CN112014367A - Application of AuNPs1-2 pH probe in male infertility and product - Google Patents
Application of AuNPs1-2 pH probe in male infertility and product Download PDFInfo
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- G—PHYSICS
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- 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
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
- G01N21/80—Indicating pH value
Abstract
The invention discloses application of an AuNPs1-2 pH probe in male infertility and a product thereof, belonging to the technical field of biotechnology and clinical medicine detection. The AuNPs1-2 pH probe is applied to preparing a product for detecting male infertility, wherein the product comprises a reagent for detecting the pH value in sperms, and the AuNPs1-2 pH probe is used for monitoring the local pH change of subcellular cells in the male sperms and the pH change under external stimulation, so that diagnosis reference and clinical consultation are provided for male patients with infertility with unknown clinical reasons, and the etiology and pathogenesis of the male patients with infertility are searched in an auxiliary manner.
Description
Technical Field
The invention relates to application of a pH probe in male infertility and a product thereof, in particular to application of an AuNPs1-2 pH probe in qualitative detection of male infertility and a corresponding product thereof, belonging to the technical field of biotechnology and clinical medicine detection.
Background
For many years, routine analysis of male semen has been used as a basic clinical indicator for determining male fertility. However, the actual cause of infertility of most male patients with infertility in clinic at present is still unclear, and especially, the results of conventional semen analysis of about one third of male patients with infertility in clinic are normal or close to normal, and the patients are clinically classified as "infertility with unknown cause", so that the detection and judgment of male infertility in clinic are very difficult for a long time.
The prior art "CN 200510078423.6 is a detection reagent for a male infertility detection method" discloses: preparing pentaacetoxy glucoside, preparing 2-chloro-4-nitrophenyl-alpha-tetraacetoxy glucoside from the obtained pentaacetoxy glucoside, and preparing 2-chloro-4-nitrophenyl-alpha-tetraacetoxy glucoside from the obtained 2-chloro-4-nitrophenyl-alpha-tetraacetoxy glucoside; the detection reagent is used in male infertility, especially in the detection method for identifying infertility caused by spermatogenic disorder and insemination disorder, and is suitable for automatic analysis of instruments.
The prior art "application of CN201711284133.6 EMC10 protein as biomarker in diagnosing male infertility" discloses that: the substance for improving EMC10 protein activity and/or expression can prevent male sterility, treat male sterility, promote sperm and ovum fertilization with zona pellucida removed, improve sperm and cytomembrane fusion ability, treat sperm dysfunction, promote sperm maturation in epididymis, improve sperm motility, improve protein tyrosine phosphorylation level related to sperm capacitation, promote sperm capacitation, improve sperm acrosome reaction ability, repair sperm abnormal morphology, recover sodium/potassium APT enzyme activity, maintain sperm internal ion balance, maintain sperm internal Na + balance, maintain sperm internal HCO3 balance, maintain sperm internal pH balance, and improve male fertility.
The prior art "CN 201610147268.7 a sperm quality assessment kit and its using method" discloses: the kit comprises cell lysate without enzyme activity inhibitor, detection buffer solution 1, protease inhibitor cocktail, reagent A solution, reagent B solution, bovine serum albumin, fluorescent reagent, sialidase standard stock solution and detection buffer solution 2; the detection buffer solution 1 is a phosphate buffer solution or a BWW culture solution; the reagent A solution is as follows: 1% of BCA disodium salt, 2% of anhydrous sodium carbonate, 0.16% of sodium tartrate, 0.4% of sodium hydroxide and 0.95% of sodium bicarbonate, and mixing and adjusting the pH value to 11.2-11.3; the reagent B solution is: 4% copper sulfate; the detection buffer 2 was phosphate buffer containing 0.05mmol/l sodium acetate and 0.25. mu.g/μ l bovine serum albumin. Through the detection of the sialidase activity in the sperms, diagnostic basis and clinical counseling are provided for male infertility patients with unknown clinical reasons.
Disclosure of Invention
The inventor finds in long-term research that the pH value in the sperms of male patients with infertility is smaller than that in the sperms of normal males, the sperms of the male patients with infertility are acidic, and then the molecular structure of the AuNPs1-2 pH probe is combined to change a conjugated system under different pH environments, so that different intensities and different types of fluorescence are generated, namely the smaller the pH value is, the larger the red fluorescence intensity is; the higher the pH value is, the higher the green fluorescence intensity is, wherein the AuNPs1-2 pH probe generates a relatively obvious color change effect in the range of pH4.5-8.0, while the pH value in sperms of male patients with infertility and the pH value in sperms of normal males are both in the range, and based on the above, the fluorescence conjugate system in the AuNPs1-2 pH probe molecules in an acid environment is used as red fluorescence, and the pH value in sperms of normal males is used as a detection index, so that the more obvious red fluorescence intensity corresponding to the pH value in sperms of male patients with infertility can be obviously observed.
Aiming at the defects of the prior art and clinical practical requirements, the invention provides an application and a product of an AuNPs1-2 pH probe in male infertility, wherein the AuNPs1-2 pH probe (nano-scale pH probe) is used for monitoring the local pH change of subcellular cells in male sperms and the pH change under external stimulation, so that diagnosis reference and clinical consultation are provided for male patients with infertility of unknown clinical reasons, and the etiology and pathogenesis of the male patients with infertility are searched in an auxiliary manner.
In order to achieve the technical purpose, the following technical scheme is proposed:
the application of the AuNPs1-2 pH probe in male infertility comprises the application of the AuNPs1-2 pH probe in preparing products for detecting male infertility. The product takes the pH value in the normal male sperm as a detection index, and if the fluorescence of the sperm sample detected based on the AuNPs1-2 pH probe is different from the fluorescence of the normal male sperm detected based on the AuNPs1-2 pH probe, namely the pH value of the sperm sample is different from the detection index, the sperm sample is judged to be from a male patient with infertility; otherwise, the sperm sample is judged to be normal male.
Further, the intracytoplasmic pH includes the pH of the intracytoplasmic local part of the sperm under normal conditions, or the pH of the intracytoplasmic local part of the sperm under external stimuli; wherein, the subcellular includes lysosome, mitochondria, etc., and the external stimulus is progesterone stimulus, NNC 55-0396 stimulus, etc.
Further, the male patients with infertility include asthenospermia, wherein the sperm forward motion of the asthenospermia is less than 32.
Further, the male patients with infertility include male patients with infertility of unknown cause. The male patients with infertility of unknown cause refer to male patients with infertility with qualified sperm detection indexes, wherein the detection indexes (sperm physicochemical characteristics) comprise: the abstinence days are 2-7 days, the color is grey white, the semen volume is more than or equal to 1.5 mL, the pH value is more than or equal to 7.2, the viscosity is less than 2cm, the liquefaction time is less than 60min, and the condensation result is negative; sperm Concentration (SC) > 15 x 10^6/mL, forward movement (PR) > 32%, sperm motility (motility) > 40%, survival rate (eosin-nigrosine staining) > 58%, round cell concentration (round cell) < 5 x 10^6/mL, white blood cell concentration (WBC) < 1.0 x 10^ 6/mL; percentage of normal morphology sperm (PNS) > 4%.
The AuNPs1-2 pH probe product for male infertility, comprising reagents for detecting the pH value in sperm.
Further, the product is a kit, and the kit comprises: polylysine, AuNPs1-2 pH probe and PBS buffer with pH 7.
Further, the PBS buffer solution comprises PO with the concentration of 0.01mol/L4 3-And 0.8% of NaCl by mass fraction.
By adopting the technical scheme, the beneficial technical effects brought are as follows:
1) the invention applies the AuNPs1-2 pH probe technology to the in vitro germ cells, namely the sperms, provides diagnosis reference and clinical consultation for male patients with infertility of clinically unknown reasons for the first time, and also assists in searching the etiology and pathogenesis of the male patients with infertility;
2) the invention can observe the difference of the pH value in the sperm of the normal male and the pH value in the sperm of the male patient with infertility visually, vividly and effectively;
3) in the invention, the AuNPs1-2 pH probe generates a relatively obvious color change effect in a pH range of 4.5-8.0, and meanwhile, compared with a BCECF method commonly used in the prior art for measuring the pH value in the sperm, the technical scheme has the characteristics of double color development, small damage to a sperm sample, high color development sensitivity, easier observation and the like.
Drawings
FIG. 1 is a fluorescence diagram of AuNPs1-2 pH probe with different pH environment for changing conjugated structure and generating different colors;
FIG. 2 is a diagram of the effect of normal male sperm under a 40X laser confocal microscope;
FIG. 3 is a diagram of the effect of normal male sperm under a 100X confocal laser microscope;
FIG. 4 is a diagram of the effect of normal male sperm and male infertility sperm under a 40X confocal laser microscope (Merge);
FIG. 5 is a diagram of the effect of normal male sperm and male infertility sperm under a 40X confocal laser microscope (Red channel);
FIG. 6 is a statistical plot of the difference in pH between normal male sperm and male sterile patient sperm;
FIG. 7 is a graph of results of eosin staining comparing sperm motility using AuNPs1-2 pH probe and BCECF method for determining the pH in sperm (wherein arrows point to dead sperm);
FIG. 8 is a chart comparing the difference in sperm motility after measuring the pH in sperm using the AuNPs1-2 pH probe and BCECF method using eosin staining;
FIG. 9 is a graph showing the results of eosin staining comparing the pH in sperm with AuNPs1-2 pH probe and BCECF method under a 40X laser confocal microscope.
Detailed Description
In the following, the technical solutions in the embodiments of the present invention are clearly and completely described, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The application of the AuNPs1-2 pH probe in male infertility comprises the application of the AuNPs1-2 pH probe in preparing products for detecting male infertility. The product takes the pH value in the normal male sperm as a detection index, and if the fluorescence of the sperm sample detected based on the AuNPs1-2 pH probe is different from the fluorescence of the normal male sperm detected based on the AuNPs1-2 pH probe, namely the pH value of the sperm sample is different from the detection index, the sperm sample is judged to be from a male patient with infertility; otherwise, the sperm sample is judged to be normal male.
The intracytoplasmic pH includes a pH that is normally local to a subcellular within the sperm, or, a pH that is local to a subcellular within the sperm under an external stimulus; wherein, the subcellular includes lysosome, mitochondria, etc., and the external stimulus is progesterone stimulus, NNC 55-0396 stimulus, etc.
Male patients with infertility include asthenospermic patients, wherein the asthenospermic patients have a forward movement of sperm of less than 32.
Male patients with infertility also include male patients with infertility of unknown cause. The male patients with infertility of unknown cause refer to male patients with infertility with qualified sperm detection indexes, wherein the detection indexes (sperm physicochemical characteristics) comprise: the abstinence days are 2-7 days, the color is grey white, the semen volume is more than or equal to 1.5 mL, the pH value is more than or equal to 7.2, the viscosity is less than 2cm, the liquefaction time is less than 60min, and the condensation result is negative; sperm Concentration (SC) > 15 x 10^6/mL, forward movement (PR) > 32%, sperm motility (motility) > 40%, survival rate (eosin-nigrosine staining) > 58%, round cell concentration (round cell) < 5 x 10^6/mL, white blood cell concentration (WBC) < 1.0 x 10^ 6/mL; percentage of normal morphology sperm (PNS) > 4%.
Example 2
The AuNPs1-2 pH probe product for male infertility, comprising reagents for detecting the pH value in sperm.
The product is a kit, which comprises: polylysine, AuNPs1-2 pH probe and PBS buffer solution, wherein the pH value of the PBS buffer solution is 7, and the PBS buffer solution comprises PO with the concentration of 0.01mol/L4 3-And 0.8% of NaCl by mass fraction.
Example 3
Based on example 2, this example provides a method for using a product of AuNPs1-2 pH probe in male infertility, which specifically includes the following steps:
firstly, coating a 3.5 cm glass bottom culture dish (only on a glass slide at the center of the culture dish) with 200 mu L of polylysine, standing for 10 min in a dark place, discarding liquid, sucking dry, placing in room temperature, and drying overnight at 18-26 ℃ or drying for 1 h at 60 ℃;
washing the normal male semen and the male infertility patient semen by adopting a PBS buffer solution respectively, wherein the method specifically comprises the following steps:
adding 1-2 mL of semen into 2-3 mL of PBS buffer solution, centrifuging for 10 min at 520G, removing supernatant, and washing until the supernatant is nearly transparent;
the semen of normal male and the semen of male infertility patient (specifically, semen of weak sperm patient) are provided directly from human sperm bank, and the types of semen (normal and weak sperm) are consistent with clinical judgment index;
respectively diluting normal male sperm and male infertility patient sperm washed by the PBS buffer solution by using the PBS buffer solution as a solvent, wherein the sperm is diluted to an appropriate concentration (preferably to free single sperm under a microscope) to obtain a sperm diluent;
respectively placing 200 μ L sperm diluent in 1.5 ml LP tube, respectively adding 5 μ L AuNPs1-2 pH probe (provided by key laboratory of chemical and technical education department of Sichuan university college of chemistry), incubating at room temperature in dark for 5-10 min, centrifuging at 520G for 10 min, removing supernatant, adding 200 μ L PBS buffer solution, resuspending, and mixing sperm in PBS buffer solution;
fourthly, 3 μ L of the prepared co-stained normal male sperm is dropped on a glass bottom culture dish and photographed under a 40X laser confocal microscope (Olympus Micro), and the obtained result is shown in figure 2; the photographs were taken under a 100 Xlaser confocal microscope (OlympusMicro), and the results are shown in FIG. 3. In addition, referring to fig. 2 to 3, it can be seen that: more sperms can be seen by taking a picture under a 40X laser confocal microscope (OlympusMicro), so that the fluorescence intensity level in the whole sperms can be counted conveniently.
And fifthly, dripping 3 mu L of prepared male patient sperm with the co-infection infertility on a glass bottom culture dish, and taking a picture under a 40X laser confocal microscope (OlympusMicro).
The results show that: after the treatment with the AuNPs1-2 probe, the pH in the sperm of the male infertility patients is less than 7 (acid), and the acidic fluorescence conjugate system is red fluorescence, so that the red fluorescence intensity of the pH in the sperm of the male infertility patients is more significant (as shown in table 1 below), and after the treatment with statistical software, the two red fluorescence intensities have significant statistical difference (as shown in fig. 6).
Example 4
Based on example 3, the inventors also designed an eosin staining method to compare sperm motility after measuring the pH in sperm by using AuNPs1-2 pH probe and BCECF method, wherein the step of measuring the pH in sperm by BCECF method specifically comprises:
firstly, coating a 3.5 cm glass bottom culture dish (only on a glass slide at the center of the culture dish) with 200 mu L of polylysine, standing for 10 min in a dark place, discarding liquid, sucking dry, placing in room temperature, and drying overnight at 18-26 ℃ or drying for 1 h at 60 ℃;
washing normal male semen and sterile male semen by adopting a PBS buffer solution respectively, wherein the method specifically comprises the following steps:
adding 1-2 mL of semen into 2-3 mL of PBS buffer solution, centrifuging for 10 min at 520G, removing supernatant, and washing until the supernatant is nearly transparent;
the semen of the normal male and the semen of the male with sterility are both directly provided by a human sperm bank, and the types of the semen (normal and sterility) are consistent with the clinical judgment index;
respectively diluting normal male sperms and sterile male patient sperms washed by the PBS buffer solution by using the PBS buffer solution as a solvent, wherein the sperms are diluted to proper concentration (free single sperms are preferably observed under a microscope) to obtain a sperm diluent;
respectively placing 200 μ L sperm diluent in 1.5 ml LEP tube, respectively adding 5 μ L BCECF probe (purchased from Dalian Meilun biotechnology Co., Ltd.), incubating at room temperature in dark for 5-10 min, centrifuging at 520G for 10 min, removing supernatant, adding 200 μ L PBS buffer solution, resuspending, and mixing sperm in PBS buffer solution;
dripping 3 mu L of prepared co-stained normal male sperm on a glass bottom culture dish, and taking a picture under a 40X laser confocal microscope (Olympus micro);
and fifthly, dripping 3 mu L of prepared male patient sperm with the co-infection infertility on a glass bottom culture dish, and taking a picture under a 40X laser confocal microscope (OlympusMicro).
The method for measuring the sperm motility specifically comprises the following steps:
adopting eosin dye solution (purchased from Chengdu general Hua technology Co., Ltd.) as sperm motility detection reagent, taking 10uL of completely liquefied fresh semen into an EP centrifugal tube, taking 30uL of eosin dye solution, uniformly mixing with the semen, and standing for 30; taking 10uL of the mixed solution at one end of the slide, preparing the slide into a smear by pulling a piece, and drying in air.
Immediately observing under a common microscope or observing after sealing a non-aqueous sealing tablet, and counting no less than 200 sperms;
1) the sperm which is colorless and transparent or dyed into light red is live sperm, and the sperm which is dyed into red or dark red is dead sperm;
2) sperm survival = number of viable sperm/total sperm counted x 100%;
the result shows that the total number of experimental sperm samples in the BCECF method is 593, the number of the surviving sperms is 385 and the number of the dead sperms is 208 after the BCECF method is used, namely the average value of the survival rate of the sperms is 65.92 percent; the total number of experimental sperm samples in the assay using the AuNPs1-2 pH probe was 653, and after using the AuNPs1-2 pH probe, the number of surviving sperm was 458, the number of dead sperm was 195, and the average value of sperm survival rate was 70.14%, as shown in fig. 7-8, further we obtained: after the pH in the sperm is detected by using the AuNPs1-2 pH probe, the sperm motility is obviously higher than that after the pH in the sperm is detected by using a BCECF method.
In addition, the AuNPs1-2 pH probe is a two-way labeled pH probe, which shows green color if there is an alkalization process in sperm, while the BCECF probe shows only green fluorescence after use, i.e., it is not easily observed and identified by color after the related sperm are alkalized (as shown in fig. 9).
Claims (8)
- The application of the AuNPs1-2 pH probe in male infertility is characterized by comprising the application of the AuNPs1-2 pH probe in preparing products for detecting male infertility.
- 2. The use of the AuNPs1-2 pH probe according to claim 1, wherein the product uses pH in normal male sperm as a detection index, and if the fluorescence of the sperm sample detected based on the AuNPs1-2 pH probe is different from the fluorescence of normal male sperm detected based on the AuNPs1-2 pH probe, that is, the pH of the sperm sample is different from the detection index, the sperm sample is judged to be from a male patient with infertility; otherwise, the sperm sample is judged to be normal male.
- 3. The use of an AuNPs1-2 pH probe according to claim 2, wherein the intra-sperm pH comprises the normal intra-sperm subcellular local pH, or the intra-sperm subcellular local pH under external stimuli.
- 4. Use of the AuNPs1-2 pH probe according to any one of claims 1 to 3, in male infertility, wherein the male infertility patient comprises a asthenospermia patient.
- 5. The use of the AuNPs1-2 pH probe according to any one of claims 1 to 3, in male infertility, wherein the male infertility patient comprises a male patient with unexplained infertility.
- A product of AuNPs1-2 pH probe in male infertility, comprising reagents for detecting pH in sperm.
- 7. The product of AuNPs1-2 pH probe in male infertility according to claim 6, wherein the product is a kit comprising: polylysine, AuNPs1-2 pH probe and PBS buffer, the pH of PBS buffer is 7.
- 8. The product of AuNPs1-2 pH probe in male infertility according to claim 7, wherein the PBS buffer comprises PO at a concentration of 0.01mol/L4 3-And 0.8% of NaCl by mass fraction.
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