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Core-shell gold-platinum alloy nano immunochromatographic test paper for detecting tenuazonic acid

A technology of alternarinic acid and immunochromatographic test paper, which is applied in the field of immunoassay, and can solve problems such as limited sensitivity, limited probe signal, and insufficient signal

Active Publication Date: 2021-02-02
FUJIAN AGRI & FORESTRY UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, colloidal gold immunochromatography test paper also has shortcomings. In addition to being limited by the characteristics of the antibody on the probe, its sensitivity is mainly limited by the signal of the probe, especially the competition mode immunochromatography test paper.
Alternaria tenoic acid is a small molecule, and its immunological analysis methods all adopt the competition mode. In fact, the less the amount of antibodies and probes used in this mode, the higher the sensitivity, but it is easy to cause insufficient signal and affect the results. Interpretation
From the perspective of the probe signal, the present invention strives to provide a solution to the problem of low sensitivity of the competitive mode immunochromatography test paper, and at the same time, establishes a rapid detection method for Alternaria tenoic acid

Method used

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  • Core-shell gold-platinum alloy nano immunochromatographic test paper for detecting tenuazonic acid
  • Core-shell gold-platinum alloy nano immunochromatographic test paper for detecting tenuazonic acid
  • Core-shell gold-platinum alloy nano immunochromatographic test paper for detecting tenuazonic acid

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Experimental program
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Embodiment 1

[0041] The preparation of embodiment 1 hybridoma cell line 6D5

[0042] 1. Preparation of artificial complete antigen

[0043] Dissolve 0.5 mg TA in 1 mL of pyridine, add 1.5 mg of carboxymethylhydroxylamine hemihydrochloride (CMO), mix well, stir at room temperature at low speed for 24 h, blow dry with nitrogen, and then dissolve in 1 mL of dimethyl formaldehyde Add 0.6 mg NHS and 0.75 mg DCC in sequence, mix quickly and uniformly, and react under low-speed stirring for 12 h in the dark at room temperature to obtain a hapten activation solution; dissolve 5 mg of hemocyanin KLH in 8 mL of 10× PBS (pH 7.4), under low-speed stirring, add the activation solution drop by drop, stir at low speed and avoid light for 6 h. The reaction solution was centrifuged at 12000 r / min for 30 min, dialyzed with 1×PBS (pH 7.4) for three days, and the dialysate was changed every 4 hours. After dialysis, the PEG20000 was concentrated to obtain the immune antigen TA-O-KLH. The preparation process ...

Embodiment 2

[0054] Example 2 Preparation and Characterization of Anti-Alternaria Acid Monoclonal Antibody

[0055] 1. Preparation and purification of ascites

[0056] 1) Preparation of ascites: the hybridoma cell line 6D5 in the logarithmic growth phase was divided into 1×10 6cell / injected into the abdominal cavity of 8-week-old Bal b / c female mice sensitized with incomplete Freund's adjuvant, observe the mice one week later, extract the ascites when the mouse's abdomen is enlarged and feel tense, and place it at 2500 r / min Centrifuge for 5 minutes, remove cells, etc., take the supernatant and centrifuge at 4°C, 12000 r / min for 20 minutes, and divide into three layers after centrifugation (from bottom to top are aggregates, the middle layer containing a large amount of antibodies, and the lipid layer), and take middle layer.

[0057] 2) Antibody purification: The caprylic acid-ammonium sulfate method was used for crude extraction combined with Protein G affinity chromatography for purif...

Embodiment 3

[0071] Example 3 Core-shell gold-platinum alloy nano Au@Pt 0.4 preparation and characterization

[0072] 1. Preparation and optimization of core-shell gold-platinum alloy nanoparticles

[0073] 20nm colloidal gold (gold nano-particle, GNP) was prepared by reducing chloroauric acid with sodium citrate, and 100mL ddH 2 O, heat to boiling, add 2mL 1% trisodium citrate, mix well, quickly add 1mL 1% HAuCl at one time 4 , keep boiling and stirring until the color is stable, cool at room temperature, ddH 2 O was adjusted to 100mL. Prepare 200mL of the above colloidal gold, divide it into 10 equal parts, each 20mL, add 2.00 mL of 30 mM ascorbic acid (L-AA), mix well, stir vigorously at 25°C, according to the molar ratio of platinum to gold in the system ( r = n Pt / n Au ) is 0, 0.04, 0.08, 0.12, 0.2, 0.3, 0.4, 0.6, 0.8 and 1, add 1% of different volumes (0, 10, 20, 30, 50, 75, 100, 150, 200, 250 μL) h 2 PtCl 6 , kept stirring for 30min, and respectively set the volume to 24m...

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Abstract

The invention belongs to the field of immunodetection, and relates to a core-shell gold-platinum alloy nano immunochromatographic test paper for detecting tenuazonic acid, which has a detection threshold (eliminating T line) of 25 ng / mL and a visual detection limit (vLOD) of 0.39 ng / mL. Compared with the conventional colloidal gold test paper, the detection threshold (eliminating the T line) is reduced by 4 times, and the visual detection limit (vLOD) is reduced by 32 times. The invention provides a high-sensitivity rapid detection method of alternaria tenuissima ketonic acid.

Description

technical field [0001] The invention belongs to the field of immunological detection, and in particular relates to a core-shell type gold-platinum alloy nano-immunochromatographic test paper for detecting alternarinic acid. Background technique [0002] Every year, human and animal poisoning safety incidents caused by mycotoxin pollution are common, and the loss of grain and food caused by mycotoxin pollution is too numerous to enumerate, which has attracted widespread attention from the international community. Mycotoxin is a kind of secondary metabolite produced by fungal metabolism. At present, there are at least about 400 different mycotoxins in nature, a class of heterocyclic compounds with molecular weights ranging from 50 Da to greater than 500 Da. Mycotoxins are often highly toxic, acute and chronic toxic, potentially carcinogenic and even lethal. Alternaria tenuazonic acid (Tenuazonic Acid, TA) is a mycotoxin mainly produced by fungi of the genus Alternaria, which ...

Claims

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Application Information

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IPC IPC(8): G01N33/58G01N33/577G01N33/558G01N33/53
CPCG01N33/587G01N33/577G01N33/558G01N33/5308
Inventor 汪世华蔡培原王荣智凌素美贾坤志
Owner FUJIAN AGRI & FORESTRY UNIV
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