CN109799224A - Quickly detect the method and application of protein concentration in Chinese medicine extract - Google Patents
Quickly detect the method and application of protein concentration in Chinese medicine extract Download PDFInfo
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Abstract
The invention discloses the methods and application of protein concentration in quick detection Chinese medicine extract, this method comprises: the collection of (a) calibration set sample；(b) concentration of protein in Coomassie Brilliant Blue detection calibration set sample is utilized；(c) measurement of calibration set sample Raman spectrum；(d) foundation of calibration model；(e) detection of sample to be tested.The method provided by the invention that protein concentration in Chinese medicine extract is quickly detected based on Raman spectrum, easy to operate, quick, compared with traditional method of protein measurement (chemical staining method), without preparing with rower song, minute is short；Simultaneously without using every chemical reagent and solvent, there is environment friendly.There is preferable correlation between the Raman signatures spectrum of red sage root extract and wherein protein concentration in embodiment, measurement error is less than 10%.This method controls the quality of traditional Chinese medicine intermediate by real-time monitoring quality index, and then guarantees the safety and validity of traditional Chinese medicine.
Description
Technical field
The present invention relates to production process quality-monitoring is related to, protein concentration in Chinese medicine extract is especially quickly detected
Method and application.
Background technique
Protein is a kind of impurity common in traditional Chinese medicine injection, measures its content to control traditional Chinese medicine injection quality, guarantor
Barrier traditional Chinese medicine injection uses safe significant.Cited Protein quantitative analysis means include kelvin in " Chinese Pharmacopoeia "
Nitriding, forint phenol reagent process, biuret method, Coomassie Brilliant Blue, diquinoline -4 2,2'-, dicarboxylic acids (BCA) method and ultraviolet -
Visible spectrophotometry.Above method applicable object is not quite similar, and UV-VIS spectrophotometry is only applicable to protein purification
Detection, Kjeldahl's method sensitivity is lower and complex system in chaff interferent it is more, remaining reagent method be required to colour reagent with
It is further measured with colorimetric method again after example reaction, it is complicated for operation, it is time-consuming more long, there is higher want to the level of operator
It asks, and has certain pollution to environment, other nonprotein constituents may also have interference to measurement result in complex system.
Raman spectrum analysis technology is a kind of Molecular spectral analytical methods based on Raman scattering, and signal derives from
The vibration and rotation of material molecule.Raman spectrum is different from molecule infrared spectroscopy, and polar molecule and nonpolar molecule can generate
Raman spectrum.The application range of Raman spectrum is throughout the every field such as chemistry, physics, biology and medicine, Raman spectrum pair
There is very big value in qualitative analysis, quantitative analysis and measurement molecular structure.Raman spectrum analysis method do not need to sample into
Row pre-treatment, sample requirements are few, in the analysis process with easy to operate, minute is short, consumption chemicals is few, detection spirit
The advantages that sensitivity is high.
Raman spectroscopy is applied to Chinese materia medica preparation production process intermediate quality real-time monitoring and quickly detects another
Advantage is that the Raman spectrum of sample is extremely weak by the interference of water, can almost not have to consider hydrone during actual measurement
The influence generated to sample spectra is vibrated, therefore is used directly for the quick inspection without any pretreated Chinese medicine extracting solution
It surveys.
Traditional method of protein measurement is complicated for operation, processing speed is slower, needs to pre-process and additional reagent, molten
Agent does not meet the theory of green manufacturing, is unfavorable for the realization that medicine intermediate is let pass in real time.Because in a kind of quickly detection of the invention
The method of protein concentration is of great significance in medicament extracting solution.
Summary of the invention
The object of the present invention is to provide the methods of protein concentration in quick detection Chinese medicine extract.Operation of the present invention
Simply, quickly, without pretreatment and additional reagent, solvent, meet the theory of green manufacturing, it is real-time to be conducive to medicine intermediate
The realization of clearance；Furthermore method of the invention can effectively solve traditional Chinese medicine injection monitoring of residual protein after refinement treatment and ask
Topic, makes the safety of traditional Chinese medicine injection be guaranteed, and can be used for traditional Chinese medicine injection production overall process intermediate protein concentration
Monitoring and control.
Technical solution of the present invention: the method for quickly detecting protein concentration in Chinese medicine extract, by measuring in be measured
The dense of wherein protein is calculated using the calibration model of building in the Raman spectrum data of the protein concentration of medicament extracting solution
Degree.
In quick detection Chinese medicine extract above-mentioned in the method for protein concentration, the method is mentioned by collecting Chinese medicine
Liquid calibration samples are taken, the Raman spectrum data of calibration samples is measured, and measure the concentration of protein in calibration samples, according to for school building
The concentration of protein and corresponding Raman spectrum data establish calibration model in positive sample, by the drawing of Chinese medicine extract to be measured
In the calibration model of graceful spectroscopic data input building, the concentration of wherein protein can be calculated.
In quick detection Chinese medicine extract above-mentioned in the method for protein concentration, the method is mentioned by collecting Chinese medicine
Liquid calibration samples are taken, are surveyed using the Raman spectrum data of Raman spectroscopy measurement calibration samples, and using Coomassie Brilliant Blue
The conduct reference point for determining protein in calibration samples establishes correction sample using Partial Least Squares or principal component regression method respectively
In this between Raman spectrum data and protein concentration relationship calibration model, by the Raman spectrum data of Chinese medicine extract to be measured
It inputs in the calibration model of building, the concentration of wherein protein can be calculated.
In quick detection Chinese medicine extract above-mentioned in the method for protein concentration, the Raman spectrum number of the calibration samples
According to being measured by Raman spectroscopy, calibration samples is put into the quartz colorimetric utensil of 10mm light path, is placed in Raman
On the matched cell rack of spectrometer, spectral scan is carried out, obtains calibration samples Raman spectrum data, wherein Raman spectrometer swashs
Optical wavelength 785nm, Raman shift range 173.7-3200.8cm-1, resolution ratio 4.5cm-1, laser intensity 50%, the time of integration
10000ms。
In quick detection Chinese medicine extract above-mentioned in the method for protein concentration, the Raman of the Chinese medicine extract to be measured
Spectroscopic data is measured by Raman spectroscopy, Chinese medicine extract is put into the quartz colorimetric utensil of 10mm light path, and
It is placed on the matched cell rack of Raman spectrometer, carries out spectral scan, obtain the Raman spectrum data of Chinese medicine extract, each
The Raman spectrum of Chinese medicine extract need to deduct dark current, scan 3 times, take average spectroscopic data；Wherein Raman spectrometer laser wave
Long 785nm, Raman shift range 173.7-3200.8cm-1, resolution ratio 4.5cm-1, laser intensity 50%, the time of integration
10000ms。
In quick detection Chinese medicine extract above-mentioned in the method for protein concentration, the calibration model is applied chemistry meter
Technology is measured, the Multivariate Correction regression model of protein concentration in Chinese medicine extract is established using multivariate data processing software,
The multivariate data analysis method is principal component regression method or Partial Least Squares, and it is true to intersect progress internal verification using leaving-one method
Fixed best number of principal components, the coefficient of determination >=0.95 of the calibration model.Protein in quick detection Chinese medicine extract above-mentioned
It is described to establish the verifying that calibration model further includes calibration model, the verifying in the method for concentration are as follows: to take unknown extracting solution, survey
Its fixed Raman signatures spectroscopic data, is inputted in calibration model, calculates the concentration of protein in sample, with the sample according to examining
The protein concentration of Mas bright blue method measurement compares, it is desirable that measurement error is less than 10%.
In quick detection Chinese medicine extract above-mentioned in the method for protein concentration, it the described method comprises the following steps:
(a) Chinese medicine extract calibration samples are collected；
(b) calibration samples protein concentration reference point measures: using using egg in Coomassie Brilliant Blue measurement calibration samples
White matter concentration；
(c) measurement of calibration samples Raman spectrum: calibration set sample is placed in the matched cell rack of Raman spectrometer
It is scanned, obtains calibration samples Raman signatures spectroscopic data；
(d) establish calibration model: using multivariate data analysis method, establish in calibration set sample protein concentration with it is right
The mathematical model of relationship, as calibration model between the Raman signatures spectrum answered；
(e) Chinese medicine extract to be measured detection: acquiring the Raman signatures spectroscopic data of Chinese medicine extract to be measured, and by this spectrum
Data input in calibration model, calculate protein concentration in sample to be tested.
The method of protein concentration protein compression in red sage root extract in a kind of quick detection Chinese medicine extract above-mentioned
The application of degree quickly measured.
The present invention is using the protein concentration in Coomassie Brilliant Blue measurement calibration set sample, according to " Chinese Pharmacopoeia " 2015
Method described in the 4th general rule 0731 of version, according to the alkalinity in Coomassie brilliant blue G250 in an acidic solution and protein molecule
Amino acid (arginine) and aromatic amino acid, which combine, forms blue complex, in a certain range its shade and protein
Concentration is proportional, makees standard curve with protein control product solution, using the content of protein in colorimetric method for determining test sample.It examines
The interfering substance (detergent, Triton X-100, lauryl sodium sulfate) of Mas bright blue method theoretically should not be in traditional Chinese medicine extraction
Exist in liquid, therefore the applicability with higher for the measurement of protein concentration in Chinese medicine extract.
(1) preparation of acid dyeing liquid: taking Coomassie brilliant blue G250 0.1g, after adding ethyl alcohol 50ml to dissolve, adds phosphoric acid
100ml is diluted with water to 1000ml, mix, filtration, take filtrate to get.
(2) preparation of reference substance solution: seralbumin reference substance 10mg is taken, 10ml is diluted with water to, is made every milliliter
The standard items mother liquor of the 1mg containing protein；Standard items mother liquor 1ml is taken, 10ml is diluted with water to, is made every milliliter containing protein
The standard solution of 0.1mg.
(3) production of standard curve: standard solution 0.0ml, 0.1ml, 0.2ml, 0.4ml obtained by precision measurement (2),
0.6ml, 0.8ml, 1.0ml distinguish in the tool plug test tube that tagging is 0~No. 6, after adding water to 1.0ml, then are separately added into (1)
Gained acid dyeing liquid 5.0ml, mixes immediately, and according to UV-VIS spectrophotometry, absorbance is measured at 595nm wavelength；Together
When using No. 0 test tube as blank.Equation of linear regression is calculated with the absorbance (y) of reference substance solution concentration (x) corresponding thereto.
(4) preparation of sample solution: precision weighs calibration set sample solution 0.5g, is diluted with water to 10mL, is that sample is dilute
Release liquid.
(5) in sample solution protein concentration measurement: precision measures gained sample diluting liquid 1.0mL in (4), with (3)
Operation is measured in, from the protein concentration calculated in sample diluting liquid in equation of linear regression, multiplied by extension rate to get school
Protein concentration in positive collection sample solution.
The present invention uses Raman spectroscopy acquisition correction collection sample characteristics spectrum, obtains chemical substance in sample solution
Global Information.Raman spectrum has very big value for qualitative analysis, quantitative analysis and measurement molecular structure.Raman spectrum analysis
Method do not need to sample carry out pre-treatment, sample requirements are few, in the analysis process have it is easy to operate, minute is short,
Consume the advantages that chemicals is few, detection sensitivity is high.Raman spectroscopy is applied to weight among Chinese materia medica preparation production process
Real-time monitoring and another advantage quickly detected are that the Raman spectrum of sample is extremely weak by the interference of water, in actual measurement process
In can almost not have to consider that hydrone vibrates the influence that generate sample spectra, therefore be used directly for without any pre- place
The quick detection of the Chinese medicine extracting solution of reason.
Raman spectrometer optical maser wavelength 785nm used, Raman shift range 173.7-3200.8cm-1, resolution ratio 4.5cm-1, laser intensity 50% (sound end power is about 300mW), time of integration 10000ms.Calibration set sample solution is placed in 10mm
It in the quartz colorimetric utensil of light path, is placed on the matched cell rack of Raman spectrometer, carries out spectral scan, obtain the Raman of sample
Characteristic spectrum data.
Applied Chemometrics technology of the present invention establishes protein in Chinese medicine extract using multivariate data processing software
The Multivariate Correction regression model of concentration.The multivariate data analysis method is principal component regression method (PCR method) or offset minimum binary
Method (PLS method).Progress internal verification is intersected using leaving-one method and determines best number of principal components, when the coefficient of determination of calibration model arrives
0.95 or more, it is believed that model has built up completion, i.e., positive model for school building the coefficient of determination >=0.95.
The foundation of the calibration model further includes the verifying of calibration model, the verifying are as follows: takes agnoprotein matter concentration
Chinese medicine extract measures its Raman spectrum under same parameter in above process, is inputted in above-mentioned calibration model, calculates
Protein concentration in middle extracting solution, compared with the sample is according to the protein concentration of said determination, it is desirable that measurement error is less than
10%.As verifying collection sample protein matter concentration need to fall in corresponding calibration set sample protein concentration variation range it
It is interior.
Above-mentioned calibration model can according to actual needs, increase calibration set and verifying collection, to model constantly update with it is perfect.
The present invention is by collecting a certain number of Chinese medicine extract samples as calibration samples collection, with certain acquisition mode
Scanning obtains the Raman spectrogram of calibration samples collection.Using the sample protein concentration result that Coomassie Brilliant Blue measures as reference
Value, using Partial Least Squares or principal component regression method, establishes between the Raman spectrum of Chinese medicine extract and protein concentration and closes
The multivariate calibration model of system.The Raman spectrum for measuring the Chinese medicine extract of agnoprotein matter concentration in the same way, using
The concentration of wherein protein can be quickly calculated in the calibration model of building.
The present invention also provides application of the method in the determination of protein concentration of red sage root extract.
Compared with prior art, the beneficial effect that the present invention has includes:
(1) in Raman light spectrum quick test Chinese medicine extract provided by the invention protein concentration method, and it is traditional
Protein determination development process is compared, easy to operate, quick, without pretreatment and additional reagent, solvent, meets green manufacturing
Theory is conducive to the realization that medicine intermediate is let pass in real time.
(2) the method can effectively solve the monitoring problem of traditional Chinese medicine injection residual protein after refinement treatment, make
The safety of medicine injection is guaranteed, and can be used for the monitoring and control of traditional Chinese medicine injection production overall process intermediate protein concentration
System, it is potential to obtain widely promoting and applying in the production process of Chinese materia medica preparation.
The following are inventor's specific experiment processes:
To sympathize red injection be by danshensu and Ligustrazine Hydrochloride be main pharmacodynamics ingredient compound injection.Red rooted salvia warp
Resulting red sage root extract is intermediate after extraction, purification and ultrafiltration, and quality is directly related with red injection quality is finally sympathized,
The content for measuring wherein related substances is necessary quality control method.
1. the collection of calibration set sample:
In red sage root extract production process, the process for refining intermediate of different batches, different disposal process, gained are collected
Solution is calibration set sample.
2. the measurement of protein concentration reference point in calibration set sample:
Using the gross protein in Coomassie Brilliant Blue measurement sample；
It is bright according to coomassie in an acidic solution according to method described in " Chinese Pharmacopoeia " 2015 editions the 4th general rules 0731
Blue G250 forms blue complex in conjunction with basic amino acid (arginine) and aromatic amino acid in protein molecule, one
Determine its interior shade of range and protein concentration is proportional, standard curve is made with protein control product solution, using colorimetric method
Measure the content of protein in test sample.Interfering substance (detergent, Triton X-100, the dodecyl of Coomassie Brilliant Blue
Sodium sulphate) do not contain in this law institute test sample sheet, therefore more closed for the measurement of the protein concentration of this law object
It is suitable.
(1) preparation of acid dyeing liquid: taking Coomassie brilliant blue G250 0.1g, after adding ethyl alcohol 50ml to dissolve, adds phosphoric acid
100ml is diluted with water to 1000ml, mix, filtration, take filtrate to get.
(2) preparation of reference substance solution: taking seralbumin matter 10mg, be diluted with water to 10ml, is made every milliliter containing egg
The standard items mother liquor of white matter 1mg；Standard items mother liquor 1ml is taken, 10ml is diluted with water to, is made every milliliter of 0.1mg's containing protein
Standard solution.
(3) production of standard curve: standard solution 0.0ml, 0.1ml, 0.2ml, 0.4ml obtained by precision measurement (2),
0.6ml, 0.8ml, 1.0ml distinguish in the tool plug test tube that tagging is 0~No. 6, after adding water to 1.0ml, then are separately added into (1)
Gained acid dyeing liquid 5.0ml, mixes immediately, and according to UV-VIS spectrophotometry, absorbance is measured at 595nm wavelength；Together
When using No. 0 test tube as blank.Equation of linear regression, mark are calculated with the absorbance (y) of reference substance solution concentration (x) corresponding thereto
Directrix curve is as shown in Figure 4.
(4) preparation of sample solution: precision weighs calibration set sample solution 0.5g, is diluted to 10mL with water, is that sample is dilute
Release liquid.
(5) in sample solution protein concentration measurement: precision measures gained sample diluting liquid 1.0mL in (4), with (3)
Operation is measured in, from the protein concentration calculated in sample diluting liquid in equation of linear regression, multiplied by extension rate to get school
Protein concentration in positive collection sample solution.
3. the measurement of calibration set sample Raman spectrum:
Calibration set sample is placed in the matched cell rack of Raman spectrometer and is scanned, calibration set sample Raman is obtained
Characteristic spectrum data；
Raman spectrometer optical maser wavelength 785nm used, Raman shift range 173.7-3200.8cm-1, resolution ratio 4.5cm-1, laser intensity 50% (sound end power is about 300mW), time of integration 10000ms.Calibration set sample solution is placed in 10mm
In the quartz colorimetric utensil of light path, it is placed on the matched cell rack of Raman spectrometer and carries out spectral scan, obtain the Raman of sample
Characteristic spectrum data.
4. the foundation of calibration model:
Using multivariate data analysis method, protein concentration and corresponding Raman signatures spectrum in calibration set sample are established
Between relationship mathematical model；
Use BWIQTMSoftware (U.S. B&W Tek Opto-Electronics) handles initial data.
A large amount of sample message is contained in Raman spectrum, is difficult therefrom to select information relevant to specific physicochemical property,
And the spectrum difference of different samples is very small, is with the naked eye difficult to differentiate；Meanwhile instrument state, sample state and measuring condition
Difference cause Raman spectrum that subtle translation or rotation occurs.Therefore, when establishing Raman spectrum calibration model, for eliminating
The irrelevant information of spectrum and the preprocess method of noise just seem very crucial and necessary.
There are many common preprocessing procedures.Mean value centralization (Mean centering), standardization
(autoscaling) and normalization (Normalization) algorithm etc. is for reducing until eliminate some redundancies, thus
While reducing sample room correlation, the difference being also capable of increasing between sample, and then reach the reproducibility for improving model and pre-
The effect of survey ability；Smoothing processing algorithm such as Savitzky-Golay convolution smooth (SG Smooth), rolling average are smooth
(Moving Average Smooth, MAS) etc. is to eliminate the common method of noise, and basic assumption is the noise that spectrum contains
For zero-mean random white noise, noise raising signal-to-noise ratio can be reduced if repeatedly measuring and being averaged；Fourier transformation (Fourier
Transform, FT) it can be used to implement the smooth of spectrum, noise reduction, data compression, information extraction etc.；Standard normal transformation
(Standard Normal Variate, SNV) be used to reduce granular size unevenly and particle surface non-specificity scatter shadow
It rings；Multiplicative scatter correction (Multiplicative Scatter Correction, MSC) can eliminate in diffusing reflection spectrum due to
The mirror-reflection of sample and it is uneven caused by noise, eliminate spectrum baseline drift phenomenon and spectrum it is not repeated；Single order
Derivative (First-order derivative algorithm, 1ST) and second derivative algorithm (Second-order DA
derivative algorithm,2ndIt DA) is common baseline correction and spectrally resolved preprocess method in spectrum analysis；Just
Signal correction (Orthogonal Signal Correction, OSC) is handed over can effectively to take out included in spectroscopic data
Various interfering noise signals.Due to the variation of instrument, sample and measuring condition, the variation of spectrum is not regular to be followed, therefore
There is no general assembled schemes for pretreatment.When actual treatment, above-mentioned several method can be combined according to certain order and be used for
Concrete condition concrete analysis.Meanwhile the factors such as spectral wavelength variable quantity is more, and intensity is weak and overlapping is serious are to prediction result
It adversely affects, therefore the suitable characteristic wave bands of screening are modeled and are also necessary preprocessing means.
Most-often used Multivariate Correction algorithm have multiple linear regression (Multivariate Linear Regression,
MLR), principal component regression (Principle Component Regression, PCR), Partial Least Squares Regression (Partial
Least Squares Regression, PLSR) etc..Wherein, principal component regression (PCR) method is that one kind can effectively solve variable
Between synteny problem modeling method, this method establishes connection not instead of not directly between Y matrix and X matrix, in X matrix
Information recombinated after extract most accurate spectral information, comprehensive recombination variable, which can will have, to be reflected to target components more
The overlay information of weight synteny is separated, while being selected again the information of spectrum, by useful information from interference noise
Middle extraction is come, and the precision and predictive ability of model are improved.PLSR algorithm is that more mature now, most widely used recurrence is calculated
Method not only decomposes spectrum data matrix X when calculating, and has also carried out principal component analysis to concentration matrix Y.
The quality of calibration model must be judged established model by the prediction of the sample to forecast set, it is corresponding
Model performance evaluation index has the coefficient of determination (R2), cross-validation mean square deviation (Root mean square error of
Cross validation, RMSECV), prediction mean square deviation (Root mean square error of prediction,
RMSEP the performance and prediction effect of established model are investigated).
Sample set Raman spectrum data is handled using different preprocess methods, is established using PLS method and PCR method
Calibration model between red sage root extract characteristic spectrum information and protein concentration is verified using the full interior extrapolation method of leaving-one method.
The preprocess method combinations of different models, modeling algorithm, number of principal components, calibration set coefficient of determination R2And RMSECV and verifying
The RMSEP of collection is as shown in table 1.
1 distinct methods of table model the calibration model between resulting red sage root extract characteristic spectrum information and protein concentration
Performance
The comparison of the model performance obtained through multiple preprocess methods, it was demonstrated that successively smooth, first derivation, standard by SG
Treated that spectrum can establish performance model the most steady, R for normal state, rolling average window2, RMSECV is minimum, RMSEP compared with
Small, there are extraordinary correlations between the Raman spectrum and protein concentration of red sage root extract.It is pre- with the quantitative calibration models
Survey protein concentration and Coomassie Brilliant Blue measurement reference point between correlativity figure referring to attached drawing 4, as seen from the figure with
Correlation is good between upper model predication value and reference point, and model has preferable performance.
5. the detection of sample to be tested:
The Raman signatures spectroscopic data of sample to be tested is acquired, and this spectroscopic data is inputted in " 4 " in gained calibration model,
Calculate protein concentration in sample to be tested.Table 2 is the protein concentration measured value and predicted value of 3 samples to be measured.
2 red sage root extract of table verifying collection sample protein concentration measured value and predicted value
By prediction result it is found that this law model built is in unknown red sage root extract sample compared with measured result
Protein concentration has preferable predictive ability.
In conclusion operation of the present invention is simple, quick, without pretreatment and additional reagent, solvent, meet green manufacturing
Theory, be conducive to the realization that medicine intermediate is let pass in real time；Furthermore method of the invention can effectively solve traditional Chinese medicine injection warp
The monitoring problem of residual protein after refinement treatment, makes the safety of traditional Chinese medicine injection be guaranteed, can be used for traditional Chinese medicine injection
Produce the monitoring and control of overall process intermediate protein concentration.
Detailed description of the invention
Fig. 1 is the Raman spectrogram of red sage root extract calibration set sample；
Fig. 2 is spectrogram of the red sage root extract calibration set sample after preferred pretreatment；
Fig. 3 is the linear regression curves that Coomassie Brilliant Blue measures protein concentration in seralbumin matter standard solution；
Fig. 4 is the correlativity figure in red sage root extract between the reference point and model predication value of protein concentration.
Specific embodiment
Below with reference to embodiment, the present invention is further illustrated, but is not intended as the foundation limited the present invention.
Embodiment.The method for quickly detecting protein concentration in Chinese medicine extract, passes through and measures Chinese medicine extract to be measured
The concentration of wherein protein is calculated using the calibration model of building for the Raman spectrum data of protein concentration.
The method is to measure the Raman spectrum data of calibration samples, and survey by collecting Chinese medicine extract calibration samples
The concentration for determining protein in calibration samples, according to the concentration of protein in positive sample for school building and corresponding Raman spectrum data
Calibration model is established, by the calibration model of the Raman spectrum data input building of Chinese medicine extract to be measured, can be calculated
The wherein concentration of protein.
The method is by collecting Chinese medicine extract calibration samples, using the drawing of Raman spectroscopy measurement calibration samples
Graceful spectroscopic data, and use Coomassie Brilliant Blue measures the conduct reference point of protein in calibration samples, using offset minimum binary
Method or principal component regression method establish the straightening die of relationship between Raman spectrum data and protein concentration in calibration samples respectively
Wherein protein can be calculated by the calibration model of the Raman spectrum data input building of Chinese medicine extract to be measured in type
Concentration.
The Raman spectrum data of the calibration samples is measured by Raman spectroscopy, and calibration samples are put into
It in the quartz colorimetric utensil of 10mm light path, is placed on the matched cell rack of Raman spectrometer, carries out spectral scan, obtain correction
Sample Raman spectrum data, wherein Raman spectrometer optical maser wavelength 785nm, Raman shift range 173.7-3200.8cm-1, differentiate
Rate 4.5cm-1, laser intensity 50%, time of integration 10000ms.
The Raman spectrum data of the Chinese medicine extract to be measured is measured by Raman spectroscopy, by traditional Chinese medicine extraction
Liquid is put into the quartz colorimetric utensil of 10mm light path, is placed on the matched cell rack of Raman spectrometer, is carried out spectral scan, is obtained
The Raman spectrum data of Chinese medicine extract is taken, the Raman spectrum of each Chinese medicine extract need to deduct dark current, scan 3 times, make even
Equal spectroscopic data；Wherein Raman spectrometer optical maser wavelength 785nm, Raman shift range 173.7-3200.8cm-1, resolution ratio
4.5cm-1, laser intensity 50%, time of integration 10000ms.
The calibration model is Applied Chemometrics technology, establishes Chinese medicine extract using multivariate data processing software
The Multivariate Correction regression model of middle protein concentration, the multivariate data analysis method are principal component regression method or offset minimum binary
Method intersects progress internal verification using leaving-one method and determines best number of principal components, the coefficient of determination >=0.95 of the calibration model.Institute
State the verifying for establishing that calibration model further includes calibration model, the verifying are as follows: take unknown extracting solution, measure its Raman signatures spectrum
Data are inputted in calibration model, are calculated the concentration of protein in sample, are measured with the sample according to Coomassie Brilliant Blue
Protein concentration compares, it is desirable that measurement error is less than 10%.
It the described method comprises the following steps:
(a) Chinese medicine extract calibration samples are collected；
(b) calibration samples protein concentration reference point measures: using using egg in Coomassie Brilliant Blue measurement calibration samples
White matter concentration；
(c) measurement of calibration samples Raman spectrum: calibration set sample is placed in the matched cell rack of Raman spectrometer
It is scanned, obtains calibration samples Raman signatures spectroscopic data；
(d) establish calibration model: using multivariate data analysis method, establish in calibration set sample protein concentration with it is right
The mathematical model of relationship, as calibration model between the Raman signatures spectrum answered；
(e) Chinese medicine extract to be measured detection: acquiring the Raman signatures spectroscopic data of Chinese medicine extract to be measured, and by this spectrum
Data input in calibration model, calculate protein concentration in sample to be tested.
In above-mentioned quick detection Chinese medicine extract the method for protein concentration in red sage root extract protein concentration it is fast
The application of speed measurement.
Claims (9)
1. quickly detecting the method for protein concentration in Chinese medicine extract, it is characterised in that: pass through and measure Chinese medicine extract to be measured
Protein concentration Raman spectrum data, the concentration of wherein protein is calculated using the calibration model of building.
2. the method for protein concentration in quick detection Chinese medicine extract according to claim 1, it is characterised in that: described
Method is to measure the Raman spectrum data of calibration samples, and measure in calibration samples by collecting Chinese medicine extract calibration samples
The concentration of protein establishes straightening die according to the concentration of protein in positive sample for school building and corresponding Raman spectrum data
Wherein protein can be calculated by the calibration model of the Raman spectrum data input building of Chinese medicine extract to be measured in type
Concentration.
3. the method for protein concentration in quick detection Chinese medicine extract according to claim 2, it is characterised in that: described
Method is that the Raman spectrum data of calibration samples is measured using Raman spectroscopy by collecting Chinese medicine extract calibration samples,
And use Coomassie Brilliant Blue measures the conduct reference point of protein in calibration samples, is returned using Partial Least Squares or principal component
Gui Fa establishes the calibration model of relationship between Raman spectrum data and protein concentration in calibration samples respectively, by Chinese medicine to be measured
In the calibration model of the Raman spectrum data input building of extracting solution, the concentration of wherein protein can be calculated.
4. the method for protein concentration in quick detection Chinese medicine extract according to claim 3, it is characterised in that: described
The Raman spectrum data of calibration samples is measured by Raman spectroscopy, and calibration samples are put into the quartz of 10mm light path
It in cuvette, is placed on the matched cell rack of Raman spectrometer, carries out spectral scan, obtain calibration samples Raman spectrum number
According to wherein Raman spectrometer optical maser wavelength 785nm, Raman shift range 173.7-3200.8cm-1, resolution ratio 4.5cm-1, laser
Intensity 50%, time of integration 10000ms.
5. the method for protein concentration, feature in quick detection Chinese medicine extract according to claim 1-4
Be: the Raman spectrum data of the Chinese medicine extract to be measured is measured by Raman spectroscopy, by Chinese medicine extract
It is put into the quartz colorimetric utensil of 10mm light path, is placed on the matched cell rack of Raman spectrometer, carry out spectral scan, obtain
The Raman spectrum data of Chinese medicine extract, the Raman spectrum of each Chinese medicine extract need to deduct dark current, scan 3 times, are averaged
Spectroscopic data；Wherein Raman spectrometer optical maser wavelength 785nm, Raman shift range 173.7-3200.8cm-1, resolution ratio 4.5cm-1, laser intensity 50%, time of integration 10000ms.
6. the method for protein concentration, feature in quick detection Chinese medicine extract according to claim 1-3
Be: the calibration model is Applied Chemometrics technology, is established in Chinese medicine extract using multivariate data processing software
The Multivariate Correction regression model of protein concentration, the multivariate data analysis method are principal component regression method or offset minimum binary
Method intersects progress internal verification using leaving-one method and determines best number of principal components, the coefficient of determination >=0.95 of the calibration model.
7. the method for protein concentration, feature in quick detection Chinese medicine extract according to claim 1-3
It is: described to establish the verifying that calibration model further includes calibration model, the verifying are as follows: take unknown extracting solution, measure its Raman
Characteristic spectrum data, are inputted in calibration model, the concentration of protein in sample are calculated, with the sample according to Coomassie brilliant blue
The protein concentration of method measurement compares, it is desirable that measurement error is less than 10%.
8. the method for protein concentration, feature in quick detection Chinese medicine extract according to claim 1-3
It is: the described method comprises the following steps:
(a) Chinese medicine extract calibration samples are collected；
(b) calibration samples protein concentration reference point measures: using using protein in Coomassie Brilliant Blue measurement calibration samples
Concentration；
(c) measurement of calibration samples Raman spectrum: calibration set sample is placed in the matched cell rack of Raman spectrometer and is carried out
Scanning obtains calibration samples Raman signatures spectroscopic data；
(d) establish calibration model: using multivariate data analysis method, establish in calibration set sample protein concentration with it is corresponding
The mathematical model of relationship, as calibration model between Raman signatures spectrum；
(e) Chinese medicine extract to be measured detection: acquiring the Raman signatures spectroscopic data of Chinese medicine extract to be measured, and by this spectroscopic data
It inputs in calibration model, calculates protein concentration in sample to be tested.
9. the method for protein concentration is in pellet in a kind of quick detection Chinese medicine extract according to claim 1-8
Join the application of protein concentration in extracting solution quickly measured.
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