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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.