Application of fluorescein in latent blood trace development

Abstract

The application discloses application of a fluorescein substance in latent blood trace development. The fluorescein substance is a compound shown in formula I or formula II. Based on the fluorescein substance, the application provides a latent blood trace developing agent which is a solution formed by the fluorescein substance and any one of the following solvents: water, methanol, ethanol, ethyl acetate, DMSO and DMF. The pH value of the solution is 3-5, preferably 4.3-4.9. The development method of the application is suitable for development of latent blood traces on various non-penetrating objects, has a wide range of applicable objects, has a good development effect on latent blood traces on dark objects and rough objects in particular, has strong fluorescence brightness of the lines, the lines are relatively clear and coherent, and the lines have a large contrast with the background.

Description

TECHNICAL FIELD

[0001] The application relates to application of a fluorescein substance in latent blood trace development and belongs to the technical field of forensic science. BACKGROUND

[0002] When light irradiates on certain substances, light with different wavelength and intensity from the incident light can be emitted, and when the irradiation of the incident light is stopped, the light will disappear immediately or gradually. The light is fluorescence. In 1575, N. Monardes, a botanist from Spain, first discovered and recorded the fluorescence phenomenon. In 1852, Stokes confirmed that fluorescence is emitted light after studying chlorophyll and quinine and found that the fluorescence wavelength of the substances is longer than that of the irradiation light, which is the light re-emitted after the substances absorb light energy. Bayer synthesized fluorescein in 1871, and to date, the many advantages of fluorescein are widely used in biological molecular detection, DNA hybridization analysis and antibody labeling and the like.

[0003] Fluorescein is a substance with a rigid coplanar large conjugated structure, and two benzene rings are fixed on the same plane by an oxygen bridge. The fluorescein can produce strong yellow-green fluorescence under the irradiation of laser. Fluorescein substances are good in biocompatibility and high in fluorescence quantum yield, and are often used as biological staining agents, fluorescence spectrophotometric analysis agents and oxidation-reduction indicators. With the progress of testing instrument technology, the analysis method based on fluorescence as a related testing index is gradually improved, and especially the fluorescence analysis method of modified fluorescein substances plays an irreplaceable role in biological and chemical analysis.

[0004] In recent years, the fluorescence probe method developed rapidly in the field of biological diagnosis is to use the fluorescein light-emitting principle. Fluorescein small molecule substances interact with nucleic acids, proteins or other macromolecular structures in the body, and can be used to study the properties and behaviors of the biological macromolecular substances. Through the combination of the fluorescence probe method and the laser confocal imaging technology, the substances in tissues and cells are well detected on line without destroying the activity of the biological cells. The sample loaded with the probe after the collection of the cells can be detected by a fluorescence spectrophotometer, a fluorescence enzyme label instrument or a flow cytometer. Studies have shown that the fluorescence probe method has the advantages of easy operation, high selectivity and high sensitivity compared with other detection methods, and the fluorescence probe has good biocompatibility and can also protect the detection object from being damaged, and is currently widely applied in the fields of biology, environment, medicine, chemistry and the like.

[0005] Latent blood handprint is a trace often appeared in crime scene, which has strong directionality for investigation and case solving. Blood contains abundant biological macromolecular substances, and the principle of fluorescence probe method can clearly show and extract the latent blood handprint left by the suspect, which plays an important role in case investigation and is an important start of the application of fluorescein substances in criminal investigation field. SUMMARY

[0006] The application provides an application of fluorescein substances in latent blood trace development, which can provide trace evidence for court trial; the latent blood trace (handprint) developed can have obvious contrast with background after laser excitation, and can be photographed and extracted to provide latent blood handprint trace evidence for court.

[0007] The fluorescein substance according to the application is a compound shown in formula I or formula II.

[0008]

[0009] In the formula, R1, R2, R3, R4, R5 and R6 are independently selected from hydrogen atom, halogen atom and alkyl group containing 1-4 carbon atoms;

[0010] R7 and R8 are independently selected from hydrogen atom, halogen atom, carboxyl group, isothiocyanate group, phenylsulfonyl group or substituted phenylsulfonyl group, benzoyl group or substituted benzoyl group, amino group, primary amine group containing 1-4 carbon atoms and secondary amine group containing 1-4 carbon atoms;

[0011] In formula I or formula II, each substituent is preferably:

[0012] R1, R2, R3, R4, R5 and R6 are independently selected from hydrogen atom; 2) R2, R3, R4 and R6 are independently selected from hydrogen atom, and R1 and R5 are independently selected from chlorine atom and bromine atom; 3) R2, R3, R4 and R6 are independently selected from chlorine atom and bromine atom, and R1 and R5 are independently selected from hydrogen atom;

[0013] R7 and R8 are independently selected from hydrogen atom, carboxyl group, isothiocyanate group, benzoyl group and substituted benzoyl group, and are preferably: R7 and R8 are hydrogen atom, R7 is carboxyl group and R8 is hydrogen atom, R7 is isothiocyanate group and R8 is hydrogen atom.

[0014] Based on the fluorescein substance, the application provides a latent blood trace developing agent, which is a solution formed by the fluorescein substance and any one of the following solvents:

[0015] water, methanol, ethanol, ethyl acetate, DMSO and DMF;

[0016] The pH value of the solution is 3-5, preferably 4.3-4.9.

[0017] The solvent is preferably methanol and / or ethyl acetate, and a small amount of DMF can be added appropriately when the solution cannot be dissolved completely and clarified, even with heating.

[0018] The concentration of the fluorescein-like substance in the solution can be 1×10 -7 mol / L-1×10 -6 mol / L.

[0019] The pH value of the solution can be adjusted by using a buffer, and the solution should be clear and transparent, and no solid substance should be precipitated, according to the dissolution condition.

[0020] The buffer is an acetate buffer.

[0021] The application of the fluorescein-like substance or the latent blood trace developing agent to the development of latent blood traces also belongs to the protection scope of the present application.

[0022] The latent blood trace is preferably a latent blood fingerprint.

[0023] Further, the present application provides a method for developing a latent blood trace, which comprises the step of developing by using the fluorescein-like substance or the latent blood trace developing agent.

[0024] Specifically, the developing mode can be spraying, dripping or coating, and the developing operation method can be selected according to the actual situation in work.

[0025] Specifically, after the fluorescein-like substance or the latent blood trace developing agent is applied to the object containing the latent blood trace, the object is placed at room temperature for 10-60 minutes (preferably 10-20 minutes), and then hydrogen peroxide solution is added dropwise, and then observation is performed.

[0026] The mass concentration of the hydrogen peroxide solution can be 30-35%, preferably 30%, so that the object can be wetted.

[0027] The observation is performed in the following manner:

[0028] 445nm laser excitation is used, and observation is performed through a 495nm filter wave plate.

[0029] The latent blood trace is located on a non-permeable object.

[0030] The non-permeable object is glass, plastic, ceramic or aluminum foil.

[0031] The fluorescein-like substance used in this invention possesses a carboxylic acid functional group. Upon contact with the amino group of blood cell proteins, a salt-forming reaction occurs, causing the blood cell to bind with the fluorescein as a whole. Hemoglobin in blood acts as a highly efficient catalyst for hydrogen peroxide, generating extremely strong oxidizing free radicals. The fluorescein molecules bound to the proteins are oxidized by this strong oxidant within a specific pH range, becoming fluorescent substances. Excitation with a 445nm laser produces fluorescence, and observation through a 495nm filter yields a clear yellow-green fluorescent fingerprint, providing a new method for revealing latent blood fingerprints.

[0032] The method of this invention is applicable to the development of latent blood marks on a variety of non-permeable objects. It has a wide range of applicable objects, and it has a good development effect on latent blood marks on dark and rough objects. The resulting lines have strong fluorescence brightness, clear and continuous lines, and high contrast between the lines and the background. Attached Figure Description

[0033] Figure 1 To use fluorescein 2-(6-hydroxy-3-oxo-3H-xanthon-9-yl)benzoic acid ( Figure 1 (a) 3',6'-Dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-oxanthracene]-5-carboxylic acid ( Figure 1 (b) and 3',6'-dihydroxy-5-isothiocyanate-3H-spiro[isobenzofuran-1,9'-oxanthracene]-3-one ( Figure 1 (c) The result of a latent blood fingerprint appearing on a black plastic bag and being photographed after being excited by a 445nm laser.

[0034] Figure 2 To use fluorescein 2-(6-hydroxy-3-oxo-3H-xanthon-9-yl)benzoic acid ( Figure 2 (a) 3',6'-Dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-oxanthracene]-5-carboxylic acid ( Figure 2 (b) and 3',6'-dihydroxy-5-isothiocyanate-3H-spiro[isobenzofuran-1,9'-oxanthracene]-3-one ( Figure 2 (c) The result of a latent blood fingerprint appearing on aluminum foil and being photographed after being excited by a 445nm laser.

[0035] Figure 3 To use fluorescein 2-(6-hydroxy-3-oxo-3H-xanthon-9-yl)benzoic acid ( Figure 3 (a) 3',6'-Dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-oxanthracene]-5-carboxylic acid ( Figure 3(b) and 3', 6'-dihydroxy-5-isothiocyanato-3H-spiro[isobenzofuran-l, 9'-xanthene]-3-one Figure 3 (c) the results of developing a latent blood print on a glass sheet and taking a photograph after excitation with a 445 nm laser.

[0036] Figure 4 2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid (fluorescein) was used to develop a latent blood print on a black plastic sheet. Figure 4 (a) 3', 6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-l, 9'-xanthene]-5-carboxylic acid Figure 4 (b) and 3', 6'-dihydroxy-5-isothiocyanato-3H-spiro[isobenzofuran-l, 9'-xanthene]-3-one Figure 4 (c) the results of developing a latent blood print on a black plastic sheet and taking a photograph after excitation with a 445 nm laser.

[0037] Figure 5 2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid (fluorescein) was used to develop a latent blood print on a black plastic sheet. Figure 5 (a) 3', 6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-l, 9'-xanthene]-5-carboxylic acid Figure 5 (b) and 3', 6'-dihydroxy-5-isothiocyanato-3H-spiro[isobenzofuran-l, 9'-xanthene]-3-one Figure 5 (c) the results of developing a latent blood print on a black plastic sheet and taking a photograph after excitation with a 445 nm laser.

[0038] Figure 6 The effects of a latent blood print developed on a black plastic bag with 3', 6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-l, 9'-xanthene]-5-carboxylic acid were observed after excitation with different waveband laser light sources, with the wavelengths of the excitation light sources being 405 nm, 445 nm, 470 nm, 500 nm and 533 nm from left to right. DETAILED DESCRIPTION

[0039] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0040] The materials, reagents and the like used in the following examples can be obtained from commercial sources unless otherwise specified.

[0041] The structural formula of 2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid used in the following examples is shown in Formula I, wherein R1, R2, R3, R4, R5 and R6 are hydrogen atoms, and R7 and R8 are hydrogen atoms.

[0042] The structure of 3', 6'-dihydroxy-3-oxo-3H-spiro [isobenzofuran-l, 9'-xanthene]-5-carboxylic acid used in the following examples is shown in Formula I, wherein R1, R2, R3, R4, R5 and R6 are hydrogen atoms, R7 is carboxyl, and R8 is hydrogen atom.

[0043] The structure of 3', 6'-dihydroxy-5-isothiocyanate-3H-spiro [isobenzofuran-l, 9'-xanthene]-3-one used in the following examples is shown in Formula II, wherein R1, R2, R3, R4, R5 and R6 are hydrogen atoms, R7 is isothiocyanate, and R8 is hydrogen atom.

[0044] Example 1: Use of 2-(6-hydroxy-3-oxo-3H-xanthene-9-yl) benzoic acid fluorescein to visualize latent blood fingerprints on various substrates

[0045] 1. Preparation of 2-(6-hydroxy-3-oxo-3H-xanthene-9-yl) benzoic acid fluorescein solution

[0046] Add 0.05 mg of 2-(6-hydroxy-3-oxo-3H-xanthene-9-yl) benzoic acid to a mixed solvent of 400 ml of methanol and 100 ml of ethyl acetate, heat to completely dissolve, then add 500 ml of acetic acid-sodium acetate buffer at pH 3.6, and stir until uniform. The pH of the resulting fluorescein solution is 4.3, and the concentration is 1.50 x 10 -7 mol / L.

[0047] 2. Sample preparation

[0048] Select five substrates, i.e. glass slides, aluminum foils, black plastic bags, black plastic sheets, and brown ceramic tiles. A volunteer uses degreased cotton to apply blood (human blood, 100% concentration) to the index finger, and then presses the finger onto the substrates to make latent blood fingerprints. Before each pressing, the finger is again coated with blood. The pressing pressure and the duration of the finger on the substrate are kept as constant as possible. Make 10 latent blood fingerprints on each substrate, and dry at room temperature.

[0049] 3. Use of 2-(6-hydroxy-3-oxo-3H-xanthene-9-yl) benzoic acid fluorescein to visualize latent blood fingerprints

[0050] Use the drop visualization method: add 3-5 drops of the prepared fluorescein solution to one of the fingerprints, and then add 2 drops of 30% hydrogen peroxide solution to the same location after 15 minutes at room temperature.

[0051] The bloodstains on the object were developed with 2-(6-hydroxy-3-oxo-3H-xanthene-9-yl)benzoic acid fluorescein, and then observed with the light sources and corresponding filters of the five wave bands of the DCS-4 handprint enhancement system. The results showed that the best development effect was obtained under the excitation of the light at 445 nm and the observation through the filter at 495 nm. The photographing results on different objects are shown in Figure 1 (a), Figure 2 (a), Figure 3 (a), Figure 4 (a), and Figure 5 (a).

[0052] Example 2: Development of bloodstains on various objects with 3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-5-carboxylic acid fluorescein

[0053] 1. Preparation of 3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-5-carboxylic acid fluorescein solution

[0054] 0.06 mg of 3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-5-carboxylic acid was added to a mixed solvent of 350 ml of methanol, 50 ml of DMF and 100 ml of ethyl acetate, heated to completely dissolve, and then 500 ml of acetic acid-sodium acetate buffer at pH 3.6 was added to fully stir and mix. The pH of the obtained fluorescein solution was 4.9, and the concentration was 1.35 x 10 -7 mol / L.

[0055] 2. Sample preparation

[0056] The same as in Example 1.

[0057] 3. Development of bloodstains with 3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-5-carboxylic acid fluorescein

[0058] The development was performed by spraying: after spraying the prepared fluorescein solution on a handprint, the handprint was left to stand at room temperature for about 15 minutes, and then 2 drops of 30% hydrogen peroxide solution were added at the same position.

[0059] The bloodstains on the black plastic bag were developed with 3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-5-carboxylic acid fluorescein, and then observed after excitation with the light sources of the five wave bands of the DCS-4 handprint enhancement system. The handprint effect is shown in Figure 6The wavelengths of the excitation light sources are shown from left to right as 405 nm, 445 nm, 470 nm, 500 nm, and 533 nm (corresponding to the filter wavelengths of 455 nm, 495 nm, 530 nm, 570 nm, and 590 nm).

[0060] By Figure 6 It can be seen that the best effect is obtained under the excitation of light at 445 nm, and the obtained fingerprint fluorescence is bright, the lines are clear and coherent, and the lines have a large contrast with the background.

[0061] The best effect is obtained by using the light at 445 nm in the DCS-4 fingerprint enhancement system, and observing the latent blood fingerprints on the five objects through the filter at 495 nm, and the photographing results on different objects are shown in Figure 1 (b), Figure 2 (b), Figure 3 (b), Figure 4 (b), and Figure 5 (b).

[0062] Example 3: Use of 3', 6'-dihydroxy-5-isothiocyanate-3H-spiro[isobenzofuran-1, 9'-xanthene]-3-ketone fluorescein to develop latent blood fingerprints on various objects

[0063] 1. Preparation of 3', 6'-dihydroxy-5-isothiocyanate-3H-spiro[isobenzofuran-1, 9'-xanthene]-3-ketone fluorescein solution

[0064] In a mixed solvent of 400 ml of ethanol and 100 ml of ethyl acetate, 0.06 mg of 3', 6'-dihydroxy-5-isothiocyanate-3H-spiro[isobenzofuran-1, 9'-xanthene]-3-ketone was added, heated to completely dissolve, and then 500 ml of acetic acid-sodium acetate buffer at pH 3.6 was added, and the mixture was stirred uniformly. The obtained fluorescein solution had a pH of 4.7 and a concentration of 1.54 x 10 -7 mol / L.

[0065] 2. Sample preparation

[0066] The same as in Example 1.

[0067] 3. Use of 3', 6'-dihydroxy-5-isothiocyanate-3H-spiro[isobenzofuran-1, 9'-xanthene]-3-ketone fluorescein to develop latent blood fingerprints

[0068] The development was performed by coating: after the prepared fluorescein solution was coated on a fingerprint, the sample was left to stand at room temperature for about 115 minutes, and then 30% hydrogen peroxide solution was added dropwise at the same position (wetting was sufficient).

[0069] The bloodstain handprint on the object is developed by 3', 6'-dihydroxy-5-isothiocyanate-3H-spiro [isobenzofuran-1, 9'-xanthene]-3-ketone fluorescein, and the best development effect can be obtained by exciting under 445nm light in the DCS-4 handprint enhancement system and observing through a 495nm filter. The photographing results on different objects are shown in the following table. Figure 1 (c), Figure 2 (c), Figure 3 (c), Figure 4 (c) and Figure 5 (c).

[0070] As can be seen from the above examples, the solution of the fluorescein substance is used to develop the bloodstain handprint, and the yellow-green fluorescent handprint can be obtained by observing through a 495nm filter under 445nm light source. The development method is suitable for developing bloodstain handprints on various non-penetrating objects. The method has a wide range of application objects, and has a good development effect on bloodstain handprints on dark objects and rough objects. The obtained handprint has strong fluorescence brightness, clear and coherent lines, large line-to-background contrast, and simple operation.

Claims

1. A blood trace developing agent, which is a solution formed by a fluorescein-like substance and at least two solvents selected from the group consisting of methanol, ethanol, ethyl acetate, DMSO and DMF. The fluorescein-like substance is a compound represented by Formula I or Formula II. In Formula I, each group is substituted as follows: 1) R1, R2, R3, R4, R5 and R6 are hydrogen atoms, and R7 and R8 are hydrogen atoms; or 2) R1, R2, R3, R4, R5 and R6 are hydrogen atoms, R7 is a carboxyl group, and R8 is a hydrogen atom. In Formula II, each group is substituted as follows: R1, R2, R3, R4, R5 and R6 are hydrogen atoms, R7 is an isothiocyanate group, and R8 is a hydrogen atom. The pH of the solution is 3 to 5. The pH of the solution is adjusted by a buffer. The buffer is an acetate buffer.

4. Use of a fluorescein-like substance or the blood trace developing agent according to any one of claims 1 to 3 for developing a blood trace.

2. The occult blood trace visualizer according to claim 1, characterized in that: The concentration of the fluorescein substance in the solution is 1 x 10 -7 mol / L to 1 x 10 -6 mol / L.

3. The occult blood trace visualizer according to claim 1 or 2, characterized in that: The fluorescein-like substance is a compound represented by Formula I or Formula II. In Formula I, each group is substituted as follows: 1) R1, R2, R3, R4, R5 and R6 are hydrogen atoms, and R7 and R8 are hydrogen atoms; or 2) R1, R2, R3, R4, R5 and R6 are hydrogen atoms, R7 is a carboxyl group, and R8 is a hydrogen atom. In Formula II, each group is substituted as follows: R1, R2, R3, R4, R5 and R6 are hydrogen atoms, R7 is an isothiocyanate group, and R8 is a hydrogen atom. The blood trace is a blood print.

6. A method for developing a blood trace, which comprises the step of developing a blood trace by using a fluorescein-like substance or the blood trace developing agent according to any one of claims 1 to 3, wherein after the fluorescein-like substance or the blood trace developing agent is applied to an object having a blood trace, the object is left at room temperature for 10 to 60 minutes, a hydrogen peroxide solution is added dropwise, and then the object is observed. The hydrogen peroxide solution has a mass concentration of 30% to 35%. The observation is performed in the following manner: The observation is performed by exciting with a 445 nm laser and observing through a 495 nm filter.

5. Use according to claim 4, characterized in that: The fluorescein-like substance is a compound represented by Formula I or Formula II. In Formula I, each group is substituted as follows: 1) R1, R2, R3, R4, R5 and R6 are hydrogen atoms, and R7 and R8 are hydrogen atoms; or 2) R1, R2, R3, R4, R5 and R6 are hydrogen atoms, R7 is a carboxyl group, and R8 is a hydrogen atom. In Formula II, each group is substituted as follows: R1, R2, R3, R4, R5 and R6 are hydrogen atoms, R7 is an isothiocyanate group, and R8 is a hydrogen atom. The development is performed by spraying, dropping or coating. The blood trace is a blood print. The blood trace is on a non-penetrable object. The non-penetrable object is glass, plastic, ceramic or aluminum foil. ​ ​ ​ 7. The method of claim 6, wherein: ​ 8. The method according to claim 6 or 7, characterized in that: ​ ​ ​

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