Synthesis of Fluorescents AuNCs

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The discussion centers on the synthesis of Fluorescent Gold Nanoclusters (AuNC) as part of a chemistry degree project. The researcher has successfully produced nanostructures under 5 nm but is facing issues with fluorescence emission during characterization. The synthesis involves using synthetic peptides, HAuCl4 as a gold precursor, ascorbic acid as a reducing agent, and NaOH to adjust pH. Despite initial fluorescence observed with a 365 nm lamp, no emission is detected with molecular fluorescence equipment, prompting a request for assistance in identifying potential errors or omissions in the synthesis process. The researcher seeks feedback on the excitation frequency used in the fluorescence tests.
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Hi there. I present the approach of the synthesis of AuNC with the aim that they have the fluorescence property, i am looking for some help to identify failures in my procedure and achieve success in obtaining the product. Thanks!
Hi everyone, as a degree project to obtain a chemistry degree, i am developing the Synthesis of Fluorescent Gold Nanoclusters (AuNC). I have investigated the route to obtain nanoestructures of nanometric size less than five nanometers, reaction conditions, and so far i have managed to obtain a reaction product with no surface plasmon band and in the DLS analysis i have managed to show the size less than 5 nm. However, the fluorescence spectroscopy characterization doesn't give me any fluorescence emission results from my AuNCs. So, i'm going to share with you the synthesis route that i have proposed and other details, so that you please help me identify errors or omission of steps and possible causes of why i don't obtain fluorescent NCs.

As a bingind ligand i am using synthetic peptides, the peptide sequence that i am using for the tests is: Ac-CTKKCGLWSILKGVGKI-NH2
The Au-Pep ratios is 1:3
As a gold precursor i am using HAuCl4
The solvent is water
The reducing agent is Ascorbic Acid
To increase the reaction to alkaline pH i am using NaOH

Synthesis:

The first step is to increase the pH of the peptide to generate the availability of its negative charges, then to a 3mM solution of peptide i add NaOH up to pH 12 and i take it to the ultrasonic bath equipment for 10 minutes. After 10 minutes i add the 1mM gold solution and a little amount of ascorbic acid 50mM, then 10 minutes in the ultrasonic bath equipment and finally i pass it to the agitation equipment for 24h at 37ºC.

After this procedure, i run a test with a 365 nm lamp and the product fluoresces, but when i take it to the molecular fluorescence equipment i get no emission result and i don't realize what i'm omitting in order no to get a product that fluoresces. :c

Plese help me!

Thank you so much!

Note: The reducing agent that i proposed is with the aim of not using a strong, toxic and polluting agent. The peptide is provide by the university and i cannot change it. Please, let me know if you need more details to understand what's going on with this procedure.
 
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What excitation frequency are you using for the molecular fluorescence equipment?
 
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