CN102706844A - Method for detecting content of methylene blue in blood plasma - Google Patents
Method for detecting content of methylene blue in blood plasma Download PDFInfo
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Abstract
The invention discloses a method for detecting the content of methylene blue in blood plasma. The method comprises the following steps of: (a) adding carboxymethyl-beta-cyclodextrin saturated solution into blank blood plasma, enabling the concentration of the mixed solution to reach 1-4.5mmol/L and maintaining the constant volume to obtain a blood plasma blank sample; (b) adding quantitative methylene blue solution into the blank blood plasma and adding the carboxymethyl-beta-cyclodextrin saturated solution to obtain a standard blood plasma sample containing the methylene blue with the concentration of Cs; and (c) adding the carboxymethyl-beta-cyclodextrin saturated solution in a blood plasma sample containing the methylene blue to obtain a blood plasma to-be-measured sample containing the methylene blue with the concentration of Cs; detecting the strength or the area of a fluorescence peak of the sample; and then calculating according to a reference substance comparison method to obtain the concentration Cs of the methylene blue in the blood plasma to-be-measured sample. According to the method disclosed by the invention, the lowest detectable limit is 0.012mumol/L and the unexpected effect is obtained.
Description
Technical field
The present invention relates to a kind of method that detects methylenum careuleum content in the blood plasma, belong to clinical examination, Pharmaceutical Analysis, bioanalysis and analytical chemistry field.
Background technology
In order to reduce the danger of the infective virus of transfusing blood clinically; Strengthen the security of blood transfusion; The general virus that adopts in the methylenum careuleum deactivation blood plasma, i.e. photochemical virus deactivation method is promptly through adding a certain amount of photosensitizer methylenum careuleum in blood plasma; Under illumination, produce photochemical reaction, thereby make in the blood plasma virally inactivated.In order to guarantee the security of blood plasma; Before deactivation, must guarantee to have in the blood plasma photosensitizer methylenum careuleum (being sufficiently high burst size) of enough concentration; Must guarantee the photosensitizer methylenum careuleum (promptly enough low residual quantity) of enough low concentrations in the finished product blood plasma after deactivation again; Analyze from above-mentioned blood plasma disposing technique, this obviously is mutual contradiction.
In order to solve this preferably to contradiction, use complete inactivation of virus bag usually, it is made up of methylenum careuleum stowage arrangement, illumination inactivation of virus bag, methylenum careuleum filtration unit, finished product blood plasma storage bag and corresponding pipeline thereof and switch.When blood plasma injected and pass through the methylenum careuleum stowage arrangement, methylenum careuleum wherein was released out, got into illumination inactivation of virus bag with blood plasma, after inactivation of virus, through the most methylenum careuleum of methylenum careuleum filtration unit filtering, thereby obtained the clinical finished product blood plasma of using.
Blood plasma through the filtration unit filtering; Though the concentration of the methylenum careuleum that it is residual is in safe range; But for the patient who expects after necessary a large amount of or long-term infusion blood plasma or the plasma transfusion long-term surviving; Its influence is still unknown, and particularly mutagenic risk might increase, so residual methylenum careuleum concentration is significant in the control blood plasma.
At present; The methylenum careuleum Determination on content method in the blood plasma of detecting has AAS, chemoluminescence method, high performance liquid chromatography and is the resonance rayleigh light scattering method etc. of probe with the gold nano particulate; These methods have shortcomings such as the low and/or complex operation of sensitivity, usually also need consume more a large amount of blood plasma.
Reported methylenum careuleum residual quantity detection method in the blood plasma in the version in 2012 " China's blood transfusion regulations for technical operation ", adopted solid phase extraction column that the methylenum careuleum in plasma sample and the standard items is carried out extract and separate, in conjunction with its residual quantity of spectrophotometry.Yet find that in practical application there are two significant disadvantage in this method: (1) operation is loaded down with trivial details especially, time-consuming, and the blood plasma consumption is big; (2) sample mensuration absorbance is low especially, usually less than 0.01; This and the Pharmacopoeia of the People's Republic of China " appendix IV A UV-VIS spectrophotometry " the item needed absorbance 0.3 ~ 0.7 of accurate mensuration content of defined down compare, and the residual quantity of measuring obviously is inaccurate.And in fact, when low residual amt, the reappearance of absorbance is poor especially, is indicating and is measuring the unreliable of result.This point also is consistent with bibliographical information.When being 0.13 umol/L (micromoles per liter) for common methylenum careuleum residual quantity, [detection and the quality control of inactivation of virus blood plasma methylenum careuleum content, China's blood transfusion magazine, 2011 such as Yang Xia; 24:881-883] method measure, its absorbance should be 0.0076, [solid phase extraction detects the methylenum careuleum in the inactivation of virus blood plasma to Bai Li etc.; Medicine forum magazine, 2010,31:108-109] method measure; Its absorbance should be 0.0042, [detection of methylenum careuleum content in the blood plasma, clinical blood transfusion and check such as Zhang Shuqin; 2010,12:36-38] method measure, its absorbance should be 0.0011.Therefore for actual sample, these absorbances all are so low, and obviously, the reliability that SPE-ultraviolet-visible spectrophotometry is measured the result is extremely low.
To the problems referred to above; [document 1: beta-schardinger dextrin-enhanced sensitivity fluorescence spectrophotometry detects the blood plasma methylenum careuleum to Zhou Qungang etc.; The clinical examination magazine, 2011,29 (5): 344-345] photosensitizer methylenum careuleum in a kind of beta-schardinger dextrin-enhanced sensitivity fluorescence spectrophotometry blood plasma is disclosed; With respect to said method, this method has obviously overcome above-mentioned two shortcomings.But the minimum 0.089 μ mol/L that quantitatively is limited to that this method can accurately detect is promptly a little less than common methylenum careuleum residual quantity (at present, for normally used complete inactivation of virus bag, its methylenum careuleum is residual usually about 0.13 μ mol/L).
In fact; Minimum residual quantity tends to be lower than 0.089 μ mol/L in the actual plasma sample; [methylenum careuleum/photochemical method is used in blood-plasma virus killing, Chinese public health, 2008 like document 2; 24 (11), 1365] the final residue amount of mentioning methylenum careuleum in this research in is 0.07 ~ 0.62 μ mol/L (the 4th section in an article).Moreover from long-range analysis, along with the raising that the development of technology and people require blood plasma, the methylenum careuleum residual quantity will be more and more lower.This obviously can cause the method in the document 1 to lose efficacy, and can't be used for the residual conventional prosecution of blood plasma methylenum careuleum.
Therefore, develop a kind of method that detects methylenum careuleum content in the blood plasma,, and improve detection sensitivity, have active operation significance with further reduction detectability.
Summary of the invention
The object of the invention provides a kind of method that detects methylenum careuleum content in the blood plasma, with further reduction detectability, and improves detection sensitivity.
For achieving the above object, the technical scheme that the present invention adopts is: a kind of method that detects methylenum careuleum content in the blood plasma comprises the steps:
(a) in blank plasma, add the carboxymethyl-beta-cyclodextrin saturated solution, making its concentration is 1 ~ 4.5mmol/L, and constant volume gets the blood plasma dummy, adopts fluorospectrophotometer to detect, and recording fluorescence peak intensity or fluorescence peak area is Fo;
(b) in blank plasma, add quantitative methylene blue solution; And adding carboxymethyl-beta-cyclodextrin saturated solution; It is identical with above-mentioned blood plasma dummy to add concentration, constant volume, and must contain methylenum careuleum concentration is the blood plasma master sample of Cs; Adopt fluorospectrophotometer to detect, recording fluorescence peak intensity or fluorescence peak area is Fs;
(c) in the plasma sample to be measured that contains methylenum careuleum, add the carboxymethyl-beta-cyclodextrin saturated solution; It is identical with above-mentioned blood plasma dummy to add concentration; Constant volume; Must contain methylenum careuleum concentration is the blood plasma sample to be tested of Cx, adopts fluorospectrophotometer to detect, and recording fluorescence peak intensity or fluorescence peak area is Fx;
Calculate by the reference substance relative method then and can obtain the methylenum careuleum concentration C x in the plasma sample to be measured;
Said step (a) and (b) are identical with the condition that (c) middle fluorospectrophotometer that adopts detects; Excitation wavelength is 665 ± 8nm, and the emission wavelength sweep limit is 670 ~ 715nm, excites slit width 2 ~ 10nm; Emission slit width 2 ~ 10nm; Sweep velocity 100 ~ 1000nm/min excites light path 2 ~ 10mm, emission light path 2 ~ 10mm.
In the preceding text, the said carboxymethyl-beta-cyclodextrin saturated solution that in blank plasma, adds, making its concentration is 1 ~ 4.5mmol/L, the concentration here is meant the volumetric molar concentration that adds the carboxymethyl-beta-cyclodextrin in the carboxymethyl-beta-cyclodextrin saturated solution blood plasma afterwards.
In the technique scheme, said step (a) and (b) adopt damping fluid or two to steam water with constant volume (c) to carry out said PBS damping fluid, Tris-HCl damping fluid or phosphate sodium dihydrogen buffer solution.
Preferably, said damping fluid is the PBS damping fluid.Its pH value is 7.2.
Corresponding with it another kind of technical scheme, a kind of method that detects methylenum careuleum content in the blood plasma comprises the steps:
(a) at least 5 blank plasmas, add the different methylene blue solutions of measuring respectively; And adding carboxymethyl-beta-cyclodextrin saturated solution; It is identical with above-mentioned blood plasma dummy to add concentration, and constant volume must contain the blood plasma master sample of methylenum careuleum series concentration; Adopt fluorospectrophotometer to detect, record a series of fluorescence peak intensity or fluorescence peak area;
(b) in the plasma sample to be measured that contains methylenum careuleum, add the carboxymethyl-beta-cyclodextrin saturated solution; It is identical with above-mentioned blank plasma sample to add concentration; Constant volume; Must contain methylenum careuleum concentration is the blood plasma sample to be tested of Cx, adopts fluorospectrophotometer to detect, and recording fluorescence peak intensity or fluorescence peak area is Fx;
Can obtain the methylenum careuleum concentration C x in the plasma sample to be measured by calibration curve method calculating;
Said step (a) is identical with the condition that (b) middle fluorospectrophotometer that adopts detects; Excitation wavelength is 665 ± 8nm, and the emission wavelength sweep limit is 670 ~ 715nm, excites slit width 2 ~ 10nm; Emission slit width 2 ~ 10nm; Sweep velocity 100 ~ 1000nm/min excites light path 2 ~ 10mm, emission light path 2 ~ 10mm.
In the technique scheme; In step (a) before, also carry out following steps (c): in blank plasma, add the carboxymethyl-beta-cyclodextrin saturated solution, making its concentration is 1 ~ 4.5mmol/L; Constant volume; Get the blood plasma dummy, adopt fluorospectrophotometer to detect, recording fluorescence peak intensity or fluorescence peak area is Fo.
In the preceding text, the said carboxymethyl-beta-cyclodextrin saturated solution that in blank plasma, adds, making its concentration is 1 ~ 4.5mmol/L, the concentration here is meant the volumetric molar concentration that adds the carboxymethyl-beta-cyclodextrin in the carboxymethyl-beta-cyclodextrin saturated solution blood plasma afterwards.
In the preceding text, when calculating the methylenum careuleum concentration in the plasma sample to be measured by calibration curve method, fluorescence signal wherein can be deducted substrate, also can not deduct substrate.
In the technique scheme, said step (a) and (b) adopt damping fluid or two to steam water with constant volume (c) to carry out, and said damping fluid is PBS damping fluid, Tris-HCl damping fluid or phosphate sodium dihydrogen buffer solution.
Preferably, said damping fluid is the PBS damping fluid.Its pH value is 7.2.
With respect to [documents 1: beta-schardinger dextrin-enhanced sensitivity fluorescence spectrophotometry detection blood plasma methylenum careuleum such as Zhou Qungang; The clinical examination magazine; 2011; 29 (5): 344-345] disclosed a kind of beta-schardinger dextrin-enhanced sensitivity fluorescence spectrophotometry, the present invention had both kept the advantage of said method, had overcome the shortcoming of said method simultaneously again.It has its source in, and the ultimate principle that the present invention utilized has different significantly with said method.The hydrophobic interaction that method in the document 1 is based on the beta-schardinger dextrin-inner chamber causes the fluorescence molecule methylenum careuleum to get into this inner chamber, forms super molecule inclusion compound, thereby strengthens the fluorescence of methylenum careuleum, improves its detection sensitivity, the minimum quantitative limit of reduction method.And the present invention strengthens the methylenum careuleum fluorescence except utilizing above-mentioned hydrophobic interaction; Also utilized another even more important interaction; Be carboxymethyl-beta-cyclodextrin with negative charge and methylenum careuleum the strong electrical interaction between positively charged; The degree that makes methylenum careuleum get into the hydrophobicity inner chamber significantly increases, and the ability that forms super molecule inclusion compound is stronger, thereby has caused detection sensitivity of the present invention to significantly improve; Minimum quantitative limit also significantly reduces; Evidence: LDL of the present invention has been low to moderate 0.012 μ mol/L, has obtained beyond thought effect, makes method of the present invention can be used for effective detection of blood plasma inactivation of virus photosensitizer methylenum careuleum residual quantity effectively.
Detection method of the present invention can be used for the mensuration of residual methylenum careuleum in assay and the blood plasma of methylenum careuleum in the blood plasma; The burst size that also can be used for methylenum careuleum before the blood-plasma virus killing measure with release efficiency and inactivation of virus after filter to the filtration efficiency mensuration of methylenum careuleum, also can be used for the determination of residual amount of photosensitizer methylenum careuleum in the finished product blood plasma.
Because the technique scheme utilization, the present invention compared with prior art has advantage:
1. the present invention has developed the method for methylenum careuleum content in a kind of new detection blood plasma, and evidence: LDL of the present invention has been low to moderate 0.012 μ mol/L, has obtained beyond thought effect.
2. assay method of the present invention is simple, quick; Directly measure methylenum careuleum in the blood plasma without separation, enrichment; And the blood plasma consumption is less, and the blood plasma resource of saves valuable is specially adapted to the conventional prosecution of methylenum careuleum burst size, filtering amount and residual quantity thereof in the blood plasma product inactivation of virus process.
3. typical curve linear regression property of the present invention is good, and quantitative limit is low; The present invention is through linear scope determination test; The proof methylenum careuleum is in 0.012 ~ 2.50 μ mol/L scope, and concentration and response are good linear relationship, and the plasma sample working curve is F=91.223C+4.942; The Quality Control requirement of methylenum careuleum in the blood plasma can be satisfied in r=0.9987.
4. experiment showed, that the recovery of the present invention in the range of linearity is 99.7 ~ 106.1%; The day within variance coefficient (CV) that middle concentration methylenum careuleum (1.07 μ mol/L) is measured is 0.13%, CV is 3.4% in the daytime; Have higher detection precision and accuracy.
Embodiment
Below in conjunction with embodiment the present invention is further described:
Embodiment one
A kind of method that detects methylenum careuleum content in the blood plasma comprises the steps:
In 300 μ L blank plasmas, add carboxymethyl-beta-cyclodextrin saturated solution 280 μ L, add PBS damping fluid 120 μ L, get the blood plasma dummy;
In 300 μ L blank plasmas, add 5 μ mol/L methylenum careuleum storing solutions, 56 μ L, and add carboxymethyl-beta-cyclodextrin saturated solution 280 μ L, add PBS damping fluid 64 μ L, must contain the blood plasma master sample of methylenum careuleum;
Contain to 300 μ L and to add carboxymethyl-beta-cyclodextrin saturated solution 280 μ L in the plasma sample to be measured of methylenum careuleum, add PBS damping fluid 120 μ L, the blood plasma sample to be tested;
Adopt fluorospectrophotometer to detect, testing conditions is: excitation wavelength 665nm, emission wavelength 680nm; Excite and launch slit width and be 5nm; Sweep velocity 100nm/min excites light path 10mm, emission light path 10mm; Measure the fluorescence intensity of above-mentioned sample, data are calculated methylenum careuleum concentration in the plasma sample to be measured according to the reference substance relative method in view of the above.
Adopt embodiment one described method, done a series of mensuration, and [beta-schardinger dextrin-enhanced sensitivity fluorescence spectrophotometry detects the blood plasma methylenum careuleum with document 1; The clinical examination magazine; 2011,29 (5): 344-345] disclosed beta-schardinger dextrin-enhanced sensitivity fluorescence spectrophotometry is done contrast, and the result is following:
Visible by last table, the minimum 0.012 μ mol/L that quantitatively is limited to of method of the present invention not only can accurately detect methylenum careuleum residual quantity common in the existing blood plasma 0.13 μ mol/L, also can effectively detect the actual sample of other low residual amt.And the detection method of document 1 can detect for common residual quantity 0.13 μ mol/L, but precision reduces (in a few days CV=5.7%, CV=6.3%) in the daytime.
Embodiment two
A kind of method that detects methylenum careuleum content in the blood plasma comprises the steps:
In 5 300 μ L blank plasmas, add 0.1 μ mol/L methylenum careuleum storing solution, 50 μ L respectively; 5 μ mol/L methylenum careuleum storing solutions, 28 μ L, 56 μ L; 25 μ mol/L methylenum careuleum storing solutions, 28 μ L, 42 μ L, and add carboxymethyl-beta-cyclodextrin saturated solution 280 μ L respectively add PBS damping fluid 70 μ L, 92 μ L, 64 μ L, 92 μ L and 78 μ L respectively, must contain 5 of the blood plasma master samples of methylenum careuleum;
Contain to 300 μ L and to add carboxymethyl-beta-cyclodextrin saturated solution 280 μ L in the plasma sample to be measured of methylenum careuleum, add PBS damping fluid 120 μ L, the blood plasma sample to be tested;
Adopt fluorospectrophotometer to detect, testing conditions is: excitation wavelength 665nm, emission wavelength 680nm; Excite and launch slit width to be 5nm, sweep velocity 100nm/min excites light path 10mm; Launch light path 10mm, measure the fluorescence intensity of above-mentioned sample.Its fluorescence signal is not deducted the substrate of dummy, calculates methylenum careuleum concentration in the plasma sample to be measured according to calibration curve method.
Embodiment three
A kind of method that detects methylenum careuleum content in the blood plasma comprises the steps:
In 300 μ L blank plasmas, add carboxymethyl-beta-cyclodextrin saturated solution 280 μ L, add PBS damping fluid 120 μ L, get the blood plasma dummy;
In 5 300 μ L blank plasmas, add 0.1 μ mol/L methylenum careuleum storing solution, 50 μ L respectively; 5 μ mol/L methylenum careuleum storing solutions, 28 μ L, 56 μ L; 25 μ mol/L methylenum careuleum storing solutions, 28 μ L, 42 μ L, and add carboxymethyl-beta-cyclodextrin saturated solution 280 μ L respectively add PBS damping fluid 70 μ L, 92 μ L, 64 μ L, 92 μ L and 78 μ L respectively, must contain 5 of the blood plasma master samples of methylenum careuleum;
Contain to 300 μ L and to add carboxymethyl-beta-cyclodextrin saturated solution 280 μ L in the plasma sample to be measured of methylenum careuleum, add PBS damping fluid 120 μ L, the blood plasma sample to be tested;
Adopt fluorospectrophotometer to detect, testing conditions is: excitation wavelength 665nm, emission wavelength 680nm; Excite and launch slit width to be 5nm, sweep velocity 100nm/min excites light path 10mm; Launch light path 10mm, measure the fluorescence intensity of above-mentioned sample.Fluorescence signal with after the substrate of deduction dummy calculates methylenum careuleum concentration in the plasma sample to be measured according to calibration curve method.
Claims (7)
1. a method that detects methylenum careuleum content in the blood plasma is characterized in that, comprises the steps:
(a) in blank plasma, add the carboxymethyl-beta-cyclodextrin saturated solution, making its concentration is 1 ~ 4.5mmol/L, and constant volume gets the blood plasma dummy, adopts fluorospectrophotometer to detect, and recording fluorescence peak intensity or fluorescence peak area is Fo;
(b) in blank plasma, add quantitative methylene blue solution; And adding carboxymethyl-beta-cyclodextrin saturated solution; It is identical with above-mentioned blood plasma dummy to add concentration, constant volume, and must contain methylenum careuleum concentration is the blood plasma master sample of Cs; Adopt fluorospectrophotometer to detect, recording fluorescence peak intensity or fluorescence peak area is Fs;
(c) in the plasma sample to be measured that contains methylenum careuleum, add the carboxymethyl-beta-cyclodextrin saturated solution; It is identical with above-mentioned blood plasma dummy to add concentration; Constant volume; Must contain methylenum careuleum concentration is the blood plasma sample to be tested of Cx, adopts fluorospectrophotometer to detect, and recording fluorescence peak intensity or fluorescence peak area is Fx;
Calculate by the reference substance relative method then and can obtain the methylenum careuleum concentration C x in the plasma sample to be measured;
Said step (a) and (b) are identical with the condition that (c) middle fluorospectrophotometer that adopts detects; Excitation wavelength is 665 ± 8nm, and the emission wavelength sweep limit is 670 ~ 715nm, excites slit width 2 ~ 10nm; Emission slit width 2 ~ 10nm; Sweep velocity 100 ~ 1000nm/min excites light path 2 ~ 10mm, emission light path 2 ~ 10mm.
2. the method for methylenum careuleum content in the detection blood plasma according to claim 1; It is characterized in that: said step (a) and (b) adopt damping fluid or two to steam water with constant volume (c) to carry out, and said damping fluid is PBS damping fluid, Tris-HCl damping fluid or phosphate sodium dihydrogen buffer solution.
3. the method for methylenum careuleum content in the detection blood plasma according to claim 2 is characterized in that: said damping fluid is the PBS damping fluid.
4. a method that detects methylenum careuleum content in the blood plasma is characterized in that, comprises the steps:
(a) at least 5 blank plasmas, add the different methylene blue solutions of measuring respectively; And adding carboxymethyl-beta-cyclodextrin saturated solution; It is identical with above-mentioned blood plasma dummy to add concentration, and constant volume must contain the blood plasma master sample of methylenum careuleum series concentration; Adopt fluorospectrophotometer to detect, record a series of fluorescence peak intensity or fluorescence peak area;
(b) in the plasma sample to be measured that contains methylenum careuleum, add the carboxymethyl-beta-cyclodextrin saturated solution; It is identical with above-mentioned blank plasma sample to add concentration; Constant volume; Must contain methylenum careuleum concentration is the blood plasma sample to be tested of Cx, adopts fluorospectrophotometer to detect, and recording fluorescence peak intensity or fluorescence peak area is Fx;
Can obtain the methylenum careuleum concentration C x in the plasma sample to be measured by calibration curve method calculating;
Said step (a) is identical with the condition that (b) middle fluorospectrophotometer that adopts detects; Excitation wavelength is 665 ± 8nm, and the emission wavelength sweep limit is 670 ~ 715nm, excites slit width 2 ~ 10nm; Emission slit width 2 ~ 10nm; Sweep velocity 100 ~ 1000nm/min excites light path 2 ~ 10mm, emission light path 2 ~ 10mm.
5. the method for methylenum careuleum content is characterized in that in the detection blood plasma according to claim 4, in step (a) before; Also carry out following steps (c): in blank plasma, add the carboxymethyl-beta-cyclodextrin saturated solution; Making its concentration is 1 ~ 4.5mmol/L, and constant volume gets the blood plasma dummy; Adopt fluorospectrophotometer to detect, recording fluorescence peak intensity or fluorescence peak area is Fo.
6. according to the method for methylenum careuleum content in claim 4 or the 5 described detection blood plasma; It is characterized in that: said step (a) and (b) adopt damping fluid or two to steam water with constant volume (c) to carry out, and said damping fluid is PBS damping fluid, Tris-HCl damping fluid or phosphate sodium dihydrogen buffer solution.
7. the method for methylenum careuleum content in the detection blood plasma according to claim 6 is characterized in that: said damping fluid is the PBS damping fluid.
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CN103630521A (en) * | 2013-12-05 | 2014-03-12 | 北京科技大学 | Method based on fluorescence silver nano-clusters for detecting cysteamine in blood serum |
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CN110658170A (en) * | 2019-10-16 | 2020-01-07 | 西安市中心血站 | Method for detecting methylene blue in virus inactivated plasma based on fluorescence resonance energy transfer |
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