content be 5 volume %；
3) upper human cancer cell is taken out, under Laser Scanning Confocal Microscope, then obtain described human cancer cell
Fluorescence imaging under the blue light of light field and 370mm excites, concrete outcome is shown in Fig. 9, and wherein, a-d is
Human Lung Cancer cell image in the case of light field, e, i are the conditions not adding copper carbon quantum dot and hydrogen sulfide
Under, under Laser Scanning Confocal Microscope, uviol lamp excites the base of observed fluorescence imaging, i.e. lung carcinoma cell
End color (blank), does not observes fluorescence substantially.Afterwards, add in lung carcinoma cell appropriate
Copper quantum carbon point, f, j are under Laser Scanning Confocal Microscope, and uviol lamp excites observed fluorescence imaging, can
To be clearly visible strong blue-fluorescence.Being added thereto to concentration again is 100 μMs and the sulfuration of 200 μMs
Hydrogen solution, finds from the viewed imaging of Laser Scanning Confocal Microscope, fluorescence intensity all occurs to a certain degree
Reduction, and add 200 μMs of hydrogen sulfide solutions fluorescence more weak, illustrate that carbon quantum dot can be well
Realize sensing and the imaging of hydrogen sulfide in cell.
The preferred embodiment of the present invention described in detail above, but, the present invention is not limited to above-mentioned reality
Execute the detail in mode, in the technology concept of the present invention, can be to the technical side of the present invention
Case carries out multiple simple variant, and these simple variant belong to protection scope of the present invention.
It is further to note that each the concrete technology described in above-mentioned detailed description of the invention is special
Levy, in the case of reconcilable, can be combined by any suitable means, in order to avoid need not
The repetition wanted, various possible compound modes are illustrated by the present invention the most separately.
Additionally, combination in any can also be carried out between the various different embodiment of the present invention, as long as its
Without prejudice to the thought of the present invention, it should be considered as content disclosed in this invention equally.
Claims (10)
1. the preparation method of a sulfurated hydrogen detection sensor, it is characterised in that including:
1) disodium EDTA, soluble copper salt and water are mixed to form the water-soluble of copper complex
Liquid；
2) aqueous solution of described copper complex is carried out hydro-thermal reaction, filter, be centrifuged, dialysing takes dialysis
Liquid in Dai is with the aqueous solution of the carbon quantum dot of prepared Copper-cladding Aluminum Bar；
3) mix the aqueous solution of the carbon quantum dot of described Copper-cladding Aluminum Bar with buffer solution to prepare described sulfuration
Hydrogen detection sensor.
Preparation method the most according to claim 1, wherein, in step 1) in, relative to 3.7g
Described disodium EDTA, the consumption of described soluble copper salt is 1.5-2.5g, described water
Consumption is 20-40ml；
Preferably, one or more in copper chloride, copper sulfate and copper nitrate of described soluble copper salt；
It is highly preferred that described mixing at least meets following condition: mixing temperature is 20-30 DEG C, during mixing
Between be 5-10min.
Preparation method the most according to claim 2, wherein, in step 2) in, described hydro-thermal is anti-
Should at least meet following condition: reaction temperature is 180-220 DEG C, the response time is 2-6h.
Preparation method the most according to claim 3, wherein, relative to the described Copper-cladding Aluminum Bar of 1mL
The aqueous solution of carbon quantum dot, the consumption of described buffer solution is 1-3mL；
Preferably, the pH of described buffer solution is 7.2-7.6；
It is highly preferred that described buffer solution is selected from phosphate buffered solution and/or carbon acid solution；
It is further preferred that the molecular cut off of described bag filter is 500-1000.
5. a sulfurated hydrogen detection sensor, it is characterised in that described sulfurated hydrogen detection sensor passes through
In claim 1-4, the method described in any one is prepared.
6. the quantitative detecting method of a hydrogen sulfide, it is characterised in that including:
1) the hydrogen sulfide standard solution of variable concentrations is respectively placed in hydrogen sulfide as claimed in claim 5
Detect in sensor and add water and be settled to solution to be measured, detect the fluorescence intensity of solution to be measured；
2) with fluorescence intensity as vertical coordinate, the concentration of hydrogen sulfide standard solution is abscissa, sets up fluorescence
The equation of the curve of spectrum；
3) hydrogen sulfide solution of unknown concentration is placed in described sulfurated hydrogen detection sensor and the constant volume that adds water
To solution to be measured, detect the fluorescence intensity of solution to be measured, then according to described Equation for Calculating unknown concentration
The concentration of hydrogen sulfide solution.
Quantitative detecting method the most according to claim 6, wherein, described equation is
Y=-0.242x+2119.15, wherein, y is fluorescence intensity, and x is the concentration of hydrogen sulfide standard solution；
Preferably, described hydrogen sulfide standard solution is prepared for sodium sulfide solid is dissolved in water.
8. the qualitative checking method of an intracellular hydrogen sulfide, it is characterised in that including:
1) human cancer cell is placed in the buffer solution that pH is 7.2-7.6 and carries out constant temperature culture；
2) the hydrogen sulfide standard solution addition of variable concentrations is carried out secondary training to described human cancer cell
Support；
3) described human cancer cell is taken out, under Laser Scanning Confocal Microscope, then obtain described human body cancer thin
Born of the same parents' fluorescence imaging under light field and blue light excite.
Qualitative checking method the most according to claim 8, wherein, in step 1) in, described slow
Dissolved liquid is selected from phosphate buffered solution and/or carbon acid solution；
Preferably, described constant temperature culture and second incubation the most at least meet following condition: cultivate
Temperature is 35-38 DEG C, and incubation time is 25-35min, CO2Content be 4-6 volume %；
In step 3) in, the wavelength of described blue light is 365-375mm.
The most according to claim 8 or claim 9, qualitative checking method, wherein, in step 1) before,
Described qualitative checking method also includes: CO in 35-38 DEG C and air2Content be 4-6 volume %
Under conditions of, human cancer cell is placed in the culture dish containing 8 weight %-12 weight % hyclones cultivation
22-26h。
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