CN108084995A - A kind of method of CuInS/ZnS quantum dots and detection of alkaline phosphatase - Google Patents
A kind of method of CuInS/ZnS quantum dots and detection of alkaline phosphatase Download PDFInfo
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- CN108084995A CN108084995A CN201711402342.6A CN201711402342A CN108084995A CN 108084995 A CN108084995 A CN 108084995A CN 201711402342 A CN201711402342 A CN 201711402342A CN 108084995 A CN108084995 A CN 108084995A
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- 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
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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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
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- 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
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
Abstract
The present invention relates to biochemistry detection technical fields, disclose a kind of CuInS/ZnS quantum dots and the method using the quantum dots characterization alkaline phosphatase.With CuInS/ZnS quantum dots as the probe in 4 Nitrophenyl phosphate disodium system of alkaline phosphatase enzyme hydrolysis, fluorescence intensity is measured under the conditions of λ ex/ λ em=405/588nm, by the variation of fluorescence intensity and alkaline phosphatase and the linear relationship of fluorescence intensity, the content of alkaline phosphatase is obtained.The CuInS/ZnS a length of 405nm of quantum dot optimum excitation wave prepared by the present invention, the maximum substrate absorbance overlap of peaks with PNP can generate inner filtering effect, so as to achieve the purpose that detect ALP.Detection sensitivity is high, and strong antijamming capability, sample requirement is low, can realize the direct detection to blood serum sample.
Description
Technical field
The present invention relates to semiconductor-quantum-point synthesis, analytical chemistry, biochemistry detection technical field, more particularly to one kind
CuInS/ZnS quantum dots and by the use of the quantum dot as the method for namo fluorescence probe detection of alkaline phosphatase.
Background technology
At present, the method that ALP is used in clinical detection serum is colorimetric method, this method is with 4-NPP (PNPP)
For substrate, under ALP existence conditions, PNPP and ALP reactions generate free phenol (p-nitrophenol PNP) and phosphoric acid, and phenol is in alkalescence
It is acted in solution with 4-AA, aoxidizes to form red quinones through the potassium ferricyanide, the red depth is lived with ALP
Property is directly proportional.Under the conditions of 37 DEG C, per 100mL serum and substrate-function 15 minutes, the ALP activity for generating 1mg phenol was 1 Jin Shi
Unit (U).With distilled water zeroising, each pipe absorbance (ultraviolet specrophotometer) is read at 510nm.This method needs sample
It measures greatly, sample pre-treatments are complicated, and sensitivity is low, poor selectivity, antijamming capability deficiency.Due in clinical sample (such as whole blood)
Complex component testing result can be interfered, therefore be only used for ingredient it is relatively easy and develop the color be not easily susceptible to disturb
System the pretreatments such as is required for sample is centrifuged, can not be directly used in analysis actual blood sample before detection.Bottom
Aldehydes matter must not be contained in object solution, blood serum sample requirement is 100 μ L；Monitoring lower-cut is unknown.
The content of the invention
In view of this, the object of the present invention is to provide a kind of method of quick detection of alkaline phosphatase, this method can be straight
It connects and human serum is measured, often surveying a sample only needs 20 μ l serum, and detection sensitivity is high, and strong interference immunity is easy to operate,
Quick measure can be achieved.
Another object of the present invention, which also resides in, provides above-mentioned CuInS/ZnS quantum dots and preparation method thereof.
In order to achieve the above object, technical scheme is as follows：
A kind of preparation method of CuInS/ZnS quantum dots, which is characterized in that comprise the following steps：
(1) soluble copper source compound, indium source compound and sulfydryl aliphatic acid are mixed with water, adjusting pH value to 8~10,
Obtain mixed liquor；
In the mixed liquor, Cu2+And In3+Molar ratio be 0.015~0.045：0.015~0.120；Cu2+And In3+'s
The sum of amount of substance and the molar ratio of sulfydryl aliphatic acid are 0.03~0.06：0.24~0.90；
(2) under stirring condition, the mixed liquor that the step (1) obtains is mixed with sulphur source compound, Microwave-assisted firing
Solution temperature is warming up to 90~100 DEG C within 4min after making mixing, keeps 1~10min of temperature, obtains containing CuInS cores
Reaction solution；
(3) after the reaction solution that step (2) obtains is cooled to less than 50 DEG C, zinc source solution and sulphur source are added in into reaction solution
Solution, 1~10min of microwave heating reaction, obtains CuInS/ZnS quantum dots at 90~100 DEG C.
Preferably, in step (1), copper source compound is cupric iodide or copper chloride, and the indium source compound is acetic acid
Indium or inidum chloride, the sulfydryl aliphatic acid are the sulfydryl aliphatic acid that backbone length is 2~8 carbon atoms.
In step (2), the sulphur source compound is Na2S。
It is furthermore preferred that in step (1), the Cu2+Molar content be 0.015~0.045mmol；It is described in step (2)
Na2The molar content of S is 0.02~0.06mmol.
Preferably, in step (3), zinc source solution is Zn (Ac)2Solution, the sulphur source solution are Na2S solution.
Preferably, copper source compound further includes before being mixed：Copper source compound is dissolved in ammonia spirit.
Present invention additionally comprises the CuInS/ZnS quantum dots that above-mentioned any one technical solution the method is prepared,
It is characterized in that, for the CuInS/ZnS quantum dots using CuInS as core, ZnS is shell, the optimal excitation of the CuInS/ZnS quantum dots
Wavelength is 405nm, launch wavelength 588nm.
The present invention also provides a kind of method of namo fluorescence probe detection of alkaline phosphatase, with CuInS/ZnS quantum dots
As the probe in alkaline phosphatase enzyme hydrolysis 4-NPP system, measured under the conditions of λ ex/ λ em=405/588nm
The fluorescence intensity of hydrolyzation system by the fluorescence intensity and alkaline phosphatase of measure and the linear relationship of fluorescence intensity, obtains
The content of alkaline phosphatase；
The optimum excitation wave a length of 405nm, launch wavelength 588nm of the CuInS/ZnS quantum dots.
Preferably, the CuInS/ZnS quantum dots are in the alkaline phosphatase enzyme hydrolysis 4-NPP system
Mass concentration be 0.5~2mM.
Preferably, in the system of the alkaline phosphatase enzyme hydrolysis 4-NPP, substrate 4- Nitrophenyl phosphates two
The molar concentration of sodium is 0.5~2mM.
Preferably, MgSO is also contained in the system of the alkaline phosphatase enzyme hydrolysis 4-NPP4。
Compared with prior art, the present invention has the following advantages：
The present invention is prepared in the method for CuInS/ZnS quantum dots, Cu2+:In3+Molar ratio be 0.015~0.045:
0.015~0.120.Since quantum dot has dimensional effect and quantum confined effect, by control two kinds of sun of different proportion from
Sub- presoma can form different sizes, the quantum dot of different spectral quality, and the above-mentioned copper and indium molar ratio in the present invention synthesizes
The quantum dot maximum excitation wavelength arrived is Chong Die with the maximal ultraviolet absorption peak (405nm) of PNP substrates in 405nm, so as to build one
Detection method of the kind based on inner filtering effect.Reaction temperature prepared by the CuInS/ZnS quantum dots of the present invention is 90~100 DEG C, instead
Be 1~10min between seasonable, can be obtained under above-mentioned reaction time and reaction temperature excitation wavelength for 405nm, stabilization, it is glimmering
The stronger CuInS/ZnS quantum dots of luminous intensity.
The method of namo fluorescence probe detection of alkaline phosphatase of the present invention, with CuInS/ZnS quantum dots as alkaline phosphatase
Probe in enzyme hydrolysis 4-NPP system measures fluorescence intensity under the conditions of λ ex/ λ em=405/588nm, leads to
The linear relationship of fluorescence intensity and alkaline phosphatase and fluorescence intensity is crossed, obtains the content of alkaline phosphatase.The CuInS/
The optimum excitation wave a length of 405nm, launch wavelength 588nm of ZnS quantum dot.The detection mechanism of this method is to utilize inner filtering effect
Realize the detection to alkaline phosphatase (ALP), specific mechanism is illustrated as follows：ALP hydrolysis 4-NPPs (PNPP)
To paranitrophenol (PNP), PNP has strong ultraviolet absorption peak at 405nm.The optimal of the CuInS/ZnS quantum dots of the present invention swashs
Wavelength and the substrate absorption maximum overlap of peaks of PNP are sent out, so as to generate inner filtering effect.Using PNPP as substrate, CuInS/ZnS is spy
In the system of pin, ALP hydrolyzes exciting lights of the PNP generated due to absorbing 405nm, causes CuInS/ZnS that fluorescent quenching occurs,
So as to achieve the purpose that detect ALP.
The present invention eliminates the extraneous factors such as oxidation, illumination in the design of inspection principle and caused may disturb first,
Inner filtering effect with the specificity for substantially increasing detection method.It is low to sample requirement in the consumption of sample, it need not complexity
Pretreatment process, detection can be directly seen to serum, dosage is few, it is only necessary to which 20 μ L (one bleeds) blood serum sample can not only mitigate
The body ＆ mind burden of patient, and for needing to obtain a variety of Serum informations from same sample in clinical research and diagnosis
Practical application for, have important practical value.Easy experimental implementation, solution is prepared and fast reaction process, can be with
Manpower consumption is greatlyd save, improves detection efficiency.Good sensitivity and the rate of recovery, it is ensured that the accuracy of inspection result, it can
Realize the quick measure to alkaline phosphatase.
Description of the drawings
Fig. 1 is that ALP detects working curve and standard curve in embodiment 4；
Fig. 2 detects ALP vigor for CuInS/ZnS quantum dots in PNPP systems；
Fig. 3 is interference result of the interfering ion to ALP detection methods of the present invention；
Fig. 4 is interference albumen or amino acid etc. to the interference result to ALP detection methods of the present invention.
Specific embodiment
The present invention provides a kind of preparation methods of CuInS/ZnS quantum dots, comprise the following steps：
(1) copper source compound, indium source compound and sulfydryl aliphatic acid with water are mixed, adjusts pH value to 8~10, mixed
Close liquid；
Wherein, Cu2+、In3+Molar ratio is 0.015~0.045：0.015~0.120；Cu2+、In3+Mole the sum of and mercapto
The molar ratio of base aliphatic acid is 0.03~0.06：0.24~0.90；
(2) under stirring condition, the mixed liquor that step (1) obtains is mixed with sulphur source compound, Microwave-assisted firing makes to mix
Solution temperature is warming up to 90~100 DEG C within 4min after conjunction, keeps 1~10min of temperature, obtains the reaction containing CuInS cores
Liquid；
(3) after the reaction solution that step (2) obtains is cooled to less than 50 DEG C, zinc source solution and sulphur source are added in into reaction solution
Solution, 1~10min of microwave heating reaction, obtains CuInS/ZnS quantum dots at 90~100 DEG C.
In the present invention, copper source compound is preferably cupric iodide or copper chloride, and the indium source compound is preferably acetic acid
Indium or inidum chloride, the sulfydryl aliphatic acid are preferably sulfydryl aliphatic acid of the backbone length for 2~8 carbon atoms, more preferably mercapto
Base propionic acid (MPA).In the present invention, it is preferred to sulfydryl aliphatic acid and water mix respectively with copper source compound, indium source compound.Respectively
During mixing, preferably the amount of sulfydryl aliphatic acid and water each accounts for the 30%~60% of its total amount.
In the present invention, Cu2+、In3+Molar ratio be 0.015~0.045：0.015~0.120, more preferably 0.02~
0.04：0.02~0.10.The quantum dot maximum excitation wavelength that above-mentioned copper and indium molar ratio in the present invention synthesizes in 405nm,
It is Chong Die with the maximal ultraviolet absorption peak (405nm) of PNP substrates, and then generate inner filtering effect.In mixed liquor of the present invention, Cu2+
Molar content be preferably 0.015~0.045mmol, more preferably 0.020~0.040mmol.
In the present invention, Cu2+、In3+Mole the sum of with the molar ratio of sulfydryl aliphatic acid be 0.03~0.06：0.24~
0.90；More preferably 0.04~0.05：0.4~0.6.Sulfydryl aliphatic acid plays the role of stabilizer ligand in the reaction, stablizes
Agent ligand can be understood as a kind of bridging agent, that is, using sulfydryl and the combination on metal ion surface, increase the polynary amount
The stability of son point.
Since mercaptopropionic acid has reproducibility, preferably first copper source compound is dissolved in ammonia spirit before reaction,
Cu2+(copper ion) and excess of ammonia water react, and form four ammino copper (II) complex ions, and ammino-complex is relatively stablized, not with sig water
Effect, it is possible thereby to ensure Cu2+Not by sulfydryl fatty acid reduction in synthetic reaction, from the influence of pH value of solution.In the present invention,
It is preferred that the mass concentration of ammonium hydroxide is 20~35%, more preferably 23~30%.The Cu2+Molar ratio with ammonium hydroxide is preferably 1~
4：4~9；More preferably 2~3：6~8.
In the present invention, adjust the mixture ph of the compound containing copper and indium cation and sulfydryl aliphatic acid and water to 8~
10, more preferably 9, make nuclear reaction carry out at the appropriate ph.In the present invention, the pH of mixture is preferably adjusted with NaOH solution
Value, the concentration of further preferred NaOH solution is 0.5~2M.
In the present invention, under stirring condition, the mixed liquor that step (1) obtains is mixed with sulphur source compound, at 90~100 DEG C
1~10min of lower reaction, obtains the reaction solution containing CuInS cores.It is preferred that the sulphur source compound is Na2S, further preferably
Na2S solution.The Na2S in S solution2-Molar content be preferably 0.02~0.06mmol, more preferably 0.03~
0.04mmol.The present invention is not particularly limited the additive amount of sulphur source compound, preferably Cu2+、In3+The sum of the amount of substance with
The molar ratio of S is 0.02~0.14 in sulphur source compound：0.03~0.04, more preferably 0.04~0.06：0.03~0.04.This
Invention is not particularly limited the condition of stirring, is preferably stirred under 200~500 rpms of rate, more preferably stirs
Rate is mixed as 300~400 rpms.
In the present invention CuInS karyomorphisms into reaction temperature use Microwave-assisted firing so that the temperature of reaction solution rises rapidly
Temperature is to required temperature.In the present invention, the reaction solution of copper and indium and sulphur source compound is warming up to reaction temperature within 4min, more preferably
To be warming up to reaction temperature in 1~3min；The temperature of the reaction is 90~100 DEG C, more preferably 95 DEG C.When using microwave
During auxiliary heating, if power is too low, required temperature can not be quickly warming up to (in 4min), the extension of heating-up time to react molten
Cationic presoma in liquid continues to be reduced, and causes initial Cu2+:In3+Ratio changes, and can not finally obtain ideal
Quantum dot；If power is too big, heating is too fast, and ageing time is inadequate, and crystal is not grown fully, can not equally obtain quantum dot.
Preferred microwave power is 500~900W in the present invention, more preferably 700~800W, can realize be warming up to 90 in 4min~
100℃。
In the present invention, the reaction time for preparing CuInS cores is 1~10min.Reaction time too short quantum dot yield bottom, it is glimmering
Luminous intensity is weak, and overlong time can make quantum dot synthesis failure, completely without fluorescence.In the present invention, preferred reaction time for 3~
8min, more preferably 5min.CuInS cores are obtained after the completion of reaction.
After the reaction solution containing CuInS cores is cooled to less than 50 DEG C, adds in zinc source solution into reaction solution and sulphur source is molten
Liquid reacts 1~10min at 90~100 DEG C, obtains CuInS/ZnS quantum dots.Mode of heating uses microwave radiation technology in this step
Heating, preferable reaction temperature are 93~97 DEG C, and the reaction time is preferably 3~8min, more preferably 5min.The present invention is to this step
In heating rate be not particularly limited, reaction temperature is preferably warming up in 4min.
In the present invention, zinc source and sulphur source and the ratio of CuInS cores are without considered critical.In the present invention, zinc source solution is
Zn(Ac)2Solution, the sulphur source solution are Na2S solution is respectively that quantum dot provides zinc and sulphur source, and then forms CuInS/ZnS
Quantum dot.It is preferred that Zn+And S2-Molar ratio be 1~2：1~4, more preferably 1：1.It is preferred that Zn in zinc source and sulphur source solution+And S2-
Total addition for 0.01~0.05mmol, more preferably 0.02~0.04mmol.
The CuInS/ZnS quantum dots that the present invention is prepared, optimum excitation wave a length of 405nm, launch wavelength 588nm.
Its maximum excitation wavelength is Chong Die with the maximal ultraviolet absorption peak (405nm) of PNP substrates, therefore can generate inner filtering effect, will
CuInS/ZnS quantum dots, which are used as, to be applied in alkaline phosphatase enzyme hydrolysis 4-NPP system, and then for detecting alkali
Acid phosphatase.
The present invention provides a kind of method of namo fluorescence probe detection of alkaline phosphatase, in alkaline phosphatase enzyme hydrolysis 4- nitre
In the system of base benzenephosphonic acid disodium, with CuInS/ZnS quantum dots as probe, measured under the conditions of λ ex/ λ em=405/588nm
Fluorescence intensity by the variation of fluorescence intensity and alkaline phosphatase and the linear relationship of fluorescence intensity, obtains alkaline phosphatase
Content；The optimum excitation wave a length of 405nm, launch wavelength 588nm of the CuInS/ZnS quantum dots.
Alkaline phosphatase (ALP) the hydrolysis 4-NPP of the present invention obtains paranitrophenol (PNP), and PNP exists
There is strong ultraviolet absorption peak at 405nm.The maximum excitation wavelength of CuInS/ZnS quantum dots of the present invention and the maximum substrate of PNP
Overlap of peaks is absorbed, so as to generate inner filtering effect.Using PNPP as substrate, CuInS/ZnS is ALP hydrolysis productions in the system of probe
Exciting lights of the raw PNP due to absorbing 405nm causes CuInS/ZnS that fluorescent quenching occurs, so as to reach the mesh of detection ALP
's.
In the present invention, in the system of the alkaline phosphatase enzyme hydrolysis 4-NPP, 4-NPP
As substrate, hydrolysis under the action of alkaline phosphatase obtains paranitrophenol.It is produced by CuInS/ZnS quantum dots and paranitrophenol
Raw inner filtering effect generates fluorescence, according to the strong and weak content for measuring alkaline phosphatase of fluorescence intensity.Alkaline phosphatase of the present invention
In the system for hydrolyzing 4-NPP, the molar concentration of substrate 4-NPP is preferably 0.5~2mM, more
Preferably 1mM.Preferably also contain MgSO in the system of alkaline phosphatase enzyme hydrolysis 4-NPP of the present invention4。MgSO4Make
For the kinases of alkaline phosphatase, promote the dephosphorylized progress of hydrolysis.Those skilled in the art can be according to this field
Routine operation adds MgSO4.In the specific embodiment of the invention, preferably MgSO4Concentration in hydrolyzation system is 0.1 μM.
In the system of alkaline phosphatase enzyme hydrolysis 4-NPP of the present invention, the CuInS/ZnS quantum dots
As probe in above-mentioned system, alkaline phosphatase is checked by the fluorescence intensity of quantum dot.It is preferred that CuInS/ZnS amounts
Mass concentration of the son point in hydrolyzation system is 0.05~2.0mg/mL, further preferably 0.1~1.0mg/mL.
The present invention is not particularly limited the method for fluoroscopic examination, is using the conventional fluorescent detection method in this field
Can, fluoroscopic examination is preferably carried out in fluorescence pond.
The present invention is by setting series of alkaline phosphatase concentration, using the above method, in alkaline phosphatase enzyme hydrolysis 4- nitros
CuInS/ZnS quantum dots are added in the system of benzenephosphonic acid disodium as probe, in detection architecture under difference alkaline phosphatase concentrations
Fluorescence intensity, so as to obtain the linear relationship of alkaline phosphatase and fluorescence intensity.Obtained linear equation is：(F0- F)=
38.148 [ALP]+253.2, wherein F be add in ALP detection architecture fluorescence intensity level, F0It is glimmering to be free of the detection architecture of ALP
Light intensity value, correlation coefficient r=0.9984.
Understand to make the object, technical solutions and advantages of the present invention clearer, with reference to embodiment to the present invention into
Row detailed description, but they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
CuInS/ZnS quantum dots are synthesized using Microwave-assisted firing method
At room temperature by 0.025mmol InCl3It is dissolved in 20mL distilled waters, uses with 0.2mmol mercaptopropionic acids (MPA)
1mol/L NaOH solutions adjust pH value to 8；Add 0.5mL 0.015mmol CuCl2Ammonia spirit and 0.3mmol
MPA；Then 0.04mmol Na are added in vigorous stirring2S solution, using Microwave-assisted firing under 800W power in 1min
Solution temperature is warming up to 100 DEG C, 5min is kept, obtains CuInS cores.Stop after heating is cooled to less than 50 DEG C, then to molten
1mL 0.02M Zn (Ac) are slowly added into liquid2Solution and 1mL 0.02MNa2S solution, the again microwave heating under 800W, 1min
It is interior that temperature is warming up to 100 DEG C, 5min is kept, to the end of reaction, obtains final product CuInS/ZnS quantum dots.
Embodiment 2
CuInS/ZnS quantum dots are synthesized using Microwave-assisted firing method
At room temperature by 0.06mmol InCl3It is dissolved in 20mL distilled waters, uses with 0.5mmol mercaptopropionic acids (MPA)
1MNaOH solution adjusts pH value to 10；Add 0.5mL 0.025mmol CuCl2Ammonia spirit and 0.4mmol MPA；So
Add in 0.02mmolNa with vigorous stirring afterwards2S solution, using Microwave-assisted firing under 700W power in 3min by solution temperature
Degree is warming up to 95 DEG C, keeps 8min, obtains CuInS cores.After stopping heating being cooled to less than 50 DEG C, then slowly add into solution
Enter 1mL 0.01M Zn (Ac)2Solution and 1mL 0.01MNa2S solution, the again microwave heating under 700W, 3min are interior by temperature liter
Temperature keeps 8min to 95 DEG C, to the end of reaction, obtains final product CuInS/ZnS quantum dots.
Embodiment 3
CuInS/ZnS quantum dots are synthesized using Microwave-assisted firing method
At room temperature by 0.12mmol InCl3It is dissolved in 20mL distilled waters, uses with 0.8mmol mercaptopropionic acids (MPA)
1MNaOH solution adjusts pH value to 9；Add 0.5mL 0.05mmol CuCl2Ammonia spirit and 0.6mmol MPA；Then
0.05mmolNa is added in vigorous stirring2S solution, using Microwave-assisted firing under 600W power in 3min by solution temperature
90 DEG C are warming up to, 10min is kept, obtains CuInS cores.After stopping heating being cooled to less than 50 DEG C, then slowly add into solution
Enter 1mL 0.01M Zn (Ac)2Solution and 1mL 0.02MNa2S solution, the again microwave heating under 600W, 3min are interior by temperature liter
Temperature keeps 10min to 90 DEG C, to the end of reaction, obtains final product CuInS/ZnS quantum dots.It has changed
Embodiment 4
The foundation of CuInS/ZnS quantum dots characterization ALP standard curves
Prepare the ALP standard solution of following concentration：0.01st, 0.025,0.05,0.1,1,2,5,10,20,50 and 100U L-1。
The ALP standard solution of the 20 above-mentioned various concentrations of μ L is taken, is added to containing 1mM PNPP and 0.1 μM of MgSO4(ALP swashs
Enzyme) 1mL 0.1mg mL-1In CuInS/ZnS quantum dot solutions, after reacting 30min, with 1cm fluorescence pond, in λ ex/ λ em=
Under the conditions of 405/588nm, measure fluorescence intensity and record fluorescence spectrum, in 0.01~100.00U L-1In the range of drawing it is bent
Line.
Working curve the result is shown in Figure 1.Show fluorescence intensity with ALP concentration in 1.00~20.00U L by Fig. 1-1In the range of
In a linear relationship, linear equation is：(F0- F)=38.148 [ALP]+253.2, wherein F be the detection architecture fluorescence for adding in ALP
Intensity value, F0To be free of the detection architecture fluorescence intensity level of ALP.Correlation coefficient r=0.9984, detection are limited to 0.001U L-1。
Embodiment 5
The detection of blood serum sample
After 20 μ L human serum samples is taken to dilute 50 times, 0.2~10U L are added in-1ALP solution, after abundant mixing.Take 20 μ L
Above-mentioned mixed liquor is added directly into containing 1mM PNPP and 0.1 μM of MgSO4The 1mL 0.1mg mL of (ALP kinases)-1CuInS/
In ZnS quantum dot solution, after reacting 30min, with 1cm fluorescence pond, in λex/λemUnder the conditions of=405/588nm, it is strong to measure fluorescence
It spends and records fluorescence spectrum.It the results are shown in Table 1.Table 1 the results show that the rate of recovery of the method for the present invention in 81.5~94.8% scopes,
Illustrate the method accurately and reliably, can be used for clinical analysis.
The measure (n=3) of ALP activity in 1 human serum of table
Embodiment 6
The antijamming capability experiment of CuInS/ZnS quantum dots characterizations ALP methods of the present invention
(1) antijamming capability of internal ion that may be present is tested：20 μ L human serum samples are taken, use pH7.4
Tris-HCl solution dilute 50 times after, the dilution of equal portions 1mL is taken, respectively at adding in 0.1 μM in the dilution of every part of 1mL
Containing Mg2+、Na+、K+、Ca2+、Cd2+、Mn2+、Cu2+And Fe3+Solution, after abundant mixing, 30min is reacted, with 1cm fluorescence pond, in λ
Under the conditions of ex/ λ em=405/588nm, measure fluorescence intensity and record fluorescence spectrum.The result is shown in Fig. 3.Abscissa is interfering ion
And ALP, ordinate F0/F(F0For blank system fluorescence intensity, F is the fluorescence intensity for adding in disturbance object).
(2) antijamming capability of albumen that may be present, enzyme, amino acid and other substances in vivo is tested：Take 20 μ
L human serum samples after diluting 50 times with the Tris-HCl solution of pH7.4, take the dilution of equal portions 1mL, respectively at every part of 1mL's
10U L are added in dilution-1Tyrosinase (Tyr), horseradish peroxidase (HRP), glucose oxidase (GOX), half Guang ammonia
Sour (Cys), acetylcholinesterase (AChE), ascorbic acid (AA), glutathione (GSH), bovine serum albumin(BSA) (BSA) and IgG
Solution, after abundant mixing, react 30min, with 1cm fluorescence pond, under the conditions of λ ex/ λ em=405/588nm, it is strong to measure fluorescence
It spends and records fluorescence spectrum.The result is shown in Fig. 4.Abscissa is interference albumen, enzyme, amino acid, other substances and ALP, ordinate F0/
F(F0For blank system fluorescence intensity, F is the fluorescence intensity for adding in disturbance object).
Interference experiment the result shows that, the detection method antijamming capability based on inner filtering effect established is good, deposits in vivo
Each ion and the substances such as albumen, amino acid do not interfere with the measurement result of ALP, it is direct actual blood sample can be met
The needs of measure.
Embodiment 7
The inspection method of the present invention and the detection method of the prior art compare
The fluorescence probe system detection method reported in the prior art is as follows：
[1]W.Na,S.Liu,X.Liu,X.Su,Ultrasensitive detection of amifostine and
alkaline phosphatase based on the growth ofCdS quantum dots,Talanta 144(2015)
1059-1064.
[2]L.Zhang,J.Zhao,M.Duan,H.Zhang,J.Jiang,R.Yu,Inhibition of dsDNA-
templated copper nanoparticles by pyrophosphate as a label-free fluorescent
strategy for alkaline phosphatase assay,Anal Chem 85(2013)3797-3801.
[3]Y.Chen,W.Li,Y.Wang,X.Yang,J.Chen,Y.Jiang,et al.,Cysteine-directed
fluorescent gold nanoclusters for the sensing of pyrophosphate and alkaline
phosphatase,Journal ofMaterials Chemistry C 2(2014)4080-4085.
[4]Y.Zhu,G.Wang,H.Jiang,L.Chen,X.Zhang,One-step ultrasonic synthesis
ofgraphene quantum dots with high quantum yield and their application in
sensing alkaline phosphatase,Chemical Communications 51(2015)948-951.
The effect of above-mentioned detection method and ALP fluorescence probes system of the present invention is compared, the results are shown in Table 3.
Performance comparison of the ALP fluorescence probes system of 2 present invention of table with reporting fluorescence probe system before
As can be seen from Table 2, detection method of the invention can directly measure blood serum sample, detect the corresponding time
Short, detection limit is low, ALP quickly can be measured, high sensitivity.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of preparation method of CuInS/ZnS quantum dots, which is characterized in that comprise the following steps：
(1) soluble copper source compound, indium source compound and sulfydryl aliphatic acid with water are mixed, adjusts pH value to 8~10, obtain
Mixed liquor；
In the mixed liquor, Cu2+And In3+Molar ratio be 0.015~0.045：0.015~0.120；Cu2+And In3+Substance
The sum of amount and the molar ratio of sulfydryl aliphatic acid be 0.03~0.06：0.24~0.90；
(2) under stirring condition, the mixed liquor that the step (1) obtains is mixed with sulphur source compound, Microwave-assisted firing makes to mix
Solution temperature is warming up to 90~100 DEG C within 4min after conjunction, keeps 1~10min of thermotonus, obtains containing CuInS cores
Reaction solution；
(3) after the reaction solution that step (2) obtains is cooled to less than 50 DEG C, add in zinc source solution into reaction solution and sulphur source is molten
Liquid, 1~10min of microwave heating reaction, obtains CuInS/ZnS quantum dots at 90~100 DEG C.
2. preparation method according to claim 1, which is characterized in that in step (1), copper source compound is cupric iodide
Or copper chloride, the indium source compound are indium acetate or inidum chloride, the sulfydryl aliphatic acid is that backbone length is 2~8 carbon originals
The sulfydryl aliphatic acid of son.
In step (2), the sulphur source compound is Na2S。
3. preparation method according to claim 2, which is characterized in that in step (1), the Cu2+Molar content be
0.015~0.045mmol；In step (2), the Na2The molar content of S is 0.02~0.06mmol.
4. preparation method according to claim 1, which is characterized in that in the step (3), zinc source solution is Zn
(Ac)2Solution, the sulphur source solution are Na2S solution.
5. preparation method according to claim 1, which is characterized in that copper source compound is also wrapped before being mixed
It includes：Copper source compound is dissolved in ammonia spirit.
6. the CuInS/ZnS quantum dots that Claims 1 to 5 any one the method is prepared, which is characterized in that described
For CuInS/ZnS quantum dots using CuInS as core, ZnS is shell, a length of 405nm of optimum excitation wave of the CuInS/ZnS quantum dots,
Launch wavelength is 588nm.
A kind of 7. method of namo fluorescence probe detection of alkaline phosphatase, which is characterized in that using CuInS/ZnS quantum dots as alkali
Probe in acid phosphatase hydrolysis 4-NPP system, measures hydrolysis body under the conditions of λ ex/ λ em=405/588nm
The fluorescence intensity of system by the fluorescence intensity and alkaline phosphatase of measure and the linear relationship of fluorescence intensity, obtains alkaline phosphorus
The content of sour enzyme；
The optimum excitation wave a length of 405nm, launch wavelength 588nm of the CuInS/ZnS quantum dots.
8. the method according to the description of claim 7 is characterized in that the CuInS/ZnS quantum dots are in the alkaline phosphatase
It is 0.05~2.0mg/mL to hydrolyze the mass concentration in 4-NPP system.
9. the method according to the description of claim 7 is characterized in that alkaline phosphatase enzyme hydrolysis 4-NPP
In system, the molar concentration of substrate 4-NPP is 0.5~2mM.
10. the method according to the description of claim 7 is characterized in that alkaline phosphatase enzyme hydrolysis 4-NPP
System in also contain MgSO4。
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107652966A (en) * | 2017-10-16 | 2018-02-02 | 中南民族大学 | A kind of normal pressure aqueous phase preparation method of hydrophily Cu In S/ZnS core-shell type nano crystal |
| CN109239036A (en) * | 2018-09-26 | 2019-01-18 | 北京化工大学 | A kind of nitrophenol isomers detection array |
| CN109609125A (en) * | 2019-02-20 | 2019-04-12 | 潍坊科技学院 | CDs/SiO2/ AuNCs ratio fluorescent probe, preparation method and application |
| CN109781976A (en) * | 2018-12-27 | 2019-05-21 | 中国农业大学 | Fluorescence immune analysis method based on carbon quantum dot |
| CN110927132A (en) * | 2019-12-11 | 2020-03-27 | 南京医科大学 | Preparation method of ratio-type fluorescent probe for quantitatively detecting ALP in serum |
| CN111965150A (en) * | 2020-07-30 | 2020-11-20 | 济南大学 | Method for fluorescence detection of alkaline phosphatase based on in-situ generated 2, 3-diaminophenazine |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102081040A (en) * | 2009-11-27 | 2011-06-01 | 中国科学院理化技术研究所 | Method for detecting enzymatic activity by using quantum dot fluorescence |
| CN102277158A (en) * | 2011-06-13 | 2011-12-14 | 天津大学 | CuInS2 quantum dots with water-soluble zinc blende structure, CuInS2/ZnS core shell quantum dots with water-soluble zinc blende structure and preparation method thereof |
| CN104316503A (en) * | 2014-10-29 | 2015-01-28 | 安徽师范大学 | Use and detection method of sensor based on grapheme quantum dot (GQDs) |
-
2017
- 2017-12-22 CN CN201711402342.6A patent/CN108084995B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102081040A (en) * | 2009-11-27 | 2011-06-01 | 中国科学院理化技术研究所 | Method for detecting enzymatic activity by using quantum dot fluorescence |
| CN102277158A (en) * | 2011-06-13 | 2011-12-14 | 天津大学 | CuInS2 quantum dots with water-soluble zinc blende structure, CuInS2/ZnS core shell quantum dots with water-soluble zinc blende structure and preparation method thereof |
| CN104316503A (en) * | 2014-10-29 | 2015-01-28 | 安徽师范大学 | Use and detection method of sensor based on grapheme quantum dot (GQDs) |
Non-Patent Citations (2)
| Title |
|---|
| LAN JIA ET AL.: "Fluorescence detection of alkaline phosphatase activity with β-cyclodextrin-modified quantum dots", 《CHEM. COMMUN.》 * |
| WEI-WEI XIONG ET AL.: "Aqueous Synthesis of Color-Tunable CuInS2/ZnS Nanocrystals for the Detection of Human Interleukin 6", 《ACS APPLIED MATERIALS & INTERFACES》 * |
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| CN107652966B (en) * | 2017-10-16 | 2020-04-10 | 中南民族大学 | Normal-pressure water phase preparation method of hydrophilic Cu-In-S/ZnS core-shell type nanocrystal |
| CN109239036A (en) * | 2018-09-26 | 2019-01-18 | 北京化工大学 | A kind of nitrophenol isomers detection array |
| CN109781976A (en) * | 2018-12-27 | 2019-05-21 | 中国农业大学 | Fluorescence immune analysis method based on carbon quantum dot |
| CN109609125A (en) * | 2019-02-20 | 2019-04-12 | 潍坊科技学院 | CDs/SiO2/ AuNCs ratio fluorescent probe, preparation method and application |
| CN110927132A (en) * | 2019-12-11 | 2020-03-27 | 南京医科大学 | Preparation method of ratio-type fluorescent probe for quantitatively detecting ALP in serum |
| CN110927132B (en) * | 2019-12-11 | 2022-03-25 | 南京医科大学 | Preparation method of ratio-type fluorescent probe for quantitatively detecting ALP in serum |
| CN111965150A (en) * | 2020-07-30 | 2020-11-20 | 济南大学 | Method for fluorescence detection of alkaline phosphatase based on in-situ generated 2, 3-diaminophenazine |
| CN111965150B (en) * | 2020-07-30 | 2022-12-20 | 济南大学 | Method for fluorescence detection of alkaline phosphatase based on in-situ generated 2, 3-diaminophenazine |
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