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Fluorescent probe based on double-stranded DNA protection and application of same to preparation of drug used for detecting Plasmodium falciparum lactate dehydrogenase

A fluorescent probe and base technology, applied in the field of fluorescent probes, to achieve the effects of strong stability, stable signal and broad application prospects

Inactive Publication Date: 2017-02-15
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, it is still impossible to accurately predict the fluorescence of AgNCs protected by a certain DNA sequence, which is also an important reason why DNA synthesis AgNCs has attracted many researchers.

Method used

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  • Fluorescent probe based on double-stranded DNA protection and application of same to preparation of drug used for detecting Plasmodium falciparum lactate dehydrogenase
  • Fluorescent probe based on double-stranded DNA protection and application of same to preparation of drug used for detecting Plasmodium falciparum lactate dehydrogenase
  • Fluorescent probe based on double-stranded DNA protection and application of same to preparation of drug used for detecting Plasmodium falciparum lactate dehydrogenase

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Referring to the literature (Proc. Natl. Acad. Sci. USA, 2013, 110, 15967-15972), E. coli BL21(DE3) containing the pET28a-PfLDH plasmid was cultured overnight at 37°C with shaking at 220 rpm. Inoculated at a ratio of 1:100, 37 ° C, 180 rpm shaking culture OD 600 After = 0.8-1.0, IPTG was added to induce the expression of LDH, and after induction of expression by shaking at 180 rpm at 25°C for 13 hours, the cells were collected by centrifugation at 4,000 rpm for 30 minutes at 4°C. The bacteria were resuspended in 20 mM phosphate buffer (pH=7.4), and the supernatant was collected by centrifugation after sonication.

[0024] Use Ni on the collected supernatant 2+ -NTA column purification of lactate dehydrogenase of the present invention: the supernatant is slowly flowed through Ni 2+ -NTA column, repeat 3 times, then wash the column with 3 times of column volume buffer (20mM phosphate buffer), repeat 3 times, finally use 3 times of column volume elution buffer (containin...

Embodiment 2

[0026] The preparation of the silver nanocluster fluorescent probe of DNA protection: with the template strand DNA (its nucleotide sequence as shown in SEQ ID NO.6) that contains complementary strand and the complementary strand DNA that contains rich G base (its nucleoside acid sequence as shown in SEQ ID NO.7) to prepare DNA-protected silver nanocluster fluorescent probe as an example, the specific steps are as follows figure 1 shown.

[0027] Weigh silver nitrate (AgNO 3 ) 17.0mg, add 20mL distilled water to configure 5mmol / L silver nitrate mother liquor for standby (keep away from light); weigh 358mg disodium hydrogen phosphate (Na 2 HPO 4 12H 2 O) add 50mL water configuration 20mmol / L disodium hydrogen phosphate solution; Then weigh 156mg sodium dihydrogen phosphate (Na 2 HPO 4 12H 2 O) Add 50mL water to configure 20mmol / L sodium dihydrogen phosphate solution; get 40.5mL disodium hydrogen phosphate solution and 9.5mL sodium dihydrogen phosphate solution to mix respe...

Embodiment 3

[0032] Establish a method for detecting Plasmodium falciparum lactate dehydrogenase in the solution with the silver nanocluster fluorescent probe protected by DNA: the silver nanocluster (I) fluorescent probe solution protected by double-stranded DNA prepared in Example 2 is reconstituted with phosphate buffer solution Diluted to 10 times the volume, prepared to a concentration of 1.0×10 -6 mol / L solution. Take 1mL of the fluorescent probe solution, and add the mother solution of Plasmodium falciparum lactate dehydrogenase to each 1mL of the fluorescent probe solution, so that the final concentration is 50~2000×10 -9 mol / L (50,100,150,200,250,300,350,400,450,500,600,700,800,900,1000,1200,1400,1600,1800,2000×10 -9 mol / L) (concentration was measured by the instrument nano 2000), and the fluorescence emission spectrum of the fluorescent probe solution in response to different concentrations of Plasmodium falciparum lactate dehydrogenase (excitation wavelength is 470nm) was recor...

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Abstract

The invention provides a silver nano-cluster fluorescent probe based on double-stranded DNA protection and application of the same to preparation of a drug used for detecting Plasmodium falciparum lactate dehydrogenase, belonging to the technical field of fluorescent probes. DNA used in the invention is of a double strand structure, wherein one strand is composed of a complementary strand DNA and a template strand DNA, and the other strand is composed of a complementary strand DNA and G base-rich DNA; the template strand DNA is a protective group for synthesis of a silver nano-cluster and can be coordinated with the surface of the silver nano-cluster to prevent further expansion of the silver nano-cluster; the G base-rich DNA improves the fluorescence emission intensity of the silver nano-cluster by approaching the silver nano-cluster; the complementary strand DNA has a length of 10 to 30 bases and is rich in A (adenine) and T (thymine) bases; the template strand DNA has a length of 10 to 20 bases and is rich in C (cytosine) bases; and the G base-rich DNA has a length of 10 to 25 bases and is rich in G bases. A detection method provided by the invention is fast in detection speed, simple to operate, simple in system, stable in signal, high in sensitivity and free of any pretreatment and does not need any complex detection apparatuses.

Description

technical field [0001] The invention belongs to the technical field of fluorescent probes, and in particular relates to a double-stranded DNA-based silver nanocluster fluorescent probe and its application in the preparation and detection of Plasmodium falciparum lactate dehydrogenase drugs. Background technique [0002] Malaria is a disease caused by Plasmodium infection, which seriously threatens human health. According to the World Health Organization (WHO), there were about 214 million malaria cases in the world in 2015, resulting in a total of about 438,000 deaths, of which 90% were in Africa and 7% were in Southeast Asia. [0003] At present, the detection of malaria mainly includes two methods, one is the direct microscope observation method, that is, the blood samples of patients with suspected high fever are directly observed with a microscope, this method is currently the most widely used method for detecting malaria, but this method is in good Under the optical co...

Claims

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Application Information

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IPC IPC(8): G01N21/64
CPCG01N21/6486Y02A50/30
Inventor 吴玉清王威贤李洪伟
Owner JILIN UNIV
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