Preparing method of electrochemiluminescence immunosensor

A luminescence immunity and electrochemistry technology, applied in the field of preparation of nanometer immunolabels, can solve the problems of short service life, poor stability, easy leakage of modified substances, etc., and achieve the effect of maintaining chemical properties, preventing leakage and improving sensitivity

Inactive Publication Date: 2010-05-12
SOUTHEAST UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The existing methods for immobilizing bipyridyl ruthenium generally have probl

Method used

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  • Preparing method of electrochemiluminescence immunosensor
  • Preparing method of electrochemiluminescence immunosensor
  • Preparing method of electrochemiluminescence immunosensor

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Embodiment 1

[0023] A preparation method of electrochemiluminescence immunosensor, the preparation steps are: SiO 2 Preparation of @Ru microspheres: Mix 7.5mL cyclohexane, 1.8mL n-hexanol and 1.77mL Tween-80 evenly, then add 340μL 5mg / mL Ru(bpy) 3 2+ The aqueous solution was uniformly stirred at room temperature for 30 minutes to form a stable water-in-oil system, and then 100 μL TEOS and 60 μL NH 3 ·H 2 O to initiate the polymerization reaction, and continue to stir the reaction at room temperature for 20h. After the reaction is completed, add 5mL acetone to the system to separate and precipitate the nanoparticles from the water-in-oil system, and then centrifuge at 8000r / min for 10min. See the formation of an orange-yellow precipitate, discard the supernatant, wash the precipitate several times with absolute ethanol and water, and fully remove the surfactant TEOS and the Ru(bpy) adsorbed on the surface of the nanoparticles 3 2+ , get SiO 2 @Ru microspheres; Preparation of nano-immun...

Embodiment 2

[0025] Preparation of Ab1-MUA / MU-Au modified electrode

[0026] The gold wire electrode was boiled in 2M KOH for 2h and washed. Put it into 30μL 2.5mM MUA and 30μL 7.5mM MU(V MUA / V MU =1:3) in the mixed solution for 10h. Obtain MUA / MU modified gold wire electrode and immerse in TNTU(o-(5-norbornene-2,3-dicarboximido)-N,N,N',N'-tetramethyluronium tetrafluoroborate ) in the activation solution for 15 minutes, rinsed, and put into 100 μL anti-AFP solution for 1 hour. After rinsing, put it into 100 μL of BSA (1 wt%) solution for 30 min to block the non-specific binding site, and obtain anti-AFP (Ab1) modified gold wire electrode (Ab1-MUA / MU-Au).

[0027] AFP antibody modified SiO2@Ru microspheres

[0028] 2mL SiO2 2 Dilute @Ru with ethanol to 6 mL, add 400 μL APTS and stir for 30 min, centrifuge, wash with water and ethanol several times to remove remaining APTS, and obtain APTS-modified SiO 2 @Ru. The above-mentioned nanoparticles were stirred and reacted with 5 mL of glu...

Embodiment 3

[0033] Electrochemiluminescence detection:

[0034] 1) Optimization of test conditions:

[0035] a)C 2 o 4 2- As an important reactant, its concentration has a great influence on ECL. With other conditions unchanged, the ECL signal value increases with C 2 o 4 2- The increase of the concentration first gradually increased and then reached a plateau. in C 2 o 4 2- When the concentration is small, the magnitude of the ECL signal is mainly controlled by C 2 o 4 2- concentration, so as C 2 o 4 2- Concentration increases proportionally; when C 2 o 4 2- When the concentration increases to a certain level, the magnitude of the ECL signal is mainly controlled by the constant SiO in the optimized system 2 @Ru Concentration, thus the platform. Final optimization C 2 o 4 2- The concentration is 3mmol / L.

[0036] b) Antigen incubation time: As the incubation time increases, the ECL signal increases and reaches a plateau after an incubation time of 30 minutes, and t...

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Abstract

The invention relates to a preparing technology of a nano immunological marker, in particular to a preparing method of an electrochemiluminescence immunosensor which has signal amplification function, is decorated by both SiO2 package luminescent material (Ru (bpy) 32 +) and a second antibody (Ab2) and used for low-concentration antigen detection. SiO2 core-shell type nano structure can not only well maintain the chemical property of the content, but also effectively prevent the leakage of the content. Since a plurality of marker molecules can be packaged in an SiO2 bead, the detection sensitivity is greatly improved. The grain diameter of synthesized SiO2@Ru is uniform and the SiO2@Ru has good monodispersity, therefore, identical amount of alpha-fetoprotein antibodies are fixed for each SiO2@Ru nano bead, and the detection repeatability is improved. The linear relation between ECL signal value and AFP concentration is good within the range of 0.01-20ng MmL-1, and the detection lower limit reaches 35pg mL-1.

Description

technical field [0001] The invention relates to a preparation technology of a nano-immunomarker, and relates to a nano-material with a signal amplification function made of SiO 2 Encapsulated luminous substance (Ru(bpy) 3 2+ ) and a second antibody (Ab2) co-modified electrochemiluminescent immunosensor for low-concentration antigen detection. Background technique [0002] The incidence of primary liver cancer (hepatocellular carcinoma, HCC) is gradually increasing in recent years. The prognosis of HCC is poor, and the 5-year survival rate is less than 5%. About 59.18 million people die of HCC every year in the world. Since the 1990s, the number of deaths due to HCC has ranked third among the deaths from malignant tumors in the world, and HCC in my country is the second-ranked cancer "killer", accounting for more than half of the world's HCC deaths each year. One of the reasons for the poor prognosis of liver cancer is that most patients fail to detect and diagnose early,...

Claims

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Application Information

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IPC IPC(8): G01N33/531G01N33/53G01N21/76
Inventor 周镇先钱静刘松琴
Owner SOUTHEAST UNIV
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