Hi G M Shazzad Hossain Prince . You could use mercaptosuccinic acid, thioglycolic acid or cystamine. The thiol group binds strongly to Ag.

Lipoic Acid, (also known as thioctic acid) is also a good choice [1].

You may want to use Lipoic Acid-PEG-Carboxy[2], instead as the hydrohilic PEG space makes it more water solubile, may provide some protection from the Ag surface and has anti-fouling, anti-protein binding properties.

The use of mixed sized PEGs may also be advised to improve particle colloidal stability [3].

To skip the EDC chemistry steps you could use LA-PEG-NHS [4, 5]: Attach this to the antibody and then add this to your Ag particles together with short chain LA-PEGs as in [5]. The short chain LA-PEG will "block" the surface, preventing NSB.

[1] https://pdfs.semanticscholar.org/ca33/ada38016ec58539be46ccb864ebb8d316996.pdf

[2] http://www.nanocs.net/PEG/Surface-reactive-PEG/Lipoic-acid-PEG-COOH/Lipoic-acid-PEG-COOH-5k.htm

[3] A Solution to the PEG Dilemma: Efficient Bioconjugation of Large Gold Nanoparticles for Biodiagnostic Applications using Mixed Layers, Langmuir2012284415634-15642

[4] http://www.nanocs.net/PEG/Surface-reactive-PEG/Lipoic-acid-PEG-NHS/Lipoic-acid-PEG-NHS-5k.htm

[5] Doxorubicin and Anti-PD-L1 Antibody Conjugated Gold Nanoparticles for Colorectal Cancer Photochemotherapy, Mol. Pharmaceutics2019163

Supplimentary Info may have useful details:

https://pubs.acs.org/doi/suppl/10.1021/acs.molpharmaceut.8b01157/suppl_file/mp8b01157_si_001.pdf

Dr. John Clarkson Thank you very much. Your references is of much help to my work. We used lauric acid to modify the surface of our Ag NPs and we are intending to use EDC/ NHS chemistry for conjugation to the antibody.

Dr. Steingrimur Stefansson Thank you as well for your suggestion. We used lauric acid since we had this chemical in our lab.

I am wondering to know if you can give me any hints on how to quantify/ measure the conjugated antibody-NP. I came to know about microBCA assay. I can also use florescent secondary antibody and do microscopy. But, I am wondering to know any validated methods to confirm my conjugation is successful.

Thank you again to both of you.

Regards,

Prince

Quantify and measure antibody on nanoparticles: see answers in https://www.researchgate.net/post/How_do_I_calculate_number_of_antibody_molecules_bound_to_gold_nanoparticles

the ref:

Article Quantifying Bound and Active Antibodies Conjugated to Gold N...

is FREE to download from the ACS web site https://pubs.acs.org/doi/10.1021/acsomega._8b00591

Remove the _ as this web site will not let me cite the original without auto pasting its own ref , which really should have the free download link...anyways....also see

Article A fluorescence-based method to directly quantify antibody im...

and associated thesis: https://ir.library.illinoisstate.edu/etd/668/

Jeremy Driskell pub's may be of general interest : https://www.researchgate.net/profile/Jeremy_Driskell/research

I'd also consider an anti-FITC antibody as a Model alternative to the anti-HRP used above.

If you want to "simply" optimize the bound antibody on your NP, use methods employed in lateral flow or dot assays: e.g. use a biotin labelled antigen e.g. FITC-Biotin and capture NP-antibody-Antigen-biotin complex on a poly-streptavidin test line / dot: this reporting system will let you compare different antibody loadings on your NPs & different attachment chemistries.

Different types of antibody may have slightly different loadings on your NP, however, I would not expect a huge range unless the pI of your antibody is very unusual, however there are ways around this: see Driskell pub for more :

Article Chemical Modification of Antibody Enables the Formation of S...

I'm afraid lauric acid will be a problem since it will probably not bind strongly to the Ag NPs.