CN115046984A - Method for detecting medicine in blood based on surface enhanced Raman spectroscopy technology - Google Patents
Method for detecting medicine in blood based on surface enhanced Raman spectroscopy technology Download PDFInfo
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- CN115046984A CN115046984A CN202210686886.4A CN202210686886A CN115046984A CN 115046984 A CN115046984 A CN 115046984A CN 202210686886 A CN202210686886 A CN 202210686886A CN 115046984 A CN115046984 A CN 115046984A
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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
- G01N21/62—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
- G01N21/65—Raman scattering
- G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/38—Diluting, dispersing or mixing samples
Abstract
The invention discloses a method for detecting a medicine in blood based on a surface enhanced Raman spectroscopy technology, relates to a method for detecting a medicine in blood, and aims to solve the technical problems of complex substrate preparation process, complicated sample pretreatment steps and poor enhancement effect of the existing surface enhanced Raman spectroscopy technology. The detection method comprises the following steps: firstly, preparing nano silver sol, wherein the particle size of silver nanoparticles is 40-60 nm; mixing the nano silver sol and a blood sample to be tested containing the drug, and then carrying out surface enhanced Raman spectroscopy test; and thirdly, comparing the target drug with the spectrogram of the pure drug, and judging whether the blood sample to be detected contains the target drug or not by contrasting the position of the characteristic peak. The detection response time of the method is within 10s, the sensitivity is as high as 1-20 ppm, and the method can be used for qualitative or quantitative rapid detection or real-time monitoring of natural medicine components and chemical synthesis medicine components in blood.
Description
Technical Field
The invention relates to a method for detecting medicines in blood, belonging to the technical field of biological detection by using a precision instrument.
Background
The invention of the medicine greatly promotes the medical health career of human beings and the rapid development of the farming and animal husbandry, and the monitoring of the type and the concentration of a certain medicine in blood is very important for the treatment of patients. Currently, there are three main types of methods for detecting the concentration of drugs in blood: spectroscopy, chromatography, and immunization. The sensitivity of the blood concentration of the spectrum method is high, the detection limit can be as low as 1ppb, but the influence of drug metabolites and compounds with similar structures on the detection is large; the chromatography has excellent qualitative and quantitative effects, the detection sensitivity can reach 1-10 ppb, but the chromatographic column has very high requirements on sample pretreatment, and the detection takes time; immunoassay instruments are expensive and the types of drugs to be detected are limited.
The detection of the drugs in blood is closely related to human health, although some existing detection methods have high sensitivity, the methods still have great improvement space in aspects of sample treatment, detection period, cost and operation complexity, and a rapid, accurate, simple to operate and low-cost blood concentration detection method is still lacked at present.
Surface Enhanced Raman Scattering (SERS) is a highly sensitive, nondestructive, and rapid detection technique, and has been applied to many studies in the field of life science, but most of the detected drugs have weak raman signals, so that a substrate with efficient surface enhancement and the drugs are required to generate raman enhancement for detection. The enhancement effect of the substrate directly affects the detection result of the substance to be detected. The nano silver substrate is used as an effective reinforced substrate, although the preparation method is more, the problems of complex preparation process of the nano silver substrate, complex pretreatment steps of samples, poor reinforcing effect and even generation of interference signals on Raman spectra, and influence on the reliability of final detection results exist, and meanwhile, most of the currently used surface reinforced silver substrates are solid substrates, so that the preparation time is long, the cost is high and the effect is poor.
Disclosure of Invention
The invention aims to solve the technical problems of complex substrate preparation process, complicated sample pretreatment steps and poor enhancement effect of the existing surface-enhanced Raman spectroscopy technology, and provides a method for detecting the drugs in blood based on the surface-enhanced Raman spectroscopy technology. When the reagent is used for detecting the medicines in the human blood, the human blood sample is directly detected without pretreatment, and the reagent can be used for qualitatively and quantitatively detecting the medicines in the human blood, thereby having important application value for clinical research of the medicines.
The invention discloses a detection method of a medicine in blood based on a surface enhanced Raman spectroscopy technology, which comprises the following steps:
firstly, preparing nano silver sol:
adding a silver nitrate aqueous solution into a reaction container, heating to 40-42 ℃, dropwise adding a sodium borohydride aqueous solution at 40-42 ℃ under the stirring condition, wherein the solution is changed into light yellow from colorless during the dropwise adding process, then the solution is changed into bright yellow, the dropwise adding of a reducing agent is stopped when the solution is changed into bright yellow, and the solution is continuously and slowly stirred for 10-30 minutes after the dropwise adding is finished to obtain a mixed solution; centrifuging the mixed solution, removing supernatant, sequentially cleaning the solid phase substance with acetone, ethanol and water, re-dispersing in water to obtain nano silver sol, and hermetically storing at the low temperature of-4 to-6 ℃;
secondly, detection:
mixing the nano silver sol and a blood sample containing a drug to be detected according to a volume ratio of 1: (0.1-10) uniformly mixing, and then taking the mixed solution to perform Raman spectrum test, wherein the test conditions are as follows: laser wavelength is 633nm, power is 0.8mW, and integration time is 10s, so that a surface enhanced Raman spectrum of the blood sample to be detected is obtained; meanwhile, a pure target drug in the blood sample to be detected is prepared into a solution, and detection is carried out under the same conditions to obtain a surface enhanced Raman spectrum of the pure target drug.
Thirdly, comparing and judging:
and comparing the Raman spectrogram of the blood sample to be detected with the Raman spectrogram of the pure product, and if the characteristic peak positions correspond to each other, judging that the blood sample to be detected contains the target drug, thereby completing the detection of the drug in the blood.
Furthermore, in the first step, the concentration of the silver nitrate aqueous solution is 0.04-0.16 mol/L.
Furthermore, in the first step, the concentration of the sodium borohydride aqueous solution is 0.02-0.20 mol/L.
Furthermore, in the first step, the rotation speed during stirring is 460-600 rpm.
Furthermore, in the first step, the rotation speed during the centrifugation is 3000-8000 rpm, and the centrifugation time is 10-50 min.
Furthermore, in the step one, the volume of the concentrated silver sol obtained after centrifugation is 1/300-1/50 before centrifugation.
Furthermore, in the second step, the nano silver sol and the blood sample containing the drug to be detected are mixed by a vortex mixer, and the mixing time is 20-30 s.
Further, the capillary tube used for sampling and directly measured on the Raman spectrometer described in step two has a diameter of 0.5mm and a length of 100 mm.
Furthermore, the amount of the mixed solution put into the sample cell in the step two is 10 to 50 muL; the air drying is easy when the amount is small.
Furthermore, the drugs contained in the blood sample in the second step are natural drugs or chemically synthesized drugs.
Further, the drug contained in the blood sample in the second step is penicillin, ciprofloxacin hydrochloride or cefixime.
According to the method, the adding amount of the reducing agent and the reaction temperature during reduction of the silver nitrate aqueous solution are controlled, so that the spherical nano silver particles with the particle size distribution of 40-60 nm are obtained, the silver sol containing the spherical nano silver particles has a good Raman enhancement effect on the medicine in the blood, signal interference is avoided, and reliable guarantee is provided for detecting the medicine in the blood and the concentration of the medicine in the blood by surface enhanced Raman spectroscopy.
The spherical nano silver substrate used for the surface enhanced Raman spectrum detection has the advantages that the preparation speed is high, the silver substrate does not need to be modified, a sample to be detected does not need to be processed, the spherical nano silver substrate can be used for the Raman spectrum detection after being directly mixed, the detection speed can be greatly improved, the response time of a single sample is within 10s, the sensitivity is as high as 1-20 ppm, the response speed is high, the sensitivity is high, the spherical nano silver substrate can be used for qualitative or quantitative detection of natural medicine components and chemical synthesis medicine components including antibiotics, and the detection speed is high, so that the spherical nano silver substrate can also be used for real-time monitoring.
Drawings
FIG. 1 is a transmission electron micrograph of the nano-silver sol prepared in step one of example 1;
FIG. 2 is a high-magnification transmission electron micrograph of the nano-silver sol prepared in step one of example 1;
FIG. 3 is the surface enhanced Raman spectra of the penicillin pure product, blood and penicillin-containing blood sample to be tested in example 1;
FIG. 4 is a surface enhanced Raman spectrum of a blood sample repeatedly tested for penicillin in example 1;
FIG. 5 is a surface enhanced Raman spectrum of blood samples of example 1 containing different concentrations of penicillin;
FIG. 6 is a curve fitted to blood samples containing different concentrations of penicillin in example 1;
FIG. 7 is a Raman spectrum of a sample containing 5ppm penicillin in a blood sample of example 1;
FIG. 8 is a TEM image of the nano-silver sol prepared in step one of example 2;
FIG. 9 is a high-magnification TEM photograph of the nano-silver sol prepared in step one of example 2;
FIG. 10 is a surface enhanced Raman spectrum of the ciprofloxacin hydrochloride pure product, blood and a blood sample to be tested containing ciprofloxacin hydrochloride in example 2;
FIG. 11 is a surface enhanced Raman spectrum of a blood sample containing ciprofloxacin hydrochloride from the repeated examination in example 2;
FIG. 12 is a surface enhanced Raman spectrum of blood samples containing ciprofloxacin hydrochloride at different concentrations in example 2;
FIG. 13 is a curve fitted to blood samples containing ciprofloxacin hydrochloride at different concentrations in example 2;
FIG. 14 is a Raman spectrum of a sample in which the content of ciprofloxacin hydrochloride in the blood sample in example 2 was 5 ppm.
Detailed Description
The following examples are used to demonstrate the beneficial effects of the present invention.
Example 1: the method for detecting the drugs in the blood based on the surface enhanced raman spectroscopy technology of the embodiment comprises the following steps:
firstly, preparing nano silver sol:
adding 500mL of silver nitrate aqueous solution with the concentration of 0.040mol/L into a three-necked bottle with the volume of 2000mL, placing the three-necked bottle on a magnetic stirrer, heating to 40 ℃ while stirring at the rotating speed of 460rpm, then stirring at the rotating speed of 460rpm, and dropwise adding sodium borohydride aqueous solution with the temperature of 40 ℃ and the concentration of 0.020mol/L at the dropwise adding speed of 25 mL/min; after 950mL of sodium borohydride aqueous solution is dripped, slowing down the speed of dripping the sodium borohydride aqueous solution to 15 mL/min; when the solution starts to turn into light yellow, dropwise adding a sodium borohydride aqueous solution at the speed of 30 drops/min until the solution turns into bright yellow, and stopping dropwise adding the reducing agent; after the dropwise addition is finished, continuously and slowly stirring for 10 minutes to obtain a mixed solution; centrifuging the mixed solution at the rotation speed of 5000rpm for 15min, discarding the supernatant, sequentially cleaning the solid phase with acetone, ethanol and water, re-dispersing in water to obtain 1mL of nano silver sol, and hermetically storing at the low temperature of-4 to-6 ℃;
secondly, detection:
mixing nano silver sol and a blood sample to be detected containing penicillin according to a volume ratio of 1: 2, adding the mixture into a vortex mixer for mixing for 20s, wherein the content of penicillin in a blood sample is 5ppm, taking a mixed solution by using a capillary tube with the diameter of 0.5mm, the suction amount of the mixed solution is 20 mu L, then placing the capillary tube containing the solution to be detected on a detection sample table of a model Renishaw inVia Raman spectrometer, and detecting under the conditions that the laser wavelength is 633nm, the laser power is 0.8mW and the integration time is 10s to obtain the surface enhanced Raman spectrum of the blood sample to be detected; simultaneously, the pure penicillin and the blood are detected under the same condition to obtain the surface enhanced Raman spectra of the pure penicillin and the blood, as shown in figure 3;
thirdly, comparing and judging:
surface enhanced Raman of blood sample containing penicillin to be detectedComparing the spectrum with the surface enhanced Raman spectrum of the pure penicillin, finding that the characteristic peak Raman shift of the surface enhanced Raman spectrum of the penicillin-containing blood sample to be detected is 1001.69cm -1 The characteristic peak (1001.69 cm) of the Raman spectrum of the penicillin pure product -1 ) If the penicillin contained in the blood sample is completely matched with the penicillin contained in the blood sample, the target drug penicillin can be judged to be contained in the blood sample to be detected, and the detection of the drug in the blood is completed.
The transmission electron microscope photos of the nano silver sol obtained in the first step are shown in fig. 1 and fig. 2, and as can be seen from fig. 1 and fig. 2, the nano silver is spherical, the diameter of the sphere is 40-60 nm, the size of the sphere is uniform, and the enhancement of Raman signals is facilitated.
FIG. 3 is the surface enhanced Raman spectrum of the penicillin pure product, the blood and the blood sample containing penicillin, and it can be seen from FIG. 3 that the characteristic peak Raman shift of the surface enhanced Raman spectrum of the blood sample containing penicillin is 1001.69cm -1 The characteristic peak (1001.69 cm) of the Raman spectrum of the penicillin pure product -1 ) The Raman spectrum of the blood completely matched with the detected object is not 1001.69cm at the Raman shift -1 The position of the penicillin in the blood has a characteristic peak, and the method can be used for effectively carrying out qualitative analysis on the medicinal penicillin in the blood.
Taking 5 parts of the nano-silver sol prepared in the first step of example 1, and mixing the nano-silver sol with a blood sample containing penicillin according to a volume ratio of 1: 2, uniformly mixing, wherein the content of penicillin in the blood sample is 1ppm, taking the mixed solution by using a capillary tube with the diameter of 0.5mm, the suction amount of the mixed solution is 20 mu L, placing the capillary tube containing the solution to be detected on a detection sample table of a Renishaw inVia Raman spectrometer, and detecting under the conditions that the laser wavelength is 633nm, the laser power is 0.8mW and the integration time is 10s to obtain the surface enhanced Raman spectrum of the blood sample containing penicillin, wherein the drawing is shown in figure 4; as can be seen from FIG. 4, the results of five repeated measurements are all at a Raman shift of 1001.69/cm -1 Characteristic peaks appear at the positions, and the relative standard deviation of the peak heights is 258, 216, 225, 264 and 210 is 24.8, so that the nano silver sol prepared in the embodiment 1 has good reproducibility when being used for Raman spectrum detection.
Taking 5 parts of the nano-silver sol prepared in the first step of example 1, and mixing the nano-silver sol with a blood sample containing penicillin according to a volume ratio of 1: 2, uniformly mixing, wherein the penicillin content in the blood sample is respectively 1ppm, 2ppm, 5ppm, 10ppm and 20ppm, taking the mixed solution by using a capillary tube with the diameter of 0.5mm, the suction amount of the mixed solution is 20 mu L, placing the capillary tube containing the solution to be detected on a detection sample stage of a Renishaw inVia Raman spectrometer, and detecting under the conditions that the laser wavelength is 633nm, the laser power is 0.8mW and the integration time is 10s to obtain the surface enhanced Raman spectrum of the blood sample containing the penicillin; as shown in fig. 5; as can be seen from FIG. 5, the characteristic peak of the surface enhanced Raman spectrum of penicillin (Raman shift is 1001.69 cm) -1 ) The peak height decreased with decreasing penicillin concentration, and the linear fit spectrum is shown in fig. 6, and the fitted linear equation is y 33.376x +260.54, R 2 0.9721, penicillin (1-20 ppm) in different concentrations in blood is determined to have a certain linear relation with the peak height of the characteristic peak, and quantitative analysis can be carried out. The Raman spectrum of the sample in which the amount of penicillin in the blood sample was 5ppm is shown in FIG. 7, and it can be seen from FIG. 7 that the Raman shift is 1001.69cm -1 The obtained characteristic peak height is 410.89, the concentration of penicillin in blood is calculated to be 4.35ppm by using a fitted linear equation, the recovery rate is 87.0%, and it can be determined that the method of the embodiment 1 can effectively quantify the concentration of penicillin in blood within the range of 1-20 ppm, and has the characteristics of high sensitivity (1ppm) and fast response speed (10 s).
The prepared nano silver particle substrate is relatively uniform in shape, the diameter of the sphere is 40-60 nm, the particle size range is narrow, the concentration of silver particles in the nano silver sol and the matching degree of the particle size of the silver particles and penicillin are high, the SERS signal enhancement effect is good, and the reproducibility is good; the spheres with the particle size have small and negligible effect on other substances in blood, do not influence SERS signals of penicillin, and can effectively monitor the concentration of the drugs and metabolites thereof in the blood by virtue of the high-efficiency reaction principle of Raman spectrum, so as to feed back the treatment effect of a patient in real time; the method has the characteristics of high sensitivity, high response speed, real-time monitoring of drug content in blood and the like, and can provide guiding suggestions for the dosage in the clinical research and disease treatment processes.
Comparative example 1: the comparative example is different from example 1 in that the preparation method of the nano silver sol in the first step is as follows: adding 500mL of silver nitrate aqueous solution with the concentration of 0.040mol/L into a three-necked bottle with the volume of 2000mL, placing the three-necked bottle on a magnetic stirrer, heating to 30 ℃ while stirring at the rotating speed of 460rpm, then stirring at the rotating speed of 460rpm, and simultaneously dropwise adding sodium borohydride aqueous solution with the temperature of 30 ℃ and the concentration of 0.020mol/L at the dropwise adding speed of 25 mL/min; after 950mL of sodium borohydride aqueous solution is dripped, slowing down the speed of dripping the sodium borohydride aqueous solution to 15 mL/min; when the solution starts to turn into light yellow, dropwise adding a sodium borohydride aqueous solution at the speed of 30 drops/min until the solution turns into bright yellow, stopping dropwise adding the reducing agent, and then dropwise adding 1050mL of the sodium borohydride aqueous solution; after the dropwise addition is finished, continuously and slowly stirring for 10 minutes to obtain a mixed solution; centrifuging the mixed solution at the rotation speed of 5000rpm for 15min, discarding the supernatant, sequentially cleaning the solid phase with acetone, ethanol and water, re-dispersing in water to obtain nano silver sol, and hermetically storing at the low temperature of-4 to-6 ℃;
the other steps and parameters were the same as in example 1.
Comparison of the characterization results of the transmission electron microscope of the nano-silver sol prepared in the comparative example 1 and the nano-silver sol prepared in the example 1 shows that the nano-silver sol prepared at 30 ℃ has uneven particle size, and the particle size generated in the initial stage of the reaction is not uniform with the particle size generated in the final stage due to the fact that the reaction speed is low and the particle size generated in the initial stage is continuously grown as a template, so that the signal enhancement effect is affected.
Comparative example 2: the comparative example is different from example 1 in that the preparation method of the nano silver sol in the first step is as follows:
adding 500mL of silver nitrate aqueous solution with the concentration of 0.040mol/L into a three-necked bottle with the volume of 2000mL, placing the three-necked bottle on a magnetic stirrer, heating to 50 ℃ while stirring at the rotating speed of 460rpm, then keeping the rotating speed of 460rpm, stirring while dropwise adding sodium borohydride aqueous solution with the temperature of 50 ℃ and the concentration of 0.020mol/L, wherein the dropwise adding speed is 25 mL/min; after 950mL of sodium borohydride aqueous solution is dripped, slowing down the speed of dripping the sodium borohydride aqueous solution to 15 mL/min; when the solution starts to turn into light yellow, dropwise adding a sodium borohydride aqueous solution at the speed of 30 drops/min until the solution turns into bright yellow, stopping dropwise adding the reducing agent, and then dropwise adding 1050mL of the sodium borohydride aqueous solution; after the dropwise addition is finished, continuously and slowly stirring for 10 minutes to obtain a mixed solution; centrifuging the mixed solution at the rotation speed of 5000rpm for 15min, discarding the supernatant, sequentially cleaning the solid phase with acetone, ethanol and water, re-dispersing in water to obtain nano silver sol, and hermetically storing at the low temperature of-4 to-6 ℃;
the other steps and parameters were the same as in example 1.
Comparison of the characterization results of the transmission electron microscope of the nano-silver sol prepared in the comparative example 2 and the nano-silver sol prepared in the example 1 shows that the nano-silver sol prepared at 50 ℃ has an excessively large particle size, most of the particle size generated in the initial stage is used as a template to continuously grow due to the high reaction speed, the particle size of the obtained nano-silver is more than 100nm, the signal enhancement effect is affected, and the nano-silver is easy to settle.
Example 2: the method for detecting the drugs in the blood based on the surface enhanced raman spectroscopy technology of the embodiment comprises the following steps:
firstly, preparing nano silver sol:
adding 500mL of silver nitrate aqueous solution with the concentration of 0.040mol/L into a three-necked bottle with the volume of 2000mL, placing the three-necked bottle on a magnetic stirrer, heating to 42 ℃ while stirring at the rotating speed of 500rpm, then stirring while maintaining the rotating speed of 500rpm, and dropwise adding sodium borohydride aqueous solution with the temperature of 42 ℃ and the concentration of 0.020mol/L at the dropwise adding speed of 50 mL/min; after 950mL of sodium borohydride aqueous solution is dripped, the speed of dripping the sodium borohydride aqueous solution is reduced to 40 mL/min; when the solution starts to turn into light yellow, dropwise adding a sodium borohydride aqueous solution at the speed of 30 drops/min until the solution turns into bright yellow, and stopping dropwise adding the reducing agent; after the dropwise addition is finished, continuously and slowly stirring for 30 minutes to obtain a mixed solution; centrifuging the mixed solution at the rotation speed of 5000rpm for 15min, discarding the supernatant, sequentially cleaning the solid phase with acetone, ethanol and water, and dispersing in water to obtain 1mL of nano silver sol, and hermetically storing at the low temperature of-4 to-6 ℃;
secondly, detection:
mixing the nano silver sol and a to-be-detected blood sample containing ciprofloxacin hydrochloride according to the volume ratio of 1: 2, adding the mixture into a vortex mixer for mixing for 20s, wherein the content of ciprofloxacin hydrochloride in a human blood sample is 5ppm, taking the mixed solution by using a capillary tube, the diameter of the capillary tube is 0.5mm, the suction amount of the mixed solution is 20 mu L, placing the capillary tube containing the solution to be detected on a detection sample table of a Renishaw inVia Raman spectrometer, and detecting under the conditions that the laser wavelength is 633nm, the laser power is 0.8mW and the integration time is 10s to obtain the surface enhanced Raman spectrum of the blood sample to be detected; simultaneously detecting the ciprofloxacin hydrochloride pure product and the blood under the same condition to obtain surface enhanced Raman spectra of the ciprofloxacin hydrochloride pure product and the blood, wherein the surface enhanced Raman spectra are shown in figure 10;
thirdly, comparing and judging:
comparing the surface enhanced Raman spectrum of the blood sample containing the ciprofloxacin hydrochloride to be detected with the surface enhanced Raman spectrum of the pure ciprofloxacin hydrochloride, and finding that the characteristic peak Raman shift of the surface enhanced Raman spectrum of the blood sample containing the ciprofloxacin hydrochloride to be detected is 1384.69cm -1 And characteristic peak (1384.69 cm) of Raman spectrogram of ciprofloxacin hydrochloride pure product -1 ) The Raman spectrum of the blood completely matched with the detected object is not 1384.69cm at the Raman shift -1 If the position of the target ciprofloxacin hydrochloride has a characteristic peak, the target ciprofloxacin hydrochloride can be judged to be contained in the blood sample containing the ciprofloxacin hydrochloride to be detected, and the qualitative detection of the medicine in the blood is completed.
The transmission electron microscope photograph of the nano silver sol obtained in the first step of this embodiment 2 is shown in fig. 8 and 9, and as can be seen from fig. 8 and 9, the nano silver is spherical, the diameter of the sphere is 40-60 nm, and the size of the sphere is uniform, which is beneficial to enhancing raman signals.
Taking 5 parts of the nano-silver sol prepared in the first step of the example 2, and mixing the nano-silver sol with a ciprofloxacin hydrochloride-containing blood sample according to the volume ratio of 1: 2 mixing well, wherein the content of ciprofloxacin hydrochloride in the blood sample is 1ppm, and then using a capillary with the diameter of 0.5mmTaking a mixed solution from the tube, wherein the suction amount of the mixed solution is 20 mu L, placing the mixed solution on a sample platform of a Renishaw inVia Raman spectrometer, and detecting the mixed solution under the conditions that the laser wavelength is 633nm, the laser power is 0.8mW and the integration time is 10s to obtain the surface enhanced Raman spectrum of the blood sample containing the ciprofloxacin hydrochloride, wherein the surface enhanced Raman spectrum is shown in figure 11; as can be seen from FIG. 11, the results of five repeated measurements are all at a Raman shift of 1384.69/cm -1 Characteristic peaks appear at the positions, the peak heights are 457, 522, 632, 574 and 621 respectively, and the relative standard deviation is 72.9, so that the nano silver sol prepared in the embodiment 2 has good reproducibility when being used for Raman spectrum detection.
Taking 5 parts of the nano-silver sol prepared in the first step of the example 2, and mixing the nano-silver sol with a ciprofloxacin hydrochloride-containing blood sample according to the volume ratio of 1: 2, uniformly mixing, wherein the contents of ciprofloxacin hydrochloride in the blood sample are respectively 1ppm, 2ppm, 5ppm, 10ppm and 20ppm, then respectively taking the mixed solution by using a capillary tube with the diameter of 0.5mm, wherein the suction amount of the mixed solution is 20 mu L, placing the mixed solution on a sample table of a Renishaw inVia Raman spectrometer, and detecting under the conditions that the laser wavelength is 633nm, the laser power is 0.8mW and the integration time is 10s to obtain the surface enhanced Raman spectrum of the blood sample containing ciprofloxacin hydrochloride; as shown in fig. 12; as can be seen from FIG. 12, the ciprofloxacin hydrochloride has a characteristic surface enhanced Raman spectrum peak (Raman shift of 1384.69/cm) -1 ) The peak height decreases with the decrease of the ciprofloxacin hydrochloride concentration, the linear fitting spectrum is shown in figure 13, and the fitted linear equation is that y is 40.638x +252.15, R 2 0.9448, ciprofloxacin hydrochloride (1-20 ppm) with different concentrations in blood is determined to have a certain linear relation with the characteristic peak height, and quantitative analysis can be carried out. The Raman spectrum of the sample containing 5ppm of ciprofloxacin hydrochloride in the blood sample is shown in FIG. 14, and the Raman shift is 1384.69/cm -1 The obtained characteristic peak height is 481.5, the fitted linear equation is utilized to calculate the concentration of ciprofloxacin hydrochloride in blood to be 5.64ppm, the recovery rate is 112.9%, and it can be judged that the method in the embodiment 2 can effectively quantify the concentration of ciprofloxacin hydrochloride in blood within the range of 1-20 ppm, and has the advantages of high sensitivity (1ppm), high response speedFast (10 s).
The nano silver particle substrate prepared by the embodiment 2 is relatively uniform in shape, the diameter of the sphere is 40-60 nm, the particle size range is narrow, the concentration of silver particles in the nano silver sol, the matching degree of the particle size of the silver particles and ciprofloxacin hydrochloride are high, the SERS signal enhancement effect is good, and the reproducibility is good; the spheres with the particle size have small and negligible effect on other substances in blood, do not influence the SERS signal of ciprofloxacin hydrochloride, can effectively identify the concentration of the drugs and metabolites thereof in the blood by virtue of the high-efficiency reaction principle of Raman spectrum, and can feed back the treatment effect of a patient in real time; the method has the characteristics of high sensitivity, high response speed, real-time monitoring of the drug content in blood and the like, and can provide guiding suggestions for clinical treatment dosage of patients.
Claims (9)
1. The detection method of the medicine in the blood based on the surface enhanced Raman spectroscopy technology is characterized by comprising the following steps:
firstly, preparing nano silver sol:
adding a silver nitrate aqueous solution into a reaction container, heating to 40-42 ℃, dropwise adding a sodium borohydride aqueous solution at 40-42 ℃ under the stirring condition, wherein the solution is changed into light yellow from colorless during the dropwise adding process, then the solution is changed into bright yellow, the dropwise adding of a reducing agent is stopped when the solution is changed into bright yellow, and the solution is continuously and slowly stirred for 10-30 minutes after the dropwise adding is finished to obtain a mixed solution; centrifuging the mixed solution, removing supernatant, sequentially cleaning a solid phase substance by using acetone, ethanol and water, re-dispersing in water to obtain nano silver sol, and hermetically storing at the low temperature of between 4 ℃ below zero and 6 ℃ below zero;
secondly, detection:
mixing the nano silver sol and a blood sample containing a drug to be detected according to a volume ratio of 1: (0.1-10) uniformly mixing, and then taking the mixed solution to perform Raman spectrum test, wherein the test conditions are as follows: the laser wavelength is 633nm, the power is 0.8mW, the integration time is 10s, and a surface enhanced Raman spectrogram of the blood sample to be detected is obtained; meanwhile, preparing a pure target drug in a blood sample to be detected into a solution, and detecting under the same condition to obtain a surface enhanced Raman spectrogram of the pure target drug;
thirdly, comparing and judging:
and comparing the Raman spectrogram of the blood sample to be detected with the Raman spectrogram of the pure product, and if the characteristic peak positions correspond to each other, judging that the blood sample to be detected contains the target drug, thereby completing the detection of the drug in the blood.
2. The method for detecting a drug in blood based on the surface-enhanced Raman spectroscopy of claim 1, wherein the concentration of the silver nitrate aqueous solution in the first step is 0.04-0.16 mol/L.
3. The method for detecting a drug in blood based on the surface-enhanced Raman spectroscopy of claim 1 or 2, wherein the concentration of the aqueous solution of sodium borohydride in the first step is 0.02-0.20 mol/L.
4. The method for detecting a drug in blood based on the surface-enhanced Raman spectroscopy according to claim 1 or 2, wherein in the first step, the rotation speed during stirring is 460 to 600 rpm.
5. The method for detecting a drug in blood based on the surface-enhanced Raman spectroscopy according to claim 1 or 2, wherein the rotation speed during the centrifugation in the first step is 3000-8000 rpm, and the centrifugation time is 10-50 min.
6. The method for detecting the drugs in the blood based on the surface-enhanced Raman spectroscopy technology according to claim 1 or 2, wherein the step two in which the nano-silver sol and the drug-containing blood sample to be detected are mixed by a vortex mixer for 20-30 seconds.
7. The method for detecting a drug in blood based on the surface-enhanced Raman spectroscopy according to claim 1 or 2, wherein the sample volume of the mixture solution put into the sample cell in the second step is 10 μ L to 50 μ L.
8. The method for detecting a drug in blood based on the surface-enhanced raman spectroscopy technique according to claim 1 or 2, wherein the drug contained in the blood sample in the second step is a natural drug or a chemically synthesized drug.
9. The method of claim 8, wherein the drug contained in the blood sample is penicillin, ciprofloxacin hydrochloride, or cefixime.
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