Carboxyl groups present on the glutathione-functionalized AuNCs were covalently conjugated with primary amines of streptavidin using EDC/sulfo-NHS chemistry (36 ). In the initial step, 20 mg of AuNC dispersed in 10 mM PBS was washed in a Nanosep centrifugal ultrafiltration device (molecular weight cutoff, 300 kDa; Pall Life Sciences, Ann Arbor, MI, USA). After washing, carboxyl groups present on AuNCs were activated with 10 mM EDC and 20 mM sulfo-NHS in PBS buffer for 30 min. After washing these activated particles with glycine buffer, 50 μl of streptavidin (1 mg/ml) in the carbonate buffer (pH 9.0) solution was added. The subsequent step involved incubation of the mixture for 24 hours at room temperature, followed by washing five times with glycine buffer. The resultant streptavidin-conjugated nanoclusters were diluted to 0.1 mg/ml in PBS and stored at 4°C for immunoassay experiments.
Nanosep centrifugal ultrafiltration device
Manufactured by Pall Corporation
Sourced in United States
The Nanosep centrifugal ultrafiltration device is a laboratory equipment designed for the separation and concentration of macromolecules and particles from solutions. It utilizes centrifugal force and a semi-permeable membrane to selectively retain the desired components while allowing smaller molecules to pass through.
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2 protocols using nanosep centrifugal ultrafiltration device
1
Covalent Conjugation of Streptavidin to Glutathione-AuNCs
2
Covalent Streptavidin Conjugation to Silica Nanoparticles
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In our present study 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)/Sulpho N-hydroxysuccinimide(Sulpho NHS) method was adopted to conjugate carboxyl functionalised silica nanoparticles covalently with primary amines of streptavidin as reported25 (link). In a typical synthesis, 20 mg of nanoparticles were dispersed in 10 mM phosphate (pH = 7.0) buffer and washed in a NanoSep centrifugal ultrafiltration device with a molecular weight cutoff of 300 kDa (Pall Life Sciences, Ann Arbor, MI, USA). Nanoparticles were redispersed in phosphate buffer and the solution was sonicated with a tip sonicator for 30 secs. The carboxyl groups are activated with 10 mmol/L of EDC (Thermo Scientific, USA) and 10 mmol/L sulpho-NHS (Thermo Scientific, USA) in PBS buffer for 30 min. Activated particles were washed once with glycine buffer. 50 μL of 1 mg/mL Streptavidin (Scripps Lab, USA) in the carbonate buffer (pH = 9.0) solution was added. After 3 hours of incubation at room temperature the streptavidin conjugated nanoparticles were washed 5 times with glycine buffer. They were resuspended in DMSO and stored at 4 °C.
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