Synthesis of Near-Infrared Emitting Gold Nanoclusters for Biological Applications

Summary

A reliable and easily reproducible method for preparation of functionalizable, near-infrared emitting photoluminescent gold nanoclusters and their direct detection inside HeLa cells by flow cytometry and confocal laser scanning microscopy is described.

Abstract

Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been devoted to their development. In this protocol, a recently developed, facile method for preparation of water soluble, biocompatible, and colloidally stable near-infrared emitting AuNCs have been described in detail. This room-temperature, bottom-up chemical synthesis provides easily functionalizable AuNCs capped with thioctic acid and thiol-modified polyethylene glycol in aqueous solution. The synthetic approach requires neither organic solvents or additional ligand exchange nor extensive knowledge of synthetic chemistry to reproduce. The resulting AuNCs offer free surface carboxylic acids, which can be functionalized with various biological molecules bearing a free amine group without adversely affecting the photoluminescent properties of the AuNCs. A quick, reliable procedure for flow cytometric quantification and confocal microscopic imaging of AuNC uptake by HeLa cells also been described. Due to the large Stokes shift, proper setting of filters in flow cytometry and confocal microscopy is necessary for efficient detection of near-infrared photoluminescence of AuNCs.

Introduction

In the past decade, ultrasmall (≤ 2 nm) photoluminescent gold nanoclusters (PL AuNCs) have emerged as promising probes for both fundamental research and practical applications^{1,2,3,4,5,6,7,8,9,10}. Their many desirable characteristics include high photostability, tunable emission maxima, long emission lifetimes, large Stokes shift....

Protocol

1. Preparation of near-infrared emitting AuNCs (1)

1. Add 7.8 mg (37.8 μmol) thioctic acid (TA) and 60 μL of 2 M NaOH to 23.4 mL of ultrapure water (resistivity 18.2 MΩ.cm at 25 °C) and stir (at least 1,000 rpm) until it dissolves completely (~15-20 min). For faster dissolution of TA, sonicate the mixture. For the synthesis, freshly prepared TA solution is recommended.
2. Add 10.2 μL of HAuCl₄·3H₂O (470 mg/mL) of aqueous solution to the solution.
3. After 15 min, add 480 μL of NaBH₄ (1.9 mg/mL) under vigorous stirring (at least 1,000 rpm) and stir the reaction mixture under....

Results

NIR PL AuNCs were prepared from Au³⁺ in the presence of TA, and then thiol-terminated PEG (MW 2,000) was bound on the AuNC surface to obtain 1 following the workflow shown in Figure 1. Amidic coupling between 1 and 3-(aminopropyl) triphenylphosphonium (TPP) bromide provided 2. As expected, absorption spectra (Figure 2a) indicated that AuNCs 1 and 2 do not have a chara.......

Discussion

NIR-emitting AuNCs were synthesized using a bottom-up approach in which the gold precursor solution (HAuCl₄) was treated with suitable thiol ligands, followed by reduction of Au³⁺. Reduction of metal ions in aqueous solution tend to aggregate and results in large nanoparticles rather than ultrasmall NCs²¹. To prepare ultrasmall (≤2 nm) PL AuNCs, the synthetic conditions were adjusted to prevent formation of large particles and promote formation of ultrasmall clusters. T.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride	TCI Chemicals	D1601	https://www.tcichemicals.com/eshop/en/eu/commodity/D1601/;jsessionid=3AD046E5389206AAE33C8AAB5036CDD6?gclid=CjwKCAjwiZnnBRBQEiwAcWKfYrO69K6Np3tYeSsAouqGndUvzzsy1hStBPuHG-X3cpTIsAqq9z0cDBoC76MQAvD_BwE
Bovine serum albumin	Sigma-Aldrich	A4161	https://www.sigmaaldrich.com/catalog/product/sigma/a4161?lang=en®ion=CZ
Disodium hydrogen phosphate dihydrate	PENTA s.r.o.	15130-31000	https://www.pentachemicals.eu/soubory/specifikace/specifikace_281.pdf
DL-Thioctic acid, 98%	Alfa Aesar	L04711	https://www.alfa.com/en/catalog/L04711/
Hydrochloric acid 35%	PENTA s.r.o.	19350-11000	https://www.pentachemicals.eu/soubory/specifikace/specifikace_512.pdf
Hydrogen tetrachloroaurate(III) trihydrate, ACS, 99.99% (metals basis), Au 49.0% min	Alfa Aesar	36400	https://www.alfa.com/en/catalog/036400/
O-(2-Mercaptoethyl)-O′-methylpolyethylene glycol 2000	Sigma-Aldrich	743127	https://www.sigmaaldrich.com/catalog/product/aldrich/743127?lang=en®ion=CZ
Potassium chloride	PENTA s.r.o.	16200-31000	https://www.pentachemicals.eu/soubory/specifikace/specifikace_346.pdf
Sodium borohydride	Sigma-Aldrich	452882	https://www.sigmaaldrich.com/catalog/product/aldrich/452882?lang=en®ion=CZ&gclid=CjwKCAjwiZnnBRBQEiwAcWKfYuoZKvdK_fH24F1gGugG4pamF2FFZLd36YyZmRTdGgkbm5SbyGP0jBoCoo0QAvD_BwE
Sodium chloride	PENTA s.r.o.	16610-31000	https://www.pentachemicals.eu/soubory/specifikace/specifikace_376.pdf
Sodium dihydrogenphosphate dihydrate	PENTA s.r.o.	12330-31000	https://www.pentachemicals.eu/soubory/specifikace/specifikace_124.pdf
Sodium hydroxide pellets	PENTA s.r.o.	15740-31000	https://www.pentachemicals.eu/soubory/specifikace/specifikace_307.pdf
XTT (sodium 2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium inner salt)	Thermo Fisher Scientific	X12223	https://www.thermofisher.com/order/catalog/product/X12223#/X12223