CN110658170A - Method for detecting methylene blue in virus inactivated plasma based on fluorescence resonance energy transfer - Google Patents
Method for detecting methylene blue in virus inactivated plasma based on fluorescence resonance energy transfer Download PDFInfo
- Publication number
- CN110658170A CN110658170A CN201910982767.1A CN201910982767A CN110658170A CN 110658170 A CN110658170 A CN 110658170A CN 201910982767 A CN201910982767 A CN 201910982767A CN 110658170 A CN110658170 A CN 110658170A
- Authority
- CN
- China
- Prior art keywords
- methylene blue
- virus inactivated
- inactivated plasma
- concentration
- plasma
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- 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
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
Abstract
The invention discloses a method for detecting methylene blue in virus inactivated plasma based on fluorescence resonance energy transfer, which comprises the following steps: step one, establishing a methylene blue detection standard curve to obtain a standard curve equation. Selecting virus inactivated plasma treated by methylene blue, adding a methylene blue standard solution into the virus inactivated plasma, wherein the concentration of the added methylene blue in the virus inactivated plasma is A, adding a gold nanocluster stabilized by bovine serum albumin, determining the fluorescence intensity of the virus inactivated plasma at the moment to be I ', substituting the I' into the standard curve equation in the step one to obtain the total concentration C of the methylene blue in the virus inactivated plasma, and obtaining the concentration of the original methylene blue in the virus inactivated plasma through C-A. The concentration of total methylene blue in plasma, C, was derived from C-A as the original methylene blue concentration in virus inactivated plasma. The method has few steps, and can rapidly detect methylene blue in virus inactivated plasma.
Description
[ technical field ] A method for producing a semiconductor device
The invention belongs to the technical field of blood detection, and particularly relates to a method for detecting methylene blue in virus inactivated plasma based on fluorescence resonance energy transfer.
[ background of the invention ]
In order to prevent transfusion from infecting viruses, the clinical plasma widely adopts methylene blue illumination method to inactivate the plasma viruses, which is the only single blood component virus inactivation technology approved in clinic in China. Foreign researches show that the reduction degree of the plasma virus titer is directly related to the intensity of the used visible light and the concentration of the methylene blue, but the methylene blue has the possibility of causing mutation, and if the residual quantity is too much, the appearance and the color of the plasma are obviously changed and are not easily accepted by patients, so that the method has important significance for controlling the concentration of the methylene blue in the virus inactivated plasma. In the whole blood and component blood quality standard (2012 edition), it is specified that the residual quantity of methylene blue in virus-inactivated plasma is less than or equal to 0.3 moL/L.
The existing blood supply mechanism for measuring the residual quantity of methylene blue is a solid phase extraction-ultraviolet spectrophotometry method, and relatively mature kits are also available in the market. The solid phase extraction-ultraviolet spectrophotometry method is to extract methylene blue in virus inactivated plasma by using a solid phase small column, and then to determine the residual quantity of the methylene blue by using the maximum absorption of the methylene blue at 653 nm. Although the solid phase extraction-ultraviolet spectrophotometry can be used for measuring the residual quantity of methylene blue in virus inactivated plasma, the cost is high, the operation is complicated, and the sample needs 6mL after extraction-washing-elution-centrifugation. Therefore, blood resources are wasted to a certain extent, the working strength of workers is enhanced, and the working efficiency is further influenced.
[ summary of the invention ]
The invention aims to provide a method for detecting methylene blue in virus-inactivated plasma based on fluorescence resonance energy transfer, which has fewer steps and quickly realizes the detection of the methylene blue in the virus-inactivated plasma.
The invention adopts the following technical scheme: a method for detecting methylene blue in virus inactivated plasma based on fluorescence resonance energy transfer comprises the following steps:
step one, establishing a methylene blue detection standard curve to obtain a standard curve equation.
Selecting virus inactivated plasma treated by methylene blue, adding a methylene blue standard solution into the virus inactivated plasma, adding a bovine serum albumin-stabilized gold nanocluster, and determining the fluorescence intensity of the virus inactivated plasma to be I’Substituting the I' into the standard curve equation in the step one to obtain the concentration C of the total methylene blue in the virus inactivated plasma, and obtaining the concentration of the original methylene blue in the virus inactivated plasma from C-A.
Further, the concentration A in the second step is 10-30 μ M.
Further, the specific process of the first step is as follows: mixing the blank solution with a plurality of concentrations cMBAdding different methylene blue standard solutions into corresponding bovine serum albumin-stabilized gold nanoclusters respectively, and measuring fluorescence intensity, wherein the fluorescence intensity of the blank solution is I0The fluorescence intensity of the methylene blue standard solution is I, in order0-I)/I0Establishing a standard curve with the ordinate and the methylene blue concentration as the abscissa to obtain a standard curve equation of (I)0-I)/I0＝0.018cMB+0.021, wherein the blank solution is purified water without the addition of a standard solution of methylene blue.
The invention has the beneficial effects that: the method is simple to operate, has few steps, and quickly realizes the detection of the methylene blue in the virus inactivated blood plasma.
[ description of the drawings ]
FIG. 1 is a combined graph of an ultraviolet absorption spectrum of methylene blue and a fluorescence spectrum of a gold nano-particle with stable bovine serum albumin.
FIG. 2 is a fluorescence spectrum of a gold nanocluster stabilized with bovine serum albumin to which deionized water and methylene blue solution are added, respectively.
FIG. 3 is a graph showing the effect of pH change of a buffer solution on the degree of methylene blue quenching fluorescence of gold nanoclusters.
FIG. 4 is a graph of incubation temperature versus the degree of methylene blue quenched gold nanocluster fluorescence.
FIG. 5 is a graph of incubation time versus the degree of methylene blue quenching gold nanocluster fluorescence.
FIG. 6 fluorescence detection of fresh plasma without methylene blue treatment.
FIG. 7 is a fluorescence detection chart of ordinary plasma without methylene blue treatment.
[ detailed description ] embodiments
The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.
The embodiment of the invention discloses a method for detecting methylene blue in virus inactivated plasma based on fluorescence resonance energy transfer, which comprises the following steps:
step one, establishing a methylene blue detection standard curve: adding a blank solution and a plurality of methylene blue standard solutions with different concentrations of cMB into the gold nanocluster stabilized by bovine serum albumin respectively, and measuring the fluorescence intensity of the blank solution to be I0The fluorescence intensity of the methylene blue standard solution is I, in order0-I)/I0As ordinate, methylene blue concentration cMBEstablishing a standard curve for the abscissa to obtain a standard curve equation of (I)0-I)/I0＝0.018cMB+0.021, and a linear correlation coefficient r ═ 0.996. Wherein the blank solution is deionized water.
The specific process of establishing the standard curve is as follows: taking a plurality of 50 mu L portions of bovine serum albumin stable gold nanoclusters, respectively placing the portions into corresponding 1.5mL EP tubes, wherein the portions are consistent with the number of methylene blue standard solutions with different concentrations, respectively adding the bovine serum albumin stable gold nanoclusters into 100 mu L portions of methylene blue standard solutions, the final concentrations are 1440 mu M, 720 mu M, 360 mu M, 180 mu M, 72 mu M, 36 mu M, 3.6 mu M, 1.8 mu M, 0.9 mu M, 0.36 mu M and 0.18 mu M, respectively, uniformly mixing in a vortex mode, adding a phosphate buffer solution with pH of 7.4 to fix the volume to 500 mu L, fixing the volume, placing the mixture in a 37 ℃ environment for incubation reaction for 10min, and measuring various fluorescence intensities I and the fluorescence intensity I when water is used as a blank control0。
Selecting virus inactivated plasma treated by methylene blue, adding a methylene blue standard solution into the virus inactivated plasma, determining the final concentration of the added methylene blue in the virus inactivated plasma to be A, the concentration A to be 10-30 mu M, adding bovine serum albumin stable gold nanoclusters, determining the fluorescence intensity of the virus inactivated plasma to be I ', substituting the I' into the standard curve equation in the step one, obtaining the total concentration C of the methylene blue in the virus inactivated plasma, and obtaining the original concentration of the methylene blue in the virus inactivated plasma from C-A. I.e. residual amount of methylene blue in virus inactivated plasma-total concentration of methylene blue measured-concentration of added methylene blue standard solution. In order to ensure the accuracy of the detection result, the invention selects the intermediate concentration in the linear range of methylene blue detection, namely A is 10-30 mu M to carry out the measurement of the residual quantity of methylene blue in virus inactivated plasma.
According to the method, the linear range of methylene blue detection is 0.9-36 mu M according to the standard curve equation in the step one. In the whole blood and component blood quality standard (2012 edition), the quality standard of the methylene blue residue in virus inactivated plasma is not more than 0.3 μ M, is not in the linear range of methylene blue detection, and is lower than the lowest detected linear range concentration of the method of the present invention, so that additional methylene blue standard solution is required for detection. Due to the uncertainty of the residual quantity of methylene blue in the virus inactivated plasma.
In this example, the ultraviolet absorption spectrum of methylene blue and the fluorescence spectrum of the bovine serum albumin-stabilized gold nanoclusters were measured to obtain a combined graph of the ultraviolet absorption spectrum of methylene blue and the fluorescence spectrum of the bovine serum albumin-stabilized gold nanoclusters, and as shown in fig. 1, the spectra of both were overlapped by 30% or more, so that methylene blue could be detected by fluorescence energy transfer. The principle of fluorescence resonance energy transfer detection is that the fluorescent signal is reduced or enhanced by using a detection object to participate or interfere in the energy transfer process, so that the quantification of the detection object is realized. Because the emission spectrum of the gold nanocluster stabilized by the bovine serum albumin is obviously overlapped with the absorption spectrum of methylene blue, the methylene blue is positively charged, and the gold nanocluster stabilized by the bovine serum albumin is negatively charged in a certain pH value range, the two can be mutually combined through electrostatic interaction to generate fluorescence resonance energy transfer, so that the fluorescence of the gold nanocluster is quenched.
In order to verify the feasibility of the present invention, in this embodiment, an experiment was performed, in which deionized water and methylene blue solution in equal amounts were added to the bovine serum albumin-stabilized gold nanoclusters, respectively, and fluorescence was measured, and the obtained fluorescence spectrogram is shown in fig. 2, as can be seen from fig. 2, after adding methylene blue, the fluorescence of the bovine serum albumin-stabilized gold nanoclusters is quenched to a great extent, and the detection result is not affected by the fluorescence, so that it is feasible to detect methylene blue by using the bovine serum albumin-stabilized gold nanoclusters.
In this embodiment, a methylene blue solution is added to a gold nanocluster stabilized with bovine serum albumin, a buffer solution is added to obtain a sample, and then the following verification is performed:
1. the influence of the pH change of the phosphoric acid buffer solution on the fluorescence degree of methylene blue quenched gold nanoclusters is verified: the selected pH is shown in figure 3, the fluorescence intensity of the solution is measured under different pH values, the change of the pH value has little influence on the detection result, the quenching degree is maximum when the pH value is close to the pH value of human blood, and the detection result is most accurate.
2. Effect of incubation temperature and incubation time on assay results: the results of fluorescence detection after treating the samples at different incubation temperatures are shown in fig. 4, and it can be seen that incubation of the samples at the conventional laboratory temperature has no significant effect on the detection results. The method has low requirement on external experimental conditions and is convenient to popularize. Meanwhile, the influence of the incubation time on the quenching degree is also verified, as shown in fig. 5, the influence of the incubation time on the quenching degree is not large, and the method has low requirement on time and is beneficial to the actual operation of a laboratory.
In this example, 10 different fresh plasma without virus inactivation treatment and normal plasma, which means plasma left for more than 8 hours after blood collection, were selected to verify the specificity of the method established in the present invention. Adding 100 mu L of fresh plasma or common plasma which is not subjected to virus inactivation treatment into 50 mu L of bovine serum albumin stable gold nanoclusters, uniformly mixing the plasma by vortex, placing the mixture in a dry heater at 37 ℃ for incubation reaction for 10min, and measuring fluorescence to obtain the fluorescence intensity of each plasma, wherein the measurement result of the fresh plasma is shown in figure 6. The measurement results of the normal plasma are shown in fig. 7, which shows that other components in the normal plasma are not detected when methylene blue is not added, indicating that the interference of other components is not generated when methylene blue in the plasma is detected by adopting a fluorescence detection method; and the standing time of the blood plasma has no influence on the detection result.
In order to verify that the concentration of the added methylene blue meets the detection standard, the final concentrations of the added methylene blue are respectively selected to be 20 mu M, 10 mu M and 30 mu M, the detection is carried out, and meanwhile, the relative standard deviation and the accuracy are respectively calculated, and both the relative standard deviation and the accuracy meet the requirements. The method comprises the following specific steps:
example 1
The prepared virus inactivated fresh plasma sample was left 500. mu.L. 100. mu.L of the retained virus-inactivated fresh plasma sample was added with 100. mu.L of methylene blue standard solution to a final concentration of 20. mu.M and vortexed. And then adding 50 mu L of bovine serum albumin stabilized gold nanoclusters into the sample solution, uniformly mixing by vortex, adding a phosphoric acid buffer solution with the pH value of 7.4 to fix the volume to 500 mu L, uniformly mixing by vortex, placing the reaction system in an environment at 37 ℃ for incubation reaction for 10min, and determining the fluorescence intensity to be 2176. Fluorescence intensity I measured in the blank using 100. mu.L of deionized water instead of the added methylene blue standard solution0Is 3542. Combining the above data, using the standard curve equation (I)0-I)/I0＝0.018cMB+0.021, and the residual quantity of methylene blue in the fresh plasma after virus inactivation is 0.26. mu.M.
The residual quantity of the methylene blue in one virus inactivated plasma sample is parallelly determined for three times, and the relative standard deviation RSD is calculated for judgment, wherein the RSD is 0.6 percent and meets the requirement that the RSD is within 1 percent. The accuracy of the method is compared with the result measured by a solid phase extraction-ultraviolet spectrophotometry, and then the recovery rate is calculated to be 101%, so that the requirement of the accuracy on 98-102% is met. Examples 2 and 3 were both calculated using this method.
Example 2
The prepared virus inactivated normal plasma sample was left 500. mu.L. 100 mu L of the reserved virus inactivated common plasma sample is taken, 100 mu L of methylene blue standard solution is added into the sample, the final concentration is 10 mu M, and the mixture is swirled and mixed evenly. And then adding 50 mu L of bovine serum albumin stabilized gold nanoclusters into the sample solution, uniformly mixing by vortex, adding a phosphoric acid buffer solution with the pH value of 7.4, fixing the volume to 500 mu L, uniformly mixing by vortex, placing the reaction system in an environment at 37 ℃ for incubation reaction for 10min, and determining the fluorescence intensity to be 2783. Fluorescence intensity I measured in the blank using 100. mu.L of deionized water instead of the added methylene blue standard solution0Is 3502. Combining the above data, using the standard curve equation (I)0-I)/I0Methylene blue residue in the recovered virus inactivated normal plasma was calculated to be 0.24 μ M at a recovery of 99.41% with a relative standard deviation of 0.73%, when calculated as 0.018cMB + 0.021.
Example 3
500 mu L of the prepared virus inactivated fresh plasma sample is reserved, 100 mu L of the reserved virus inactivated fresh plasma sample is taken, 100 mu L of methylene blue standard solution is added into the sample, the final concentration is 30 mu M, and the mixture is swirled and mixed evenly. And then adding 50 mu L of bovine serum albumin stabilized gold nanoclusters into the sample solution, uniformly mixing by vortex, adding a phosphoric acid buffer solution with the pH value of 7.4 to fix the volume to 500 mu L, uniformly mixing by vortex, placing the reaction system in an environment at 37 ℃ for incubation reaction for 10min, and determining the fluorescence intensity to be 1526. Wherein 100. mu.L of deionized water was used in place of the added methylene blue standard solution in the blank, the fluorescence intensity I0 was measured to be 3514. Combining the above data, using the standard curve equation (I)0-I)/I0Methylene blue residue in the fresh, virus inactivated plasma was calculated to be 0.27 μ M as 0.018cMB +0.021, with a recovery of 98.76% and a relative standard deviation of 0.81%.
The method has the advantages of strong detection selectivity, simple operation, no need of complex sample pretreatment, less sample requirement, and capability of completing the determination of methylene blue only by reserving 500 mu L of plasma sample.
Claims (3)
1. The method for detecting methylene blue in virus inactivated plasma based on fluorescence resonance energy transfer is characterized by comprising the following steps:
step one, establishing a methylene blue detection standard curve to obtain a standard curve equation;
selecting virus inactivated plasma treated by methylene blue, adding a methylene blue standard solution into the virus inactivated plasma, adding a bovine serum albumin-stabilized gold nanocluster, and determining the fluorescence intensity of the virus inactivated plasma to be I’Introduction of said I’And (4) substituting the concentration C into the standard curve equation in the step one to obtain the concentration C of the total methylene blue in the virus inactivated plasma, and obtaining the concentration of the original methylene blue in the virus inactivated plasma from C-A.
2. The method for detecting methylene blue in virus inactivated plasma based on fluorescence resonance energy transfer as claimed in claim 1, wherein the concentration A in the second step is 10-30 μ M.
3. The method for detecting methylene blue in virus inactivated plasma based on fluorescence resonance energy transfer according to claim 1 or 2, wherein the specific process of the first step is as follows: mixing the blank solution with a plurality of concentrations cMBAdding different methylene blue standard solutions into corresponding bovine serum albumin-stabilized gold nanoclusters respectively, and measuring fluorescence intensity, wherein the blank solution is deionized water, and the fluorescence intensity of the blank solution is I0The fluorescence intensity of the methylene blue standard solution is I, in order0-I)/I0Establishing a standard curve with the ordinate and the methylene blue concentration as the abscissa to obtain a standard curve equation of (I)0-I)/I0＝0.018cMB+0.021。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910982767.1A CN110658170A (en) | 2019-10-16 | 2019-10-16 | Method for detecting methylene blue in virus inactivated plasma based on fluorescence resonance energy transfer |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910982767.1A CN110658170A (en) | 2019-10-16 | 2019-10-16 | Method for detecting methylene blue in virus inactivated plasma based on fluorescence resonance energy transfer |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110658170A true CN110658170A (en) | 2020-01-07 |
Family
ID=69040961
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910982767.1A Pending CN110658170A (en) | 2019-10-16 | 2019-10-16 | Method for detecting methylene blue in virus inactivated plasma based on fluorescence resonance energy transfer |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110658170A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102706844A (en) * | 2012-06-07 | 2012-10-03 | 苏州市中心血站 | Method for detecting content of methylene blue in blood plasma |
| CN103837516A (en) * | 2014-03-20 | 2014-06-04 | 南京工业大学 | Method for rapidly detecting glucose concentration based on gold nanocluster fluorescent probe |
| CN108120701A (en) * | 2017-11-21 | 2018-06-05 | 四川师范大学 | Ratiometric fluorescent probe for Visual retrieval mercury ion and preparation method thereof |
-
2019
- 2019-10-16 CN CN201910982767.1A patent/CN110658170A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102706844A (en) * | 2012-06-07 | 2012-10-03 | 苏州市中心血站 | Method for detecting content of methylene blue in blood plasma |
| CN103837516A (en) * | 2014-03-20 | 2014-06-04 | 南京工业大学 | Method for rapidly detecting glucose concentration based on gold nanocluster fluorescent probe |
| CN108120701A (en) * | 2017-11-21 | 2018-06-05 | 四川师范大学 | Ratiometric fluorescent probe for Visual retrieval mercury ion and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 鲁林林: "金簇/碳点荧光纳米探针的制备及其在分析检测中的应用", 《中国优秀博硕士学位论文全文数据库（硕士）工程科技I辑》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JPS62244399A (en) | Competative homogenous examination method | |
| CN1866012A (en) | Quantitative and quick immune detection method and special apparatus therefor | |
| Granadillo et al. | Determination of total chromium in whole blood, blood components, bone, and urine by fast furnace program electrothermal atomization AAS and using neither analyte isoformation nor background correction | |
| CN108823280A (en) | T4 polynueleotide kinase activity test method based on silver nanoclusters fluorescence probe | |
| Kasari et al. | Time-gated luminescence assay using nonmetal probes for determination of protein kinase activity-based disease markers | |
| CN106404726B (en) | A kind of fluorescence probe based on double-stranded DNA protection and the application in preparing detection plasmodium falciparum lactic dehydrogenase drug | |
| Martins et al. | Mass spectrometry–based method targeting Ig variable regions for assessment of minimal residual disease in multiple myeloma | |
| Liu et al. | Development of an amplified luminescent proximity homogeneous assay for quantitative determination of hepatitis B surface antigen in human serum | |
| Liu et al. | Detection of endometriosis with the use of plasma protein profiling by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry | |
| CN102634572B (en) | Reagent kit for detecting mRNA (messenger ribonucleic acid) expression of fused gene E2A-PBX1 via fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) | |
| EP3037807B1 (en) | Method and system for detecting a target within a population of molecules | |
| KR20040029076A (en) | A method for the determination of multiple analytes | |
| CN110658170A (en) | Method for detecting methylene blue in virus inactivated plasma based on fluorescence resonance energy transfer | |
| Qian et al. | Fluorescence resonance energy transfer detection methods: Sensitized emission and acceptor bleaching | |
| Yuwen et al. | A Quencher‐Based Blood‐Autofluorescence‐Suppression Strategy Enables the Quantification of Trace Analytes in Whole Blood | |
| Jiang et al. | Single nanoparticle analysis for homogeneous immunoassay of CA19-9 for serological evaluation | |
| CN111763713B (en) | Method and kit for detecting miRNA-21 based on target isothermal cycle amplification and nucleic acid test strip technology for non-diagnostic purposes | |
| JP5596124B2 (en) | Method and apparatus for detecting drugs in a sample | |
| Hong et al. | Simultaneous detection of three gynecological tumor biomarkers in clinical serum samples using an ICP-MS-based magnetic immunoassay | |
| CN105842219A (en) | Tyrosinase-assisted fluorescence enhanced tyrosine protein kinase activity analysis method | |
| CN105928912A (en) | Heparin detection method | |
| CN110317855A (en) | The fluorescence detection method of identification IDO1 enzymatic activity and screening IDO1 enzyme inhibitor based on cell | |
| CN106645719A (en) | Uses of cyanine dye in thrombin detection, thrombin detection kit, and thrombin detection method | |
| CN105838790B (en) | A kind of silver nanoclusters sensor and preparation method thereof and the application in detection viral gene | |
| CN108051412A (en) | A kind of method of tetranitro aluminium phthalocyanine near-infrared fluorescent detection ascorbic acid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |