CN111007059A - Blood stain color developing agent and preparation and use methods thereof - Google Patents
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Abstract
The invention relates to a blood stain color developing agent and a preparation and use method thereof, belonging to the technical field of color developing agents. Solves the technical problems that the blood stain color developing agent in the prior art is easy to fade and false positive is easy to appear. The blood stain color developing agent consists of a reagent A and a reagent B; the reagent A contains organic solid, protective agent and water, wherein the organic solid is prepared by sulfonation and salting out after the polycondensation reaction of p-hydroxybenzaldehyde and N-ethyl-N-benzylaniline according to the mass ratio of 1: 2, the concentration of the organic solid is 1-4 wt%, and the concentration of the protective agent is 0.2-2 wt%; the reagent B contains hydrogen peroxide and water, and the concentration of the hydrogen peroxide is 3-5 wt%. The bloodstain color developing agent is convenient to use, can show special blue when meeting bloodstains, is not easy to cause false positive problem, can not fade along with time extension, can not damage DNA in the bloodstains when in use, can not diffuse the bloodstains, can display potential blood fingerprints, and has wide application range.
Description
Technical Field
The invention belongs to the technical field of color-developing agents, and particularly relates to a blood stain color-developing agent and a preparation and use method thereof.
Background
The blood stain color developing agent is a reagent for developing blood stains, is commonly used for rapidly detecting potential blood stains and blood fingerprints on the spot of a case in public security system criminal investigation, and provides basis for rapidly restoring the case and issuing the field. Due to the particularity of the use scenario, such display actually has the following basic requirements that firstly the reagent is required to be simple and reliable to operate in the use process, and secondly the reagent is required not to damage DNA remained in blood stains.
The blood stain color developing agent in the prior art mainly depends on a luminol luminescence method, a benzidine color developing method and a phenolphthalein color developing method. The luminol luminescence method mainly depends on the luminescence principle, a color developing agent needs to be repeatedly sprayed on the blood stain in a dark scene, the blood stain is detected by emitting bright light, and the fluorescence can be removed after a long time. The benzidine color development method depends on that benzidine generates a turquoise blue reaction when meeting some bloodstains, but the color change reaction can be caused by other substances and is not specific to the bloodstains, such as iron rust and ferric chloride which are contacted with the benzidine, and false positive can be generated. The phenolphthalein color development method also has a problem of false positive as in the benzidine color development method.
In view of the above, there is a need for developing a blood stain developer which can develop color quickly, is not easily discolored for a long time, is not easily false positive, and does not destroy DNA remaining in blood stains.
Disclosure of Invention
The invention provides a blood stain color-developing agent and a preparation and use method thereof, aiming at solving the technical problems that the blood stain color-developing agent is easy to fade and false positive is easy to appear in the prior art.
The technical scheme adopted by the invention for solving the technical problems is as follows.
The invention provides a blood stain color developing agent, which consists of a reagent A and a reagent B;
the reagent A contains organic solid, protective agent and water, wherein the organic solid is prepared by sulfonation and salting out after the polycondensation reaction of p-hydroxybenzaldehyde and N-ethyl-N-benzylaniline according to the mass ratio of 1: 2, the concentration of the organic solid is 1-4 wt%, and the concentration of the protective agent is 0.2-2 wt%;
the reagent B contains hydrogen peroxide and water, and the concentration of the hydrogen peroxide is 3-5 wt%.
Preferably, the protective agent is one or a mixture of several of sodium citrate, ascorbic acid and sodium hypophosphite, and the concentration of each protective agent is 0.2-1 wt%.
The invention also provides a preparation method of the blood stain color developing agent, which comprises the following steps: and subpackaging the reagent A and the reagent B to obtain the bloodstain color developing agent.
Preferably, the preparation method of the reagent A comprises the following steps:
dissolving p-hydroxybenzaldehyde and N-ethyl-N-benzyl aniline in a solvent according to the mass ratio of 1: 2, carrying out condensation reaction under the protection of inert atmosphere and/or reducing gas, and evaporating the solvent at low temperature after the reaction is finished to obtain a reaction intermediate;
step two, mixing the reaction intermediate obtained in the step one with a sulfonated substance according to the stoichiometric ratio of 1: 1.5-2 under the protection of inert atmosphere and/or reducing gas, and carrying out sulfonation treatment to obtain a sulfonated mixture;
the sulfonate is fuming sulfuric acid or concentrated sulfuric acid;
step three, adjusting the pH value of the sulfonation mixture obtained in the step two to be neutral, and salting out under the protection of inert atmosphere to obtain an organic solid;
and step four, dissolving the organic solid obtained in the step three in water, adding a protective agent, and uniformly mixing to obtain a reagent A.
More preferably, in the first step, the solvent is ethanol.
More preferably, in the first step, the condensation reaction is carried out at 50-100 ℃ for 2-20 h.
More preferably, in the first step, the equipment used for low-temperature evaporation is a rotary evaporator.
More preferably, in the first step, the second step and the third step, the inert atmosphere is one or a mixture of nitrogen, argon and helium, and the reducing gas is hydrogen.
More preferably, in the second step, the temperature of sulfonation treatment is 40-120 ℃ and the time is 2-10 h.
More preferably, in the third step, the pH is adjusted to be neutral within 5min by using a sodium hydroxide solution or a potassium hydroxide solution.
More preferably, in the third step, the substance used for salting out is one or a mixture of sodium sulfate, potassium sulfate, sodium chloride and potassium chloride.
The invention also provides a use method of the blood stain color developing agent, which comprises the following steps: mixing the reagent A and the reagent B according to the volume ratio of (70-95) to (30-5), spraying the mixture on the surface of the blood stain, and waiting for 1-20s, wherein the potential blood stain can show color.
Compared with the prior art, the invention has the beneficial effects that:
1. the blood stain color developing agent provided by the invention is convenient to use, can show special blue when meeting blood stains, is not easy to have false positive problem, can be darker and darker along with the lapse of time, cannot fade, and cannot damage DNA in the blood stains in the whole process.
2. The blood stain color developing agent provided by the invention has the advantages that the organic solid can adsorb protein, the protein in the blood stain is fixed after the blood stain is encountered, and the diffusion of the blood stain in the process of identifying the blood stain is avoided, so that the potential blood fingerprint can be displayed.
3. The blood stain color developing agent provided by the invention is wide in application range, and is particularly suitable for detecting potential blood stains on the spot in any scene.
Drawings
In order to more clearly illustrate the technical solution of the present invention, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious to those skilled in the art that other drawings can be obtained based on these drawings without inventive efforts.
FIG. 1 is a graph showing the effect of a blood stain developer according to example 1 of the present invention on a white cloth after one year of development of a blood stain;
FIG. 2 is a graph showing the effect of the bloodstain developer of example 1 of the present invention on A4 paper after one year of development of bloodstains;
FIG. 3 is a diagram showing the effect of the blood stain developer of embodiment 1 of the present invention after one year of blood stain development on the surface of a nylon woven bag;
FIG. 4 is a graph showing the effect of the blood stain developer according to example 1 of the present invention on the tire surface after one year of development of blood stains;
FIG. 5 is a graph showing the effect of the bloodstain developer of example 1 of the present invention on the black coated paper after one year of bloodstain development;
FIG. 6 is a graph showing the effect of the blood stain developer on developing blood fingerprints according to example 1 of the present invention;
FIG. 7 shows the result of DNA typing of a blood stain on a white cloth before spraying the blood stain-developing agent of example 1;
FIG. 8 shows the result of DNA typing of a blood stain on a white cloth after spraying the blood stain-developing agent of example 1;
in fig. 9, a and b are graphs showing the effect of the blood stain developer of example 1 sprayed on the surfaces of iron powder and ferric chloride, respectively;
FIG. 10 is a graph showing the effect of the blood stain developer of example 2 of the present invention on a white wall surface after one year of development of blood stains.
Detailed Description
For a further understanding of the invention, preferred embodiments of the invention are described below in conjunction with the detailed description, but it is to be understood that the description is intended to further illustrate features and advantages of the invention, and not to limit the claims of the invention.
The blood stain color developing agent consists of a reagent A and a reagent B;
wherein, the reagent A contains organic solid, protective agent and water, the organic solid is prepared by sulfonation and salting out after the polycondensation reaction of p-hydroxybenzaldehyde and N-ethyl-N-benzylaniline according to the mass ratio of 1: 2;
the reagent B contains hydrogen peroxide and water;
the reagent B is used as a reducing substance protection reagent A, so that the further oxidation of the reagent A is avoided, and the storage time of the color developing agent is prolonged.
In the above technical scheme, the concentration of the organic solid in the reagent A is 1-4 wt%, preferably 2-3 wt%.
In the technical scheme, the protective agent is preferably one or a mixture of several of sodium citrate, ascorbic acid and sodium hypophosphite, the total concentration of the protective agent in the reagent A is 0.2-2 wt%, and the concentration of each protective agent is preferably 0.2-1 wt%.
In the technical scheme, the concentration of hydrogen peroxide in the reagent B is 3-5 wt%, preferably 3-4 wt%.
The invention also provides a preparation method of the blood stain color developing agent, which comprises the following steps: namely, the reagent A and the reagent B are subpackaged to obtain the bloodstain color developing agent.
In the above technical scheme, the preparation method of the reagent A comprises the following steps:
dissolving p-hydroxybenzaldehyde and N-ethyl-N-benzyl aniline in a solvent according to the mass ratio of 1: 2, carrying out condensation reaction under the protection of inert atmosphere and/or reducing gas, and evaporating the solvent at low temperature after the reaction is finished to obtain a reaction intermediate;
step two, mixing the reaction intermediate obtained in the step one with a sulfonated substance according to a stoichiometric ratio of 1: 1.5-2 under the protection of an inert atmosphere and/or a reducing gas, and carrying out sulfonation treatment to obtain a sulfonated mixture;
step three, adjusting the pH value of the sulfonation mixture obtained in the step two to be neutral, and salting out under the protection of inert atmosphere to obtain an organic solid;
and step four, dissolving the organic solid obtained in the step three in water, adding a protective agent, and uniformly mixing to obtain light brown liquid, namely the reagent A.
In the above technical solution, in the step one, the solvent is not particularly limited as long as it can perform a dissolving function and can be removed by low-temperature evaporation, and preferably, the solvent is ethanol.
In the above technical scheme, in the first step, the condensation reaction is preferably carried out at 50-100 ℃ for 2-20h, more preferably at 80-90 ℃ for 10-15 h.
In the above technical solution, in the first step, the equipment used for low temperature evaporation is usually a rotary evaporator.
In the technical scheme, in the first step, the second step and the third step, the inert atmosphere is one or a mixture of nitrogen, argon and helium respectively and independently, and the reducing gas is hydrogen. The product can be prevented from being oxidized by using inert or reducing gas protection.
In the second step, the sulfonated substance is fuming sulfuric acid or concentrated sulfuric acid.
In the above technical scheme, in the second step, the temperature of the sulfonation treatment is preferably 40-120 ℃, the time is preferably 2-10h, the temperature is more preferably 80-100 ℃, and the time is more preferably 4-5 h.
In the above technical scheme, in the third step, the pH is preferably adjusted to be neutral by adopting a sodium hydroxide solution or a potassium hydroxide solution, and the adjustment process is generally completed within 5 min.
In the technical scheme, in the third step, the substances adopted for salting out are preferably one or a mixture of more of sodium sulfate, potassium sulfate, sodium chloride and potassium chloride.
The use method of the blood stain color developing agent comprises the following steps: mixing the reagent A and the reagent B, spraying the mixture on the surface of a blood stain, and waiting for 1-20s, wherein the potential blood stain shows blue color, wherein the volume ratio of the reagent A to the reagent B is (70-95) to (30-5), and preferably (80-90) to (20-10).
The invention will be further explained with reference to the drawings and examples. The reagents used in the following examples are all commercially available.
Example 1
Subpackaging the reagent A and the reagent B to obtain a bloodstain color developing agent;
the preparation method of the reagent A comprises the following steps:
dissolving p-hydroxybenzaldehyde and N-ethyl-N-benzyl aniline in ethanol according to the mass ratio of 1: 2, reacting for 15 hours at 80 ℃ under the protection of nitrogen, and evaporating ethanol at low temperature by using a rotary evaporator after the reaction is finished to obtain a reaction intermediate;
step two, under the protection of nitrogen, mixing the reaction intermediate obtained in the step one with fuming sulfuric acid according to the stoichiometric ratio of 1: 1.5, and reacting for 5 hours at 80 ℃ to obtain a sulfonated mixture;
step three, adjusting the pH value of the sulfonation mixture obtained in the step two to be neutral within 5min by using a sodium hydroxide solution, adding sodium sulfate under the protection of nitrogen, and salting out to obtain an organic solid;
step four, dissolving the organic solid obtained in the step three in water to prepare a 2% aqueous solution, adding 1% of sodium citrate, 0.3% of ascorbic acid and 0.3% of sodium hypophosphite, and uniformly mixing to obtain a reagent A;
the reagent B is a hydrogen peroxide solution with the concentration of 3 percent.
The blood stain developer of example 1 was examined for its color development: the results of mixing 90mL of the reagent A with 10mL of the reagent B and spraying the mixture on the surfaces of white cloth with potential bloodstains, A4 paper, a nylon woven bag, a tire and black coated paper respectively show that the potential bloodstains can be displayed in blue within 5s (even if the potential bloodstains are still displayed under extremely low blood concentration, the potential bloodstains are not influenced by the contact time of the bloodstains and air basically), and the blue color can not be faded after the mixture is placed for one year, as shown in figures 1-5 (in figure 1, C is a control group which has no bloodstains and has the proportions of 1: 1, 1: 2, 1: 5, 1: 10, 1: 20, 1: 100, 1: 200 and 1: 1000 which respectively show that blood is diluted by 1, 2, 5, 10, 20, 100, 200 and 1000 times; in figure 2, C is a control group which shows that no bloodstains are present, the row 1 shows that the bloodstains are in contact with air for 30 days, the row 2 shows that the bloodstains are in contact with air for 24 hours, and the row 3 shows that the blood stains are fresh, the ratios 1: 100, 1: 200, 1: 500, 1: 1000, 1: 2000, 1: 10000 represent 100, 200, 500, 1000, 2000, 10000 times of blood dilution, respectively). 90mL of reagent A and 10mL of reagent B are mixed and sprayed on the surface of the paper with the potential blood fingerprints (half is shielded, and half is sprayed with the blood stain developer), and the result shows that the potential blood fingerprints can be displayed in blue within 5s, the blood fingerprints are not diffused, and the textures are clear, as shown in figure 6.
In the above experiment, the blood spots on the white cloth before the blood stain developer of example 1 was sprayed and the blood spots on the white cloth after the blood stain developer of example 1 was sprayed were extracted by the chelex-100 method, and typing was detected by a fusion kit, and as shown in fig. 7 and 8, it can be seen from fig. 7 and 8 that there was no significant difference between the typing of the blood spots after the developer was sprayed and the typing of the blood spots without the developer, and intact DNA could still be extracted. It was demonstrated that the color-developing agent of the present invention does not destroy DNA in use.
The blood stain developer of example 1 was examined for its color development: the results of mixing 90mL of reagent a with 10mL of reagent B and spraying the mixture on the surfaces of iron powder and ferric chloride are shown as a and B in fig. 9, respectively, and it can be seen from fig. 9 that the blood stain developer in example 1 has no color change when encountering iron powder and ferric chloride, which indicates that the blood stain developer of the present invention has no false positive problem.
Example 2
Subpackaging the reagent A and the reagent B to obtain a bloodstain color developing agent;
the preparation method of the reagent A comprises the following steps:
dissolving p-hydroxybenzaldehyde and N-ethyl-N-benzyl aniline in ethanol according to the mass ratio of 1: 2, reacting for 10 hours at 90 ℃ under the protection of nitrogen, and evaporating ethanol at low temperature by using a rotary evaporator after the reaction is finished to obtain a reaction intermediate;
step two, under the protection of nitrogen, mixing the reaction intermediate obtained in the step one with fuming sulfuric acid according to the stoichiometric ratio of 1: 1.5, and reacting for 4 hours at 100 ℃ to obtain a sulfonated mixture;
step three, adjusting the pH value of the sulfonation mixture obtained in the step two to be neutral within 5min by using a sodium hydroxide solution, adding sodium sulfate under the protection of nitrogen, and salting out to obtain an organic solid;
step four, dissolving the organic solid obtained in the step three in water to prepare a 3% aqueous solution, adding 1.2% of sodium citrate, 0.2% of ascorbic acid and 0.2% of sodium hypophosphite, and uniformly mixing to obtain a reagent A;
the reagent B is a hydrogen peroxide solution with the concentration of 4 percent.
The blood stain developer of example 2 was tested for color development: mixing 80mL of reagent A with 20mL of reagent B, and respectively spraying the mixture on the surfaces of white cloth with potential bloodstains, A4 paper, nylon woven bags, tires and black coated paper, wherein the results show that the potential bloodstains can be blue within 5s, and the blue color still does not fade after the mixture is placed for one year; mixing 80mL of reagent A and 20mL of reagent B, spraying the reagent on a potential blood fingerprint on a white lime wall, and displaying that the fingerprint shows blue within 5s and the lines are clear, and after the reagent is placed for one year, the lines are still clear, the color development is lasting, and the background is not colored, as shown in figure 10.
In the above experiment, the blood spots on the white cloth before the blood stain developer of example 2 was sprayed and the blood spots on the white cloth after the blood stain developer of example 2 was sprayed were extracted by the chelex-100 method, and typing was detected by a fusion kit, and the results showed that there was no significant difference between the typing of the blood spots after the developer was sprayed and the typing of the blood spots without the developer, and complete DNA could still be extracted. It was demonstrated that the color-developing agent of the present invention does not destroy DNA in use.
The blood stain developer of example 2 was tested for color development: and mixing 80mL of the reagent A with 20mL of the reagent B, and spraying the mixture on the surfaces of iron powder and ferric chloride respectively, wherein the result shows that the blood stain color developing agent in the embodiment 2 does not have color change when meeting the iron powder and the ferric chloride, which indicates that the blood stain color developing agent does not have the problem of false positive.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. The blood stain color developing agent is characterized by consisting of a reagent A and a reagent B;
the reagent A contains organic solid, protective agent and water, wherein the organic solid is prepared by sulfonation and salting out after the polycondensation reaction of p-hydroxybenzaldehyde and N-ethyl-N-benzylaniline according to the mass ratio of 1: 2, the concentration of the organic solid is 1-4 wt%, and the concentration of the protective agent is 0.2-2 wt%;
the reagent B contains hydrogen peroxide and water, and the concentration of the hydrogen peroxide is 3-5 wt%.
2. The blood stain developer according to claim 1, wherein the protective agent is one or a mixture of sodium citrate, ascorbic acid and sodium hypophosphite, and the concentration of each protective agent is 0.2-1 wt%.
3. The method for producing a blood stain-developing agent according to claim 1 or 2, wherein the blood stain-developing agent is obtained by separately packaging the reagent a and the reagent B.
4. The method for preparing the blood stain color developing agent according to claim 3, wherein the method for preparing the reagent A comprises the following steps:
dissolving p-hydroxybenzaldehyde and N-ethyl-N-benzyl aniline in a solvent according to the mass ratio of 1: 2, carrying out condensation reaction under the protection of inert atmosphere and/or reducing gas, and evaporating the solvent at low temperature after the reaction is finished to obtain a reaction intermediate;
step two, mixing the reaction intermediate obtained in the step one with a sulfonated substance according to the stoichiometric ratio of 1: 1.5-2 under the protection of inert atmosphere and/or reducing gas, and carrying out sulfonation treatment to obtain a sulfonated mixture;
the sulfonate is fuming sulfuric acid or concentrated sulfuric acid;
step three, adjusting the pH value of the sulfonation mixture obtained in the step two to be neutral, and salting out under the protection of inert atmosphere to obtain an organic solid;
and step four, dissolving the organic solid obtained in the step three in water, adding a protective agent, and uniformly mixing to obtain a reagent A.
5. The method for preparing the blood stain color developing agent according to claim 4, wherein in the first step, the solvent is ethanol; in the first step, the second step and the third step, the inert atmosphere is one or a mixture of several of nitrogen, argon and helium respectively and independently, and the reducing gas is hydrogen.
6. The method for preparing the blood stain developer according to claim 4, wherein in the step one, the condensation reaction is carried out at 50-100 ℃ for 2-20 h.
7. The method for preparing the blood stain developer according to claim 4, wherein in the second step, the temperature of the sulfonation treatment is 40-120 ℃ and the time is 2-10 h.
8. The method for preparing the blood stain developer according to claim 4, wherein in the third step, the pH is adjusted to be neutral within 5min by using a sodium hydroxide solution or a potassium hydroxide solution.
9. The method for preparing the blood stain developer according to claim 4, wherein in the third step, the salting-out is performed by one or a mixture of sodium sulfate, potassium sulfate, sodium chloride and potassium chloride.
10. The use method of the blood stain developer according to claim 1 or 2, characterized in that the reagent A and the reagent B are mixed according to the volume ratio of (70-95) to (30-5), sprayed on the surface with the potential blood stains, and the potential blood stains are waited for 1-20s to show color.
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|---|---|---|---|---|
| CN112710653A (en) * | 2020-12-10 | 2021-04-27 | 达州职业技术学院 | Special reagent for rapidly detecting potential bloodstains and preparation method thereof |
| CN113125424A (en) * | 2021-03-12 | 2021-07-16 | 广东省大湾区华南理工大学聚集诱导发光高等研究院 | Compound reagent for portable occult blood trace visualization and preparation method and use method thereof |
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Application publication date: 20200414 |