Potential fingerprint rapid fluorescence visualization display method based on aggregation-induced emission

Abstract

The invention belongs to the technical field of potential fingerprint and DNA (deoxyribonucleic acid) detection, and particularly relates to an aggregation-induced emission-based potential fingerprint rapid fluorescence visualization display method. The method comprises the following steps: preparing a tracer agent by adopting a fluorescent probe, tracing latent fingerprints or saliva and sweat, and then extracting DNA (Deoxyribonucleic Acid) in a trace sample; the fluorescent probe is a terpyridyl zinc complex and / or an imidazole phosphine bipyridyl zinc complex; the fluorescent probe does not emit fluorescence, condensed-state luminescence is generated after the fluorescent probe is combined with fingerprints, saliva or sweat, fluorescence interference recognition of a substrate is avoided, DNA sample extraction and chain extension processes are not affected, meanwhile, the tracer is a pure water phase, damage of an organic solvent to traces of the fingerprints, the saliva or the sweat and DNA fragments is avoided, and the detection requirement is better met.

Description

Technical Field

[0001] The invention belongs to the technical field of latent fingerprint and DNA detection, and in particular relates to a method for rapid fluorescence visualization of latent fingerprints based on aggregation-induced luminescence. Background Art

[0002] Tracing latent fingerprints or saliva or sweat and extracting DNA samples from them is a complicated process. With the development of technology, the tracing and extraction methods of latent fingerprints or saliva or sweat have been continuously improved, playing a huge role in solving cases and confirming the identities of suspects.

[0003] Latent fingerprints are usually attached to the surface of objects due to the natural oils, sweat or other substances of fingerprints, but because they are invisible, specific tracing methods are required. At present, the conventional color development methods for latent fingerprints include powder spraying, fumigation and chemical methods. The powder spraying method is the simplest and most commonly used method, but it has limited effect on complex surfaces or old fingerprints and is easy to contaminate evidence. The color developing reagents used in the fumigation method and chemical method, such as iodine, silver nitrate, ninhydrin and other chemical reagents, may cause damage to the skin, eyes, mucous membranes or DNA. At the same time, the fluorescent color of many latent fingerprint color developing reagents is similar to the fluorescent color of the substrate itself, which reduces the contrast and resolution. These traditional methods all have the disadvantages of low contrast, low sensitivity, poor selectivity, strong background interference, and high toxicity.

[0004] Molecular fluorescence sensors have made a lot of progress in the rapid, real-time, and highly sensitive identification of guest molecules and fluorescence imaging. Fluorescence visualization identification can be performed through strategies such as fluorescence enhancement / quenching and ratio changes to improve the sensitivity of identification. At the same time, the instruments and equipment involved in the fluorescence method are easy to operate and require a short time, which further improves the efficiency of molecular identification. Therefore, the field of latent fingerprint visualization identification using molecular fluorescence sensors has developed rapidly.

[0005] Tweezer-type metal organic complexes have excellent fluorescence properties and modifiability, and are widely used in the fields of bioimaging, chemistry, and materials science. Tweezer-type Zn(II) metal organic complexes have good water solubility and avoid the use of organic solvents. The lipophilic R group in the developer molecule can combine with the oil in the fingerprint component. After the developer interacts with the fingerprint, the degree of molecular aggregation increases, resulting in an increase in the molecular fluorescence intensity (AIE effect). Therefore, after the developer is selectively adsorbed on the fingerprint, it emits fluorescence under ultraviolet light excitation to achieve the purpose of color development.

[0006] The methods for extracting DNA from latent fingerprints are mainly based on standard DNA extraction techniques, such as organic extraction, salting out, and silica gel column purification. These methods extract DNA from skin cells in fingerprints by chemical or physical means, and then amplify and detect it. Organic extraction methods often use phenol and chloroform to separate DNA. Phenol and chloroform are toxic and carcinogenic chemicals. The DNA extracted by this method may contain some proteins, RNA and other contaminants, and the purity is low, which affects subsequent analysis. The salting out method has cumbersome steps and poor applicability, and is limited to the type of sample. The silica gel column method has a high purification cost and usually requires a certain amount of sample DNA. Low-concentration DNA may not be effectively recovered. In recent years, DNA extraction technology in fingerprints has been significantly developed, especially in the field of forensics. These advances have greatly improved the efficiency and accuracy of DNA extraction and analysis from low-amount or degraded DNA samples. With the advancement of PCR technology, high-sensitivity PCR amplification technology has been able to amplify very small amounts of DNA, and even when there is only trace or degraded DNA, effective STR analysis can be performed. As a vital part of crime scene investigation and forensic evidence collection, like latent fingerprints, saliva and sweat can be amplified by real-time PCR to improve the detection sensitivity of subsequent analysis. Summary of the invention

[0007] The purpose of the present invention is to provide a rapid fluorescence visualization method for latent fingerprints based on aggregation-induced luminescence, which has fast speed, high sensitivity and strong individual recognition ability.

[0008] The present invention provides a method for rapid fluorescence visualization of latent fingerprints based on aggregation-induced luminescence, which includes using a new type of fluorescent probe to prepare a tracer, tracing latent fingerprints or saliva or sweat, and then extracting DNA from trace samples; the fluorescent probe is two bipyridine metal chelates, which are D (donor)-A (acceptor) type bipyridine Zn (II) complexes, and its structural formula is as follows:

[0009]

[0010] The compound represented by the structural formula (I) is a terpyridine zinc complex, and the compound represented by the structural formula (II) is an imidazole phosphine bipyridine zinc complex. The fluorescent probe can be used alone, and the terpyridine zinc complex probe has better water solubility than the imidazole phosphine bipyridine zinc complex probe.

[0011] When the fluorescent probe combines with fingerprints, saliva or sweat, it will produce condensed light emission, making the DNA sample easier to detect, thereby identifying and extracting usable data from trace DNA samples. It has high sensitivity, accurate individual recognition capabilities, and a faster processing time, thereby improving the efficiency of physical evidence extraction at the scene.

[0012] The fluorescent probe is used in a pure water phase and has excellent luminescence properties and extremely low detection limits; that is, the above two fluorescent probes are mixed with water to form a solution as a display agent, and the solution concentration is 0.01-10 mol / L.

[0013] The method for extracting DNA from latent fingerprints or saliva or sweat trace samples provided by the present invention uses a tracer prepared with a fluorescent probe to trace the latent fingerprint, and then prepares a sample of the DNA in the fingerprint or saliva or sweat trace sample; then amplification operation is performed in a VeriFilerTM Plus PCR amplification kit, the sample is denatured and electrophoresed after PCR cycle reaction, and the fragment size and genotyping data are analyzed using GeneMapper ID-X Software v1.5, which proves that the extraction and chain extension processes have no influence at all.

[0014] Compared with the prior art, the advantages of the present invention are:

[0015] (1) The fluorescent probe used in the present invention is dissolved in an aqueous solution, which avoids the damage of organic solvents to fingerprints, traces of saliva or sweat and DNA fragments. It can be identified by naked eyes and is easy to extract. It poses no potential threat to the health and safety of users, is easy to operate, and has a significant effect.

[0016] (2) The fluorescent probe used in the present invention has a high sensitivity and can amplify and detect very small amounts of DNA. Even if the sample size is small, it can still be successfully detected and generate useful results.

[0017] (3) The latent fingerprint or saliva or sweat tracing method of the present invention followed by DNA extraction can quickly locate the latent fingerprint or saliva or sweat sample containing DNA at the crime scene, greatly improving the efficiency of physical evidence search.

[0018] (4) The present invention uses a specially designed high-sensitivity PCR technology to amplify low amounts of DNA. This process selects short fragments of DNA for amplification to maximize the use of degraded or small amounts of DNA extracted from fingerprints or saliva or sweat.

[0019] (5) The fluorescent probe itself does not emit fluorescence. After combining with fingerprints, saliva or sweat, it produces condensed light emission, which overcomes the fluorescence interference of the substrate itself and can be easily identified. It has no effect on the DNA extraction and chain extension process of fingerprints, saliva or sweat.

[0020] (6) Compared with other DNA detection methods, the method of the present invention is simple and rapid; after DNA extraction, results can be obtained within a few hours through PCR amplification, especially with the help of efficient automated equipment, which can greatly shorten the processing time.

[0021] The invention can be used for tracing and analyzing trace DNA samples on site and for rapid extraction of DNA, which is of great significance for handling various cases, especially criminal investigation cases. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The experimental results of two probes (I) and (II) on fingerprint DNA under glass slides and A4 paper are compared. The terpyridine complex and imidazophosphine bipyridine zinc complex probes were sprayed on the glass slide and A4 paper respectively. The position of the signal peak remained unchanged before and after spraying, indicating that the probes did not damage the DNA fragments. This further proves that the two probes in the figure can be used for DNA trace sample detection.

[0023] Figure 2 The experimental results of a probe (I) on saliva DNA under a glass slide and A4 paper are compared. The imidazophosphine bipyridyl zinc complex probe was sprayed on the glass slide and A4 paper respectively. The position of the signal peak remained unchanged before and after spraying, indicating that the probe did not damage the DNA fragment. This further proves that the two probes in the figure can be used for DNA trace sample detection.

[0024] Figure 3 This is a comparison of multiple experimental results of a probe (II) on saliva DNA under a glass slide. The imidazophosphine bipyridyl zinc complex probe was sprayed on multiple glass slides. The position of the signal peak remained unchanged before and after spraying, indicating that the probe did not damage the DNA fragment. This further proves that the two probes in the figure can be used for DNA trace sample detection.

[0025] Figure 4 This is a comparison of multiple experimental results of a probe (II) on saliva DNA under A4 paper. The imidazophosphine bipyridyl zinc complex probe was sprayed on multiple groups of A4 paper. The position of the signal peak remained unchanged before and after spraying, indicating that the probe did not damage the DNA fragments, further proving that the two probes in the figure can be used for DNA trace sample detection. DETAILED DESCRIPTION

[0026] The method for extracting DNA from latent fingerprints or saliva or sweat samples provided by the present invention comprises the following specific steps:

[0027] (I) Sample preparation, including the following processes:

[0028] (1) Prepare two 50*30mm A4 paper sheets and two glass slides;

[0029] (2) Place two A4 sheets of paper and two glass slides together;

[0030] (3) Label the left A4 paper and slide as A, and the right A4 paper and slide as B;

[0031] (4) Use your index finger to press on the joints of two A4 paper pieces and two glass slides, respectively, to ensure that half of the fingerprints are left on each piece of paper and half of the fingerprints are left on each glass slide;

[0032] (5) Place A4 paper A and glass slide A separately, and spray the developer evenly on them for 5 to 30 seconds;

[0033] (6) Use a special saliva collector to collect saliva; use cotton swabs, non-woven fabrics or sweat collection patches to collect sweat from the skin surface onto A4 paper and a glass slide; perform the operations in process (5).

[0034] (II) DNA extraction, including the following process:

[0035] (1) Use a two-step dry and wet method to collect fingerprints, saliva, or sweat from the surface of each sample using a cotton swab;

[0036] (2) The DNA of the above samples was extracted using a fully automatic 24-channel micro-DNA extraction workstation (Changchun Bokun Biotechnology Co., Ltd.), with an elution volume of 20 to 100 μL;

[0037] (3) Use a pipette to take 5 to 10 μL of the extracted DNA solution for PCR amplification;

[0038] (4) Use the VeriFilerTM Plus PCR Amplification Kit (Thermo Fisher Scientific, USA) to perform amplification operations, mix 1-3 μL VeriFilerTM Plus Master Mix, 0.5-2 μL VeriFilerTM Plus Primer Mix, and 5-8 μL extracted DNA sample to make the final volume of the PCR reaction 8-15 μL;

[0039] (5) The PCR cycle operation is as follows: 50-100°C, 0.5-2 min; 50-100°C, 10 sec, 50-100°C, 60-100 sec, 1-5 cycles; 50-100°C, 5-30 sec, 50-100°C, 60-100 sec, 10-30 cycles; 50-100°C, 2-10 min; keep warm at 0-10°C.

[0040] (6) Add 0.5-5 μL of the amplified product to 5-15 μL of deionized formamide (Hi-Di TM Formamide) and 0.5-5 μL GeneScan 600LIZ Size Standard;

[0041] (7) The samples were denatured at 60-100°C for 2-10 min and then frozen on ice before electrophoresis;

[0042] (8) Fragment size and genotyping data were analyzed using GeneMapper ID-X Software v1.5, with an analysis threshold of 20 to 100 RFU.

Claims

1. A method for rapid fluorescence visualization of latent fingerprints based on aggregation-induced luminescence, characterized in that: A fluorescent probe is used to prepare a tracer to trace latent fingerprints or saliva or sweat, and then DNA in the trace sample is extracted; the fluorescent probe is two bipyridine metal chelates, which are D (donor)-A (acceptor) type bipyridine Zn (II) complexes, and its structural formula is as follows: The compound represented by the structural formula (I) is a terpyridine zinc complex, and the compound represented by the structural formula (II) is an imidazole phosphine bipyridine zinc complex.

2. The method according to claim 1, characterized in that The tracer is an aqueous solution of the fluorescent probe, and the concentration of the solution is 0.01-10 mol / L.

3. The method according to claim 1, characterized in that The specific process is as follows: use a tracer to trace latent fingerprints, and then prepare samples of DNA from fingerprints or trace samples of saliva or sweat; then perform amplification using the VeriFilerTM Plus PCR Amplification Kit, denature and electrophorese after PCR cycling, and use GeneMapper ID-XSoftware v1.5 to analyze fragment size and genotyping data.

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