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Quantum dot fluorescence probe, preparation method and application thereof, and method and kit for detecting activity of elastinase

A technology of elastase and fluorescent probe, applied in the direction of fluorescence/phosphorescence, chemical instruments and methods, measuring devices, etc., can solve the problems of low elastase activity, decreased immunity, respiratory failure, etc., and achieve high effectiveness, High affinity, high sensitivity effect

Active Publication Date: 2019-10-18
HUAZHONG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] The main function of elastase is to hydrolyze extracellular elastin, so that immune cells can quickly reach the inflammatory site to remove pathogens, but if the elastase activity is too high, it will cause a series of lung diseases, such as chronic obstructive pulmonary disease, including emphysema , chronic bronchitis and asthma can also cause pulmonary vesicle fibrosis and gradually fail the respiratory function. In addition, too much elastin will also hydrolyze immune proteins, which will reduce the body's immune ability and cause lung cancer
[0003] The current probes used to detect elastase activity are mainly through its ultraviolet substrate NMeOSuc-AAPV-pNA, fluorescent substrate NMeOSuc-AAPV-AMC, and small molecule fluorescent probes based on coumarin. There are also FRET probes based on fluorescent proteins or polypeptide substrates, but these probes have certain limitations in application. The first is the detection limit. The activity of elastase in liquid is often very low and difficult to be detected. In addition, these probes are almost difficult to be applied to cells or even in vivo levels, so the application is limited, so the development of high sensitivity and can be applied to cells and in vivo levels The probe is very necessary

Method used

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  • Quantum dot fluorescence probe, preparation method and application thereof, and method and kit for detecting activity of elastinase
  • Quantum dot fluorescence probe, preparation method and application thereof, and method and kit for detecting activity of elastinase
  • Quantum dot fluorescence probe, preparation method and application thereof, and method and kit for detecting activity of elastinase

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Embodiment approach

[0032] According to a specific embodiment of the present invention, the elastase is neutrophil elastase (a serine proteolytic enzyme secreted by neutrophils), and the amino acid sequence of the elastase-specific polypeptide is preferably as SEQ ID No: 1 (QPMAVVQSVPQK), the rhodamine derivative is preferably sulforhodamine B. Such as figure 1 As shown, the semiconductor quantum dot forms an amide bond connection with the amino group of glutamine (Q) at the 5' end of SEQ ID No: 1 through its own carboxyl group, and the 4th sulfonic acid group of sulforhodamine B is connected with SEQ ID No: 1. The amino group of lysine (K) at the 3' end of ID No: 1 forms a sulfonamide linkage. In this preferred case, the quantum dot fluorescent probe has extremely high specificity and sensitivity to elastase.

[0033] in, figure 2 shows that elastase catalyzes figure 1 The schematic diagram of the hydrolysis of the quantum dot fluorescent probe shown, under the catalysis of elastase, the po...

preparation example 1

[0090] This preparation example is used to illustrate the preparation of the quantum dot fluorescent probe of the present invention

[0091] (1) In the MES buffer solution with a pH value of 6.0, EDC and NHS were respectively contacted with the three kinds of semiconductor quantum dots customized as above for 30 minutes at room temperature to activate the carboxyl groups on the surface of the semiconductor quantum dots; wherein, The molar ratio of quantum dots to EDC is 1:4000, and the molar ratio of EDC to NHS is 2:5.

[0092] (2) Ultrafiltration is performed on the contacted product through an ultrafiltration membrane with a molecular weight cut-off of 10 kD to remove unreacted EDC and NHS, and then PBS buffer exchange is performed to make the pH of the system = 7.2.

[0093] (3) Respectively react the activated carboxyl group on the surface of the semiconductor quantum dot obtained in step (2) with the amino group of SEQ ID No: 1 connected with sulforhodamine B at 30° C. fo...

Embodiment 1

[0096] This example is used to illustrate the method for detecting elastase activity with the quantum dot fluorescent probe provided by the present invention.

[0097] Dissolve 100 μg of elastase sample in 100 mM Tris-HCl (containing 500 mM NaCl, pH=7.5) buffer solution and carry out gradient dilution, prepare different concentrations of elastase standard solution according to the concentration given in Table 1, and prepare the obtained The elastase standard solution was preheated in a 30°C constant temperature water tank; at the same time, the black flat-bottomed 96-well plate was preheated in a 30°C incubator. Subsequently, 40 μL of the preheated elastase standard solution was placed in a preheated black flat-bottomed 96-well plate, and then 120 μL of 100 mM Tris-HCl (containing 500 mM NaCl, pH=7.5) buffer solution was added, and a negative control group ( Only add 160 μl of buffer solution), under dark conditions, 40 μL of quantum dot fluorescent probe 1 was contacted with ...

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Abstract

The invention relates to the field of activity detection of elastinase, in particular to a quantum dot fluorescence probe, a preparation method and application thereof, and a method and a kit for detecting activity of elastinase. The quantum dot fluorescence probe comprises a semiconductor quantum dot of a nucleus-shell structure, a Rhodamine derivative and an elastinase specific polypeptide connected between the semiconductor quantum dot and the Rhodamine derivative; and the semiconductor quantum dot takes CdSe as a nucleus and takes ZnS as a shell. The preparation method includes the steps:EDC and NHS make contact with the semiconductor quantum dot so as to activate carboxyl on the surface of the semiconductor quantum dot; a reaction of the activated carboxyl on the surface of the semiconductor quantum dot and amidogen of the proteolytic enzyme specific polypeptide connected with the Rhodamine derivative is obtained to obtain the quantum dot fluorescence probe. When being used for detecting the activity of a proteolytic enzyme, especially the elastinase, the quantum dot fluorescence probe has higher affinity with the proteolytic enzyme, and the effectiveness of detection is higher.

Description

technical field [0001] The invention relates to the field of elastase activity detection, in particular to a quantum dot fluorescent probe, a preparation method and application of the quantum dot fluorescent probe, a method and a kit for detecting elastase activity. Background technique [0002] The main function of elastase is to hydrolyze extracellular elastin, so that immune cells can quickly reach the inflammatory site to remove pathogens, but if the elastase activity is too high, it will cause a series of lung diseases, such as chronic obstructive pulmonary disease, including emphysema , chronic bronchitis and asthma can also cause pulmonary vesicle fibrosis and gradually fail respiratory function. In addition, too much elastin will also hydrolyze immune proteins, which will reduce the body's immunity and cause lung cancer. [0003] The current probes used to detect elastase activity are mainly through its ultraviolet substrate NMeOSuc-AAPV-pNA, fluorescent substrate NM...

Claims

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Application Information

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IPC IPC(8): C09K11/88C09K11/02G01N21/64
CPCC09K11/883C09K11/025G01N21/6486
Inventor 杨光富杨文超严爱民
Owner HUAZHONG NORMAL UNIV
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