CN105403539A - Method for using frequency doubling scattering process for detecting hemoglobin by taking fluorescent carton spot as probe - Google Patents
Method for using frequency doubling scattering process for detecting hemoglobin by taking fluorescent carton spot as probe Download PDFInfo
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- CN105403539A CN105403539A CN201510727512.2A CN201510727512A CN105403539A CN 105403539 A CN105403539 A CN 105403539A CN 201510727512 A CN201510727512 A CN 201510727512A CN 105403539 A CN105403539 A CN 105403539A
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- carbon point
- fluorescent carbon
- haemoglobin
- frequency multiplication
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/47—Scattering, i.e. diffuse reflection
- G01N21/49—Scattering, i.e. diffuse reflection within a body or fluid
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Abstract
The invention discloses a method for using a frequency doubling scattering process for detecting hemoglobin by taking a fluorescent carton spot as a probe. The method comprises the following steps: stimulating a compound of fluorescent carton spots and hemoglobin under the condition of excitation wavelength being 650nm, thereby generating frequency doubling scattered light, and then detecting the strength of the frequency doubling scattered light, comparing the strength of the frequency doubling scattered light with a standard curve of the hemoglobin concentration established under a same detecting condition, thereby acquiring the hemoglobin concentration. According to the method provided by the invention, the fluorescent carton spot is taken as the probe and the characteristic that the strength of the frequency doubling scattered light increases along with the increasing of the hemoglobin concentration in the frequency doubling scattering process is utilized to perform high-sensitivity detection; the method is simple in operation, quick in detection, high in sensitivity and excellent in selectivity; the on-line in-situ quick sensitive detection can be performed on the hemoglobin in a mixed sample; and the detection limit can reach 1.27*10<-11>mol/L.
Description
Technical field
The present invention relates to a kind of detection method of hemoglobin concentration.More particularly, the present invention relates to a kind of method utilizing fluorescent carbon point frequency multiplication scattered light to detect hemoglobin concentration.
Background technology
Haemoglobin is present in red blood cell in a large number, and haemoglobin has powerful transport O
2and CO
2function.It can carry out reversible combination with oxygen.When partial pressure of oxygen is high (as in lung), haemoglobin is easily combined with oxygen, and when partial pressure of oxygen is low (as in various tissue), it easily releases again oxygen, which provides a kind of effective method by each cell from Atmospheric Transportation to health of oxygen.Meanwhile, the final product CO of metabolism transports out external by haemoglobin.
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.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.
Up to now, have been reported the concentration being used for fluorescent carbon point to detect as probe haemoglobin, it utilizes haemoglobin quencher carbon point fluorescent characteristic, detect, but its detection limit is not very low to the concentration of haemoglobin.
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.
It is that probe uses frequency multiplication scattering method to detect the method for haemoglobin with fluorescent carbon point that a further object of the invention is to provide a kind of, it utilizes haemoglobin can strengthen the character of fluorescent carbon point frequency multiplication scattered light intensity, detect fast the concentration of haemoglobin, detection limit can reach 1.27 × 10
-11mol/L.
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 the method for haemoglobin with fluorescent carbon point, comprising:
Be under the condition of 650nm in excitation wavelength, the bond of fluorescent carbon point and haemoglobin is excited, to produce frequency multiplication scattered light, the intensity detecting frequency multiplication scattered light selects optimum excitation wavelength, the typical curve of the hemoglobin concentration set up under the intensity of described frequency multiplication scattered light and same detection condition is compared, to obtain hemoglobin concentration.
Preferably, wherein, the method for building up of the typical curve of hemoglobin concentration, is specially:
Preparation is containing the haemoglobin standard solution of the variable concentrations of fluorescent carbon point, be under the condition of 650nm in optimum 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 hemoglobin concentration, obtain the typical curve of hemoglobin concentration, for detecting hemoglobin concentration in sample solution.
Preferably, wherein, containing the compound method of the haemoglobin standard solution of the variable concentrations of fluorescent carbon point, be specially:
Get the haemoglobin 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 haemoglobin standard solution of equal-volume containing the variable concentrations of fluorescent carbon point.
Preferably, wherein, the method preparing described sample solution is:
In unknown hemoglobin solutions, 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 10.7mg/mL.
Preferably, wherein, the volume of described standard solution and described sample solution is 3mL.
Preferably, wherein, the concentration of described haemoglobin 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 utilizes the characteristic that the frequency multiplication scattered light intensity of fluorescent carbon point strengthens with the increase of the concentration of hemoglobin solutions, carry out highly sensitive detection, the method is simple to operate, detect quick, highly sensitive and selectivity is good, can carry out online original position rapid sensitive to haemoglobin in biased sample to detect, detection limit can reach 1.27 × 10
-11mol/L.
2, fluorescent carbon point provided by the invention has that cytotoxicity is low, the advantage of good biocompatibility, and effectively can carry out specific binding with haemoglobin, generate a kind of bond, this bond can carry out transition under the exciting of the light of certain wavelength, and produce frequency multiplication scattered light, by detecting the intensity of frequency multiplication scattered light, and then obtain the concentration of haemoglobin.
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 hemoglobin 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 hemoglobin concentration is set up
1, preparation is containing the haemoglobin standard solution of the variable concentrations of fluorescent carbon point, is specially:
The concentration of getting different volumes is 3 × 10
-4the haemoglobin 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 haemoglobin standard solution of equal-volume containing the variable concentrations of fluorescent carbon point, in this standard solution, the concentration of haemoglobin is respectively 0,3 × 10
-10mol/L, 7 × 10
-9mol/L, 3 × 10
-8mol/L, 7 × 10
-8mol/L, 1.5 × 10
-7mol/L, is labeled as 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 650nm, standard solution is excited, thus produce frequency multiplication scattered light, the frequency multiplication scattered light intensity of examination criteria solution, frequency multiplication scattered light spectrogram as shown in Figure 1, can learn that from frequency multiplication scattered light spectrogram the concentration of the frequency multiplication scattered light signal intensity hemoglobin solutions of fluorescent carbon point increases and strengthens, and be that top has appearred in 327nm 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 hemoglobin concentration, related coefficient is 0.998, obtain the typical curve (as shown in Figure 2) of hemoglobin 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 the concentration of haemoglobin.
Three, hemoglobin concentration in sample solution is detected
1, prepare sample solution, be specially:
In unknown hemoglobin solutions, 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 the concentration of haemoglobin 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 hemoglobin concentration are compared, obtain hemoglobin 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 hemoglobin concentration is set up
1, preparation is containing the haemoglobin standard solution of the variable concentrations of fluorescent carbon point, is specially:
The concentration of getting different volumes is 3 × 10
-5the haemoglobin 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 haemoglobin standard solution of equal-volume containing the variable concentrations of fluorescent carbon point, in this standard solution, the concentration of haemoglobin is respectively 0,3 × 10
-10mol/L, 7 × 10
-9mol/L, 3 × 10
-8mol/L, 7 × 10
-8mol/L, 1.5 × 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 650nm in excitation wavelength, standard solution is excited, thus produce the frequency multiplication scattered light that wavelength is 327nm, the frequency multiplication scattered light intensity of examination criteria solution, the linear relationship between the frequency multiplication scattered light intensity of Criterion solution and hemoglobin concentration, related coefficient is 0.997, obtain the typical curve of hemoglobin concentration, for detecting hemoglobin concentration in sample solution.
Three, hemoglobin concentration in sample solution is detected
1, prepare sample solution, be specially:
In unknown hemoglobin solutions, 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 the concentration of haemoglobin 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 hemoglobin concentration are compared, obtain hemoglobin 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 hemoglobin solutions, carry out highly sensitive detection, the frequency multiplication scattered light intensity changing value of fluorescent carbon point and hemoglobin 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 haemoglobin 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 method for haemoglobin with fluorescent carbon point, it is characterized in that, comprising:
Be under the condition of 650nm in excitation wavelength, the bond of fluorescent carbon point and haemoglobin is excited, to produce frequency multiplication scattered light, detect the intensity of frequency multiplication scattered light, the typical curve of the hemoglobin concentration set up under the intensity of described frequency multiplication scattered light and same detection condition is compared, to obtain hemoglobin concentration.
2. as claimed in claim 1 is that probe uses frequency multiplication scattering method to detect the method for haemoglobin with fluorescent carbon point, and it is characterized in that, the method for building up of the typical curve of hemoglobin concentration, is specially:
Preparation is containing the haemoglobin standard solution of the variable concentrations of fluorescent carbon point, be under the condition of 650nm 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 hemoglobin concentration, obtain the typical curve of hemoglobin concentration, for detecting hemoglobin concentration in sample solution.
3. as claimed in claim 2 is that probe uses frequency multiplication scattering method to detect the method for haemoglobin with fluorescent carbon point, it is characterized in that, containing the compound method of the haemoglobin standard solution of the variable concentrations of fluorescent carbon point, is specially:
Get the haemoglobin 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 haemoglobin 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 the method for haemoglobin with fluorescent carbon point, and it is characterized in that, the method preparing described sample solution is:
In unknown hemoglobin solutions, 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 the method for haemoglobin 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 the method for haemoglobin with fluorescent carbon point, it is characterized in that, in described standard solution and described sample solution, the concentration of fluorescent carbon point is 10.7mg/mL.
7. as claimed in claim 2 is that probe uses frequency multiplication scattering method to detect the method for haemoglobin 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 the method for haemoglobin with fluorescent carbon point, and it is characterized in that, the concentration of described haemoglobin 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 the method for haemoglobin 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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Cited By (1)
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| CN114836200A (en) * | 2022-06-16 | 2022-08-02 | 郑州大学 | Composite fluorescent probe based on human serum protein, preparation method thereof and application of composite fluorescent probe in detection of ochratoxin A |
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Application publication date: 20160316 |