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Adenosine monophosphate-protected gold-silver alloy nanocluster fluorescence probe, and application thereof in detection of Plasmodium vivax lactate dehydrogenase

An adenosine monophosphate, gold-silver alloy technology, applied in the detection of Plasmodium vivax lactate dehydrogenase, in the field of gold-silver alloy nanocluster fluorescent probes, which can solve the problem of increasing the detection cost and limiting the practical application of the detection method, etc. problem, to achieve the effect of simple structure, good biocompatibility, high stability and water solubility

Active Publication Date: 2019-11-05
JILIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, researchers have developed some PLDH biosensors and improved detection selectivity by introducing single-stranded DNA aptamers, but the application of such aptamers has greatly increased the detection cost and limited the practicality of this type of detection method. application

Method used

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  • Adenosine monophosphate-protected gold-silver alloy nanocluster fluorescence probe, and application thereof in detection of Plasmodium vivax lactate dehydrogenase
  • Adenosine monophosphate-protected gold-silver alloy nanocluster fluorescence probe, and application thereof in detection of Plasmodium vivax lactate dehydrogenase
  • Adenosine monophosphate-protected gold-silver alloy nanocluster fluorescence probe, and application thereof in detection of Plasmodium vivax lactate dehydrogenase

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Experimental program
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Embodiment 1

[0029] Referring to the literature (Proc.Natl.Acad.Sci.USA, 2013, 110, 15967–15972), the E.coli BL21(DE3) containing the pET28a-PvLDH plasmid was shaken at 220 rpm in LB medium at 37°C. Shake and incubate overnight. Inoculate at a volume ratio of 1:100, culture at 37°C and shake at 180 rpm until OD 600 =0.8~1.0, adding IPTG at a volume ratio of 1:5000 to induce the expression of LDH, shaking at 180 rpm at 25°C for 13 hours to induce expression, then centrifuging at 4000 rpm for 30 minutes at 4°C to collect the bacteria. The bacteria were resuspended in 20 mM phosphate buffer (pH=7.4), and the supernatant was collected by centrifugation after sonication.

[0030] Use Ni on the collected supernatant 2+ -NTA column purification of Plasmodium vivax lactate dehydrogenase used in the present invention: the supernatant slowly flows through Ni 2+ -NTA column, repeat 3 times, then wash the column with 3 times column volume buffer (20mM phosphate buffer), repeat 3 times, finally use ...

Embodiment 2

[0032] Preparation of gold-silver alloy nanocluster fluorescent probes protected by adenosine monophosphate:

[0033] Weigh chloroauric acid (HAuCl 4 )339.79mg (1mmol / L), add 100mL distilled water to configure a concentration of 10mmol / L chloroauric acid solution (keep away from light); weigh silver nitrate (AgNO 3 ) 16.987mg (0.1mmol / L), add 10mL distilled water to configure a silver nitrate solution with a concentration of 10mmol / L (keep away from light); weigh 80mg (2 mmol / L) sodium hydroxide (NaOH) and add 2mL distilled water to configure a concentration of 1mol / L Sodium hydroxide solution; weigh 73.048mg (0.2mmol / L) of adenosine monophosphate and dissolve it in 10mL of distilled water to prepare a concentration of 20mmol / L adenosine monophosphate solution; weigh 735.25mg (2.5mmol / L) of sodium citrate and add to 5mL Prepare 500 mmol / L sodium citrate solution with distilled water; weigh 24.143 mg (0.1 mmol) aluminum chloride hexahydrate (AlCl 3 ·6H 2 O) add 10mL distille...

Embodiment 3

[0039] A method for detecting Plasmodium vivax lactate dehydrogenase with a gold-silver alloy nanocluster fluorescent probe protected by adenosine monophosphate: freeze-dry and weigh the solid of the Au-AgNCs@AMP fluorescent probe solution prepared in Example 2, Dilute with the prepared pH=6.5 MES-NaOH buffer solution, prepare a solution with a concentration of 30 μg / mL, take 1 mL of the fluorescent probe solution, and add Plasmodium vivax lactate dehydrogenation to each 1 mL of fluorescent probe solution The mother liquor of enzyme, make its final concentration be 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 130, 150, 170, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000×10 -9 mol / L (the 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 vivax lactate dehydrogenase (excitation wavelength 340nm) was recorded by a fluorescence spectrometer. Such ...

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Abstract

The invention discloses an adenosine monophosphate-protected gold-silver alloy nanocluster fluorescence probe, and an application thereof in the detection of Plasmodium vivax lactate dehydrogenase, and belongs to the technical field of fluorescence probes. The fluorescence probe has the advantages of simple structure, easiness in synthesis, strong stability and good biocompatibility, and can generate large linear fluorescence enhanced response to the Plasmodium vivax lactate dehydrogenase (PvLDH). The response has a wide detection range (0 to 1.0 *10<-6> mol / L) and a high sensitivity, and thedetection limit can reach 3.7 ng / mL. An adjuvant Al<3+> is introduced, so the probe can completely distinguish the Plasmodium vivax lactate dehydrogenase and other types of lactate dehydrogenases. Thedetection method of the invention has the advantages of high speed, simplicity in operation, stable signal, high sensitivity, no pretreatment, and no complicated detection instruments.

Description

technical field [0001] The invention belongs to the technical field of fluorescent probes, and in particular relates to a gold-silver alloy nano-cluster fluorescent probe protected by adenosine monophosphate and its application in detection of Plasmodium vivax lactate dehydrogenase. Background technique [0002] Malaria is a vector-borne infectious disease caused by mosquito bites or blood transfusion of patients with malaria parasites. It kills more than 435,000 people every year (especially children under the age of 5 are the main victims), and seriously threatens human beings. healthy. According to the World Malaria Report 2018, there were approximately 219 million malaria cases in 2017, a slight increase compared to the previous year. There are mainly four kinds of Plasmodium that can parasitize in the human body, namely Plasmodium vivax, Plasmodium malariae, Plasmodium falciparum and Plasmodium ovale, which are the culprits that cause malaria. In the early diagnosis o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C09K11/02C09K11/58G01N21/64B82Y20/00B82Y30/00
CPCB82Y20/00B82Y30/00C09K11/025C09K11/58G01N21/6428G01N2021/6417Y02A50/30
Inventor 吴玉清张春霞李洪伟
Owner JILIN UNIV
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