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    2.2. Synthesis of Afp Antibody Modified Aunps

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    Bhaskar Mohan
    This document describes the synthesis of gold nanoparticle-conjugated AFP antibody probes and CdTe quantum dot-coated silica nanoparticle probes functionalized with AFP aptamers for the detection of AFP. Key steps include: 1) Conjugating AFP antibodies to gold nanoparticles using PEG linkers. 2) Coating silica nanoparticles with CdTe quantum dots, then functionalizing with AFP aptamers using EDC/NHS chemistry. 3) Measuring fluorescence upon binding of AFP to the aptamer-functionalized probes, and addition of the gold nanoparticle-conjugated antibody probes. 4) Preparing diluted human serum samples for measurement.

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    2.2. Synthesis of Afp Antibody Modified Aunps

    Uploaded by

    Bhaskar Mohan
    45 views2 pages
    This document describes the synthesis of gold nanoparticle-conjugated AFP antibody probes and CdTe quantum dot-coated silica nanoparticle probes functionalized with AFP aptamers for the detection of AFP. Key steps include: 1) Conjugating AFP antibodies to gold nanoparticles using PEG linkers. 2) Coating silica nanoparticles with CdTe quantum dots, then functionalizing with AFP aptamers using EDC/NHS chemistry. 3) Measuring fluorescence upon binding of AFP to the aptamer-functionalized probes, and addition of the gold nanoparticle-conjugated antibody probes. 4) Preparing diluted human serum samples for measurement.

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    This document describes the synthesis of gold nanoparticle-conjugated AFP antibody probes and CdTe quantum dot-coated silica nanoparticle probes functionalized with AFP aptamers for the detection of AFP. Key steps include: 1) Conjugating AFP antibodies to gold nanoparticles using PEG linkers. 2) Coating silica nanoparticles with CdTe quantum dots, then functionalizing with AFP aptamers using EDC/NHS chemistry. 3) Measuring fluorescence upon binding of AFP to the aptamer-functionalized probes, and addition of the gold nanoparticle-conjugated antibody probes. 4) Preparing diluted human serum samples for measurement.

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    © All Rights Reserved
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    Download as docx, pdf, or txt
    45 views2 pages

    2.2. Synthesis of Afp Antibody Modified Aunps

    Uploaded by

    Bhaskar Mohan
    This document describes the synthesis of gold nanoparticle-conjugated AFP antibody probes and CdTe quantum dot-coated silica nanoparticle probes functionalized with AFP aptamers for the detection of AFP. Key steps include: 1) Conjugating AFP antibodies to gold nanoparticles using PEG linkers. 2) Coating silica nanoparticles with CdTe quantum dots, then functionalizing with AFP aptamers using EDC/NHS chemistry. 3) Measuring fluorescence upon binding of AFP to the aptamer-functionalized probes, and addition of the gold nanoparticle-conjugated antibody probes. 4) Preparing diluted human serum samples for measurement.

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    © All Rights Reserved
    Download as DOCX, PDF, TXT or read online from Scribd
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    of 2

    2.2.

    Synthesis of AFP antibody modified AuNPs


    i. Preparation of 13nm Gold Nanoparticles – As per Frens-Turkevich method (by
    reduction of HAuCl4 with citrate (C6H8O7) in aqueous solution
    ii. AFP antibody conjugated to AuNPs surfaces – Use NHS-PEG-S-S-PEG-NHS i.e.
    bifunction PEG linkers
    iii. At a molar ratio of 1:1, AFP antibody was incubated with bifunction PEG
    linkers in sterilized HEPES buffer at 4 °C in the night.
    iv. Mix it with AuNPs (6 nM) at a molar ratio of 300:1 in 1.8mM K2CO3 for 4 h.
    v. With a molar ratio of 10000:1, mPEG-SH introduced to the mixture solution
    to AuNPs to further stabilize it.
    vi. The antibody modified AuNPs got purified by centrifugation
    vii. Then it is resuspended in 0.01M PBS and stored at 4 °C.

    2.3 Preparation of AFP aptamer functional QDs probes

     Preparation of monodispersed SiO2 nanoparticles as per seed-growth methods


    i. Mix 80 mL of ethanol, 4.85 mL of H2O, and 3.6 mL of ammonia - heat
    gradually to 55 °C with stirring
    ii. Add mixture of 3.1 mL of tetraethoxysilane (TEOS) and 8 mL of ethanol and
    incubate at 55 °C for 5 h to get seeds
    iii. Firstly mix 10ml of seeds with 80 mL of ethanol, 13 mL of H2O, and 7.5 mL of
    ammonia then 1 mL of TEOS by drop and stir for 5 hrs. Repeat this until the
    addition of 4 mL TEOS.
    iv. Wash SiO2 nanoparticles with ethanol 4 times and dry it under vaccum.
    v. Diameter of SiO2 nanoparticles = 145 ± 10 nm …(by TEM)

     Synthesis of CdTe QDs


    i. Add MPA into 10mM CdCl2 solution and make final 15 mM concentration
    and then add NaOH to it to adjust pH to 11.9 …Solution a
    ii. Transfer the mixture to the flask to remove O2 and protect it with N2
    iii. Mix 0.03 g Te powder and 0.03 g NaBH4 in 4 mL of H2O and react it to
    colourless (with N2 protection) …Solution b
    iv. Add 2ml of solution b to solution a and heat the mixture to 115 °C (under N2
    protection)
    v. We will get a series of CdTe QDs with different colors with change in reaction
    time then purify QDs by ultrafiltration

     Preparation of CdTe QD-coated SiO2 nanoparticles


    i. Disperse 20 mg SiO2 nanoparticles in 2 mL ethanol and incubate with 0.4 mL
    APTS for 6 h
    ii. Wash amino-functionalized SiO2 nanoparticles with ethanol 4 times and re-
    disperse it in in a mixture of 2 mL of CdTe QDs and 1 mL EDC and stir for 12
    hrs at 4 °C.
    iii. Finally, wash CdTe QD-coated SiO2 nanoparticles 3 times with water and re-
    disperse in 1 mL H2O as QD-SNPs

     AFP aptamer functional probes


    i. Mix 1 mL of QD-SNPs with 1 mL of AFP aptamer, then add 100 μL of freshly
    prepared EDC and 100 μL of NHS to it and incubate at room temp for 2 hrs
    ii. Finally, wash the aptamer functional nanoparticles with PBS 3 times by
    centrifugation to remove free aptamer. To block the excess amino group and
    nonspecific binding sites, resuspend in 5 mL of 1% BSA solution for 2 h

    2.4 Fluorescence measurements


    i. Under excitation of 485 nm Hitachi F-4600 (set at 10 nm) and an emission
    range from 500 to 650 nm, we get the fluorescence spectra at RT
    ii. to let AFP bind to its aptamer, firstly Incubate apt-QD-SNPs with various
    concentrations of AFP at 37 °C for 40 min
    iii. After That, Centrifuge and wash the resultant complexes with PBS and then
    add 50 μL of Ab-AuNPs to it. Incubate it at 37 °C for 40 min.
    iv. Dilute the mixture with PBS to a final volume of 100 μL for fluorescence
    measurements

    2.5. Preparation of real samples


    i. Filter the human serum samples from healthy adults through a 0.45 μm
    membrane
    ii. Centrifuge it at 12,000 rpm for 20 min and dilute supernatants 10 t using 0.01M
    PBS before measurement.

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