CN105424658A - Method for detecting trypsin with fluorescence carbon dot as probe based on frequency-doubling scattering method - Google Patents
Method for detecting trypsin with fluorescence carbon dot as probe based on frequency-doubling scattering method Download PDFInfo
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- CN105424658A CN105424658A CN201510719927.5A CN201510719927A CN105424658A CN 105424658 A CN105424658 A CN 105424658A CN 201510719927 A CN201510719927 A CN 201510719927A CN 105424658 A CN105424658 A CN 105424658A
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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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Abstract
The invention discloses a method for detecting trypsin with a fluorescence carbon dot as a probe based on the frequency-doubling scattering method. The detecting method comprises the steps of exciting a fluorescence carbon dot and trypsin compound under the excitation wavelength of 690 nm so as to generate frequency-doubling scattering light, detecting the intensity of the frequency-doubling scattering light, and comparing the intensity of the frequency-doubling scattering light with a trypsin concentration standard curve built under the same detection condition so as to obtain the concentration of trypsin. According to the detecting method, the fluorescence carbon dot is taken as the probe, and high-sensitivity detection of the concentration of trypsin is achieved by means of the feature of the frequency-doubling scattering method that the intensity of the frequency-doubling scattering light of the fluorescence carbon dot increases along with the increase of the concentration of a trypsin solution. The detecting method is easy to operate, quick in detection, high in sensitivity and high in selectivity. Online in-situ quick sensitive detection of trypsin in a mixed sample can be achieved, and detection limit can reach 7.8*10-10 mol/L.
Description
Technical field
The present invention relates to a kind of detection method of trypsinase concentration.More particularly, the present invention relates to one utilizes fluorescent carbon point frequency multiplication scattered light to detect tryptic method.
Background technology
Trypsase is the one of proteinase, works as digestive ferment.Be synthesized at the precursor trypsinogen of pancreas as enzyme.As pancreatic juice composition and secrete, by enterokinase, or activation trypsase is decomposed in tryptic restriction, is endopeptidase, it can in lysine in polypeptied chain and arginine residues carboxyl side cut off.It not only plays the effect of digestive ferment, and can also limit the precursor decomposing other enzymes such as chymotrypsinogen, procarboxypeptidase, phosphatide proenzyme, plays activation.Be the proteinase that specificity is the strongest, in the amino acid range determining protein, it becomes indispensable instrument.
In recent years, fluorescent carbon point is one of carbon nanomaterial the most popular after fullerene, carbon nano-tube and Graphene.This nano material overcomes some shortcoming of traditional quantum dot, not only there is excellent optical property and small size property, and there is good biocompatibility, be easy to realize surface-functionalized, in fields such as biochemical sensitive, imaging analysis, environment measuring, photocatalysis technology and pharmaceutical carriers, there is good application potential.But the relevant report up to now, fluorescent carbon point fluorescence probe being used for trypsase detection has not yet to see.
Light scattering phenomenon is extensively present in the mechanism of light and particle, refers to when light is by optical phenomenon viewed in all directions during medium beyond incident light direction.It comes from the electric field oscillation of photoelectricity magnetic wave and dipole element that forced vibration that in the molecule that causes, electronics produces is formed.According to electromagnetic theory, the dipole element be vibrated is a secondary wave source, and the electromagnetic wave that it is launched to all directions is exactly scattering wave.Light scattering is relevant with the unevenness of medium, and other All Media except vacuum has unevenness to a certain degree, thus produces scattered light.Nowadays, light scattering technique progressively develops into a new analytical technology.The advantages such as this technology has easy, quick, highly sensitive, and instrumentation is convenient.The analysis being successfully applied to nucleic acid, protein, inorganic ions, immunity and medicine etc. in recent years measures.But up to now, using fluorescent carbon point as probe, utilizing trypsase to strengthen the characteristic of fluorescent carbon point frequency multiplication scattered light intensity, having not yet to see for detecting tryptic relevant report.
Summary of the invention
An object of the present invention is to solve at least the problems referred to above and/or defect, and the advantage will illustrated at least is below provided.
A further object of the invention is to provide a kind of fluorescent carbon point and detects tryptic new method, and the method is simple to operate, detects quick and highly sensitive, can carry out tryptic highly sensitive identification in solution, and detection limit is low.
A further object of the invention is the new opplication of research fluorescent carbon point in trypsinase concentration detects.
In order to realize according to these objects of the present invention and other advantage, providing a kind of is that probe uses frequency multiplication scattering method to detect tryptic method with fluorescent carbon point, comprising:
Be under the condition of 690nm in excitation wavelength, fluorescent carbon point and tryptic bond are excited, to produce frequency multiplication scattered light, the intensity detecting frequency multiplication scattered light selects optimum excitation wavelength, the typical curve of the trypsinase concentration set up under the intensity of described frequency multiplication scattered light and same detection condition is compared, to obtain trypsinase concentration.
Preferably, wherein, the method for building up of the typical curve of trypsinase concentration is specially:
Preparation is containing the trypsase standard solution of the variable concentrations of fluorescent carbon point, be under the condition of 690nm in excitation wavelength, the frequency multiplication scattered light intensity of examination criteria solution, linear relationship between the frequency multiplication scattered light intensity of Criterion solution and trypsinase concentration, obtain the typical curve of trypsinase concentration, for detecting trypsinase concentration in sample solution.
Preferably, wherein, the compound method containing the trypsase standard solution of the variable concentrations of fluorescent carbon point is specially:
Get the trypsase original solution of different volumes, and add the fluorescent carbon point of same volume respectively wherein and the buffer solution of different volumes is equal to the volume of mixed liquor, obtain the trypsase standard solution of equal-volume containing the variable concentrations of fluorescent carbon point.
Preferably, wherein, the method preparing described sample solution is:
In unknown trypsin solution, add the fluorescent carbon point of certain volume, make identical with the volume of fluorescent carbon point in described standard solution, add buffer solution to identical with the volume of described standard solution, obtain described sample solution.
Preferably, wherein, described buffer solution to be pH be 6 phosphate buffered solution.
Preferably, wherein, in described standard solution and described sample solution, the concentration of fluorescent carbon point is 6.1mg/mL.
Preferably, wherein, the volume of described standard solution and described sample solution is 3mL.
Preferably, wherein, the concentration of described trypsase stoste is 3 × 10
-4mol/L, 3 × 10
-5mol/L, 3 × 10
-6mol/L or 3 × 10
-7mol/L.
Preferably, wherein, the preparation method of described fluorescent carbon point is:
Step a, by 1.0g molecular weight be 1500 polyglycol and 15ml glycerine stir and be placed in microwave reactor, pyroreaction 15min at 140 DEG C;
Step b, the sample obtained by step a add the serine of 1.0g after being cooled to 50 DEG C, be then warming up to 180 DEG C of microwave reaction 10min;
The bag filter dialysis that step c, the sample obtained by step b inject molecular weight 1000 carried out evaporation and concentration after 24 hours, obtained the dislysate of 300ml;
Steps d, by the dislysate of described 300ml rotary evaporation one hour at 60 DEG C, obtain 260ml sample, described sample is fluorescent carbon point.
The present invention at least comprises following beneficial effect:
1, the present invention using fluorescent carbon point as probe, utilize the characteristic that the frequency multiplication scattered light intensity of fluorescent carbon point strengthens with the increase of the concentration of trypsin solution, highly sensitive detection is carried out to tryptic concentration, the method is simple to operate, detect quick, highly sensitive and selectivity is good, can carry out online original position rapid sensitive to trypsase in biased sample to detect, detection limit can reach 7.8 × 10
-10mol/L.
2, the cytotoxicity of fluorescent carbon point prepared by microwave reaction synthesis method of the present invention is low, reaction conditions gentleness is controlled, and productive rate is higher, and effectively can carry out specific binding with trypsase, generate a kind of bond, this bond can carry out transition under the exciting of the light of certain wavelength, and produces frequency multiplication scattered light, by detecting the intensity of frequency multiplication scattered light, and then obtain tryptic concentration.
Part is embodied by explanation below by other advantage of the present invention, target and feature, part also will by research and practice of the present invention by those skilled in the art is understood.
Accompanying drawing explanation
Fig. 1 is the frequency multiplication scattered light spectrogram of one embodiment of the present of invention Plays solution;
Fig. 2 is the typical curve of trypsinase concentration in one embodiment of the present of invention.
Embodiment
Below in conjunction with accompanying drawing, the present invention is described in further detail, can implement according to this with reference to instructions word to make those skilled in the art.
< embodiment 1>
One, the synthesis of fluorescent carbon point
By 1.0g molecular weight be 1500 polyglycol and 15ml glycerine stir be placed in microwave reactor, pyroreaction 15min at 140 DEG C, the serine of 1.0g is added after test tube is cooled to 50 DEG C, then 180 DEG C of microwave reaction 10min are warming up to, the bag filter dialysis of obtained sample being injected molecular weight 1000 carried out evaporation and concentration after 24 hours, obtain the dislysate of 300ml, by the dislysate of described 300ml rotary evaporation one hour at 60 DEG C, obtain 260ml sample, described sample is fluorescent carbon point.
Two, the typical curve of trypsinase concentration is set up
1, preparation is containing the trypsase standard solution of the variable concentrations of fluorescent carbon point, is specially:
The concentration of getting different volumes is 3 × 10
-4the trypsase original solution of mol/L, and phosphate buffered solution to the volume of mixed liquor that the pH of the fluorescent carbon point and different volumes that add same volume wherein is respectively 6 is 3mL, wherein, the concentration of fluorescent carbon point is 6.1mg/mL, obtain the trypsase standard solution of equal-volume containing the variable concentrations of fluorescent carbon point, in this standard solution, tryptic concentration is respectively 0,1 × 10
-9mol/L, 7 × 10
-9mol/L, 3 × 10
-8mol/L, 5 × 10
-8mol/L, 7 × 10
-8mol/L, 1 × 10
-7mol/L, is labeled as g, f, e, d, c, b and a successively.
2, the frequency multiplication scattered light intensity of examination criteria solution Criterion curve, is specially:
After above-mentioned standard solution is left standstill 3 minutes, under the condition of excitation wavelength 690nm, standard solution is excited, thus produce frequency multiplication scattered light, the frequency multiplication scattered light intensity of examination criteria solution, Fig. 1 is the frequency multiplication scattered light spectrogram of typical curve, can learn that from frequency multiplication scattered light spectrogram the concentration of the frequency multiplication scattered light signal intensity trypsin solution of fluorescent carbon point increases and strengthens, and be that top has appearred in 348nm place at wavelength, in frequency multiplication scattered light spectrogram, frequency multiplication scattered light intensity angle value corresponding to this top is just as the frequency multiplication scattered light intensity of standard solution, linear relationship between the frequency multiplication scattered light intensity of Criterion solution and trypsinase concentration, related coefficient is 0.999, obtain the typical curve (as shown in Figure 2) of trypsinase concentration, in typical curve, ordinate is the difference of the frequency multiplication scattered light intensity of one of them standard solution and the frequency multiplication scattered light intensity of standard solution a, horizontal ordinate is tryptic concentration.
Three, trypsinase concentration in sample solution is detected
1, prepare sample solution, be specially:
In unknown trypsin solution, add the fluorescent carbon point of certain volume, make identical with the volume of fluorescent carbon point in described standard solution, add pH be the phosphate buffered solution of 6 to 3mL, obtain sample solution.
2, detect the frequency multiplication scattered light intensity of sample solution, obtain tryptic concentration in sample solution
Under the testing conditions identical with examination criteria solution frequency multiplication scattered light intensity, detect the frequency multiplication scattered light intensity of sample solution, the described intensity of frequency multiplication scattered light and the typical curve of trypsinase concentration are compared, obtain trypsinase concentration.
< embodiment 2>
One, the synthesis of fluorescent carbon point
By 1.0g molecular weight be 1500 polyglycol and 15ml glycerine stir be placed in microwave reactor, pyroreaction 15min at 140 DEG C, the serine of 1.0g is added after test tube is cooled to 50 DEG C, then 180 DEG C of microwave reaction 10min are warming up to, the bag filter dialysis of obtained sample being injected molecular weight 1000 carried out evaporation and concentration after 24 hours, obtain the dislysate of 300ml, by the dislysate of described 300ml rotary evaporation one hour at 60 DEG C, obtain 260ml sample, described sample is fluorescent carbon point.
Two, the typical curve of trypsinase concentration is set up
1, preparation is containing the trypsase standard solution of the variable concentrations of fluorescent carbon point, is specially:
The concentration of getting different volumes is 3 × 10
-5the trypsase original solution of mol/L, and phosphate buffered solution to the volume of mixed liquor that the pH of the fluorescent carbon point and different volumes that add same volume wherein is respectively 6 is 3mL, wherein, the concentration of fluorescent carbon point is 10.7mg/mL, obtain the trypsase standard solution of equal-volume containing the variable concentrations of fluorescent carbon point, in this standard solution, tryptic concentration is respectively 0,1 × 10
-9mol/L, 7 × 10
-9mol/L, 3 × 10
-8mol/L, 5 × 10
-8mol/L, 7 × 10
-8mol/L, 1 × 10
-7mol/L.
2, the frequency multiplication scattered light intensity of examination criteria solution Criterion curve, is specially:
After above-mentioned standard solution is left standstill 2 minutes, be under the condition of 690nm in excitation wavelength, standard solution is excited, thus produce the frequency multiplication scattered light that wavelength is 348nm, the frequency multiplication scattered light intensity of examination criteria solution, the linear relationship between the frequency multiplication scattered light intensity of Criterion solution and trypsinase concentration, related coefficient is 0.998, obtain the typical curve of trypsinase concentration, for detecting trypsinase concentration in sample solution.
Three, trypsinase concentration in sample solution is detected
1, prepare sample solution, be specially:
In unknown trypsin solution, add the fluorescent carbon point of certain volume, make identical with the volume of fluorescent carbon point in described standard solution, add pH be the phosphate buffered solution of 6 to 3mL, obtain sample solution.
2, detect the frequency multiplication scattered light intensity of sample solution, obtain tryptic concentration in sample solution
Under the testing conditions identical with examination criteria solution frequency multiplication scattered light intensity, detect the frequency multiplication scattered light intensity of sample solution, the described intensity of frequency multiplication scattered light and the typical curve of trypsinase concentration are compared, obtain trypsinase concentration.
Utilize fluorescent carbon point as probe, by the characteristic that the frequency multiplication scattered light intensity of fluorescent carbon point strengthens with the increase of the concentration of trypsin solution, carry out highly sensitive detection, the frequency multiplication scattered light intensity changing value of fluorescent carbon point and trypsinase concentration are good linear relationship.The inventive method is simple to operate, detect quick, highly sensitive and selectivity is good, can carry out online original position rapid sensitive detect trypsase in biased sample.
Although embodiment of the present invention are open as above, it is not restricted to listed in instructions and embodiment utilization.It can be applied to various applicable the field of the invention completely.For those skilled in the art, can easily realize other amendment.Therefore do not deviating under the universal that claim and equivalency range limit, the present invention is not limited to specific details and illustrates here and the legend described.
Claims (9)
1. be that probe uses frequency multiplication scattering method to detect a tryptic method with fluorescent carbon point, it is characterized in that, comprising:
Be under the condition of 690nm in excitation wavelength, fluorescent carbon point and tryptic bond are excited, to produce frequency multiplication scattered light, detect the intensity of frequency multiplication scattered light, the typical curve of the trypsinase concentration set up under the intensity of described frequency multiplication scattered light and same detection condition is compared, to obtain trypsinase concentration.
2. as claimed in claim 1 is that probe uses frequency multiplication scattering method to detect tryptic method with fluorescent carbon point, and it is characterized in that, the method for building up of the typical curve of trypsinase concentration is specially:
Preparation is containing the trypsase standard solution of the variable concentrations of fluorescent carbon point, be under the condition of 690nm in excitation wavelength, the frequency multiplication scattered light intensity of examination criteria solution, linear relationship between the frequency multiplication scattered light intensity of Criterion solution and trypsinase concentration, obtain the typical curve of trypsinase concentration, for detecting trypsinase concentration in sample solution.
3. as claimed in claim 2 is that probe uses frequency multiplication scattering method to detect tryptic method with fluorescent carbon point, and it is characterized in that, the compound method containing the trypsase standard solution of the variable concentrations of fluorescent carbon point is specially:
Get the trypsase original solution of different volumes, and add the fluorescent carbon point of same volume respectively wherein and the buffer solution of different volumes is equal to the volume of mixed liquor, obtain the trypsase standard solution of equal-volume containing the variable concentrations of fluorescent carbon point.
4. as claimed in claim 3 is that probe uses frequency multiplication scattering method to detect tryptic method with fluorescent carbon point, and it is characterized in that, the method preparing described sample solution is:
In unknown trypsin solution, add the fluorescent carbon point of certain volume, make identical with the volume of fluorescent carbon point in described standard solution, add buffer solution to identical with the volume of described standard solution, obtain described sample solution.
5. is that probe uses frequency multiplication scattering method to detect tryptic method with fluorescent carbon point as described in claim 3 or 4, it is characterized in that, described buffer solution to be pH be 6 phosphate buffered solution.
6. as claimed in claim 2 is that probe uses frequency multiplication scattering method to detect tryptic method with fluorescent carbon point, it is characterized in that, in described standard solution and described sample solution, the concentration of fluorescent carbon point is 6.1mg/mL.
7. as claimed in claim 2 is that probe uses frequency multiplication scattering method to detect tryptic method with fluorescent carbon point, and it is characterized in that, the volume of described standard solution and described sample solution is 3mL.
8. as claimed in claim 3 is that probe uses frequency multiplication scattering method to detect tryptic method with fluorescent carbon point, and it is characterized in that, the concentration of described trypsase stoste is 3 × 10
-4mol/L, 3 × 10
-5mol/L, 3 × 10
-6mol/L or 3 × 10
-7mol/L.
9. as claimed in claim 1 is that probe uses frequency multiplication scattering method to detect tryptic method with fluorescent carbon point, and it is characterized in that, the preparation method of described fluorescent carbon point is:
Step a, by 1.0g molecular weight be 1500 polyglycol and 15ml glycerine stir and be placed in microwave reactor, pyroreaction 15min at 140 DEG C;
Step b, the sample obtained by step a add the serine of 1.0g after being cooled to 50 DEG C, be then warming up to 180 DEG C of microwave reaction 10min;
The bag filter dialysis that step c, the sample obtained by step b inject molecular weight 1000 carried out evaporation and concentration after 24 hours, obtained the dislysate of 300ml;
Steps d, by the dislysate of described 300ml rotary evaporation one hour at 60 DEG C, obtain 260ml sample, described sample is fluorescent carbon point.
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2015
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| WO2011127001A2 (en) * | 2010-04-05 | 2011-10-13 | University Of Massachusetts | Quantum dot-based optical sensors for rapid detection and quantitative analysis of biomolecules and biological materials |
| CN102565020A (en) * | 2012-01-13 | 2012-07-11 | 上海师范大学 | Method for quantitatively detecting protein through quantum dot resonant scattering |
| CN103364353A (en) * | 2013-07-19 | 2013-10-23 | 广西师范大学 | Aptamer nanogold resonance Rayleigh scattering spectrum method for determination of lysozyme |
| CN104568890A (en) * | 2015-01-22 | 2015-04-29 | 广西师范学院 | Method for detecting ethidium bromide by fluorescent carbon dot probe |
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