Synthesis of Fluorescents AuNCs

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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?
 

What are Fluorescent Gold Nanoclusters (AuNCs)?

Fluorescent gold nanoclusters (AuNCs) are ultra-small clusters of gold atoms, typically comprising a few to several dozen atoms. These nanoclusters exhibit unique optical properties, including fluorescence, due to quantum confinement effects. The fluorescence from AuNCs can be tuned by changing the size of the clusters and the type of ligands bound to their surface.

How are Fluorescent AuNCs synthesized?

Fluorescent AuNCs are synthesized through various chemical methods, with the most common being the reduction of gold salts in the presence of a stabilizing ligand. The choice of reducing agents and ligands, such as thiolates, phosphines, or proteins, plays a crucial role in controlling the size, stability, and optical properties of the nanoclusters. Conditions like temperature, pH, and reaction time are also critical for the precise synthesis of AuNCs.

What are the applications of Fluorescent AuNCs?

Due to their biocompatibility and strong fluorescence, AuNCs are widely used in biomedicine and bioimaging. They serve as probes for imaging in cells and tissues, sensors for detecting metal ions and molecules, and as agents for targeted drug delivery and photothermal therapy. Their applications extend to electronics and catalysis, leveraging their unique electronic properties and high surface area.

What are the main challenges in synthesizing Fluorescent AuNCs?

The synthesis of fluorescent AuNCs faces several challenges, including achieving uniform and precise control over their size and fluorescence properties. Reproducibility in synthesis can be difficult due to the sensitivity of the process to experimental conditions. Additionally, ensuring the stability of AuNCs in biological environments without losing their fluorescent properties is also challenging.

How do the properties of AuNCs change with their size and composition?

The optical and electronic properties of AuNCs are highly dependent on their size, composition, and surface chemistry. As the size of the clusters decreases, quantum confinement effects become more pronounced, leading to changes in their fluorescence emission wavelength. The type and arrangement of surface ligands can alter the electronic interactions within the clusters, further influencing their stability, solubility, and optical properties. Adjusting these parameters allows for the fine-tuning of AuNCs for specific applications.

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