Silica fluorescent immunolabeled probe based on fluorescence resonance energy transfer as well as preparation method and application of silica fluorescent immunolabelled probe

A fluorescence resonance energy and silicon dioxide technology, which is applied in the field of biological analysis and immune analysis, can solve the problems of slow carrier metabolism, toxic and side effects of organisms, etc.

Pending Publication Date: 2018-05-01
ANHUI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The metabolism after entering the organism is inseparable from the health of the organism. Excessiv

Method used

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  • Silica fluorescent immunolabeled probe based on fluorescence resonance energy transfer as well as preparation method and application of silica fluorescent immunolabelled probe
  • Silica fluorescent immunolabeled probe based on fluorescence resonance energy transfer as well as preparation method and application of silica fluorescent immunolabelled probe
  • Silica fluorescent immunolabeled probe based on fluorescence resonance energy transfer as well as preparation method and application of silica fluorescent immunolabelled probe

Examples

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Embodiment 1

[0074] A method for preparing a silica fluorescent immunolabeling probe based on fluorescence resonance energy transfer, the method comprising the following steps:

[0075] 1) Dissolve 50 mg of cetyltrimethylammonium bromide CTAB in 50 mL of deionized water under magnetic stirring at 65 °C;

[0076] 2), then add 650μL ethylene glycol and 2.1mL ammonia water at 65°C;

[0077] 3) Add TEOS 200 μL within 5 minutes after adding ethylene glycol and ammonia water and react for 30 minutes;

[0078] 4), mix 0.5mL 0.004mmol / mL BODIPY solution and 0.5mL 0.004mmol / mL Rhodamine B solution, add the mixed solution of 1mL BODIPY and Rhodamine B to step 3), and then add trimethoxymethane Silane MTMS 30μL, react for 2h;

[0079] 5) Add 90 μL mL of 3-aminopropyltriethoxysilane APTES to continue the reaction for 1.5 hours;

[0080] After the reaction is completed, stir and cool to room temperature, centrifuge and wash 3 times, and freeze-dry to obtain RB-BODIPY-FRET silica nanoparticles, which...

Embodiment 2

[0091] An application of silica fluorescent immunolabeling probe-coupled antibody based on fluorescence resonance energy transfer, the specific method is:

[0092] 1. First prepare anti-MFAP highly specific polyclonal antibody:

[0093] 1-1. First immunization: mix the immunogen with Freund's complete adjuvant in equal volume ratio, emulsify with a syringe, and then immunize New Zealand white rabbits by subcutaneous injection on the back. Subcutaneously inject 10 points, the total dose is 1.0mg / kg / time. Booster immunization after three weeks;

[0094]1-2. Booster immunization: adopt the same back subcutaneous injection method and injection dose for injection immunization, and the different adjuvant used for booster immunization is Freund's incomplete adjuvant. After that, a booster immunization was carried out every two weeks, and the titer was measured by collecting blood from the edge of the rabbit's ear in the middle week. After the titer reached 1:64000, a booster immuni...

Embodiment 3

[0102] A method for preparing silica fluorescent immunolabeling probes based on fluorescence resonance energy transfer, comprising the following steps:

[0103] 1) Dissolve 50 mg of cetyltrimethylammonium bromide CTAB in 50 mL of deionized water at 65 °C;

[0104] 2), then add 650μL ethylene glycol and 2.1mL ammonia water at 65°C;

[0105] 3) Add TEOS 200 μL within 5 minutes after adding ethylene glycol and ammonia water and react for 30 minutes;

[0106] 4), mix 0.5mL 0.005mmol / mL BODIPY solution and 0.5mL 0.005mmol / mL Rhodamine B solution, add 1mL of the mixed solution of BODIPY and Rhodamine B to step 3), and then add trimethoxymethane Silane MTMS30μL, react for 2h;

[0107] 5) Add 90 μL mL of 3-aminopropyltriethoxysilane APTES to continue the reaction for 1.5 hours;

[0108] After the reaction is completed, stir and cool to room temperature, centrifuge and wash 3 times, and freeze-dry to obtain RB-BODIPY-FRET silica nanoparticles, which are silica fluorescent immunolabe...

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Abstract

The invention provides a silica fluorescent immunolabeled probe based on fluorescence resonance energy transfer as well as a preparation method and application of the silica fluorescent immunolabelledprobe. The preparation method comprises the steps of using mesoporous silica nanoparticles as matrix, and constructing a fluorescence resonance energy transfer system (RB-BODIPY-FRET) by doping rhodamine B and BODIPY. The fluorescent labeling probe is coupled by using a sodium periodate oxidation antibody, so that a reliable basis is provided for the super-sensitive detection and immunoassay of nano-drug carriers. Compared with the prior art, the silica fluorescent immunolabeling probe is prepared by means of codoping of fluorescent dyes, i.e., the BODIPY and the rhodamine B, and fluorescenceresonance energy transfer occurs between the two fluorescent dyes, so that the fluorescence intensity is increased, and quenching is reduced; the fluorescent labeled probe is coupled by using the sodium periodate oxidation antibody, so that the coupling efficiency is improved, and the influence of a cross-linking agent on the activity of the antibody is eliminated.

Description

technical field [0001] The invention belongs to the fields of biological analysis and immunoanalysis, and in particular relates to a silica fluorescent immunolabeling probe based on fluorescence resonance energy transfer, a preparation method and an application. Background technique [0002] Polysuccinimide (PSI), N-(3-aminopropyl) imidazole (NAPI) and oleylamine (OAm) react together to obtain PSI-OAm-NAPI amphiphilic polymeric nanoparticles (MFAP), which is a kind of polymer Nano drug carrier. Polymer emulsion delivery systems have been widely used to encapsulate drugs, and many polymer nano-drug delivery systems have been approved in clinical applications or are being used in clinical trials for the treatment of cancer. The metabolism after entering the organism is inseparable from the health of the organism. Excessive drug dosage may cause slow metabolism of the carrier and cause toxic side effects on the organism. Therefore, the quantitative determination of the drug c...

Claims

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Application Information

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IPC IPC(8): G01N33/533G01N21/64
CPCG01N21/6428G01N33/533G01N2021/6439
Inventor 张明翠闫希
Owner ANHUI NORMAL UNIV
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