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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 applications1,2,3,4,5,6,7,8,9,10. Their many desirable characteristics include high photostability, tunable emission maxima, long emission lifetimes, large Stokes shifts, low toxicity, good biocompatibility, renal clearance and facile bioconjugation. PL AuNCs can provide photoluminescence from the blue to the near-infrared (NIR) spectral region, depending on the number of atoms within the cluster11 and the nature of the surface ligand12. NIR (650-900 nm) emitting AuNCs are particularly promising for long-term in vitro and in vivo imaging of cells and tissues, as they offer high signal-to-noise ratio due to minimum overlap with intrinsic autofluorescence, weaker scattering and absorption, and high tissue penetration of NIR light13,14.

In recent years, various approaches that take advantage of Au-S covalent interactions have been developed to prepare NIR-PL AuNCs capped with a variety of thiol-containing ligands13,15,16,17. For biomedical applications, AuNCs must be functionalized with a biological component to facilitate binding interactions. Thus, AuNCs with high colloidal stability that are easily functionalizable in aqueous solvent are highly desirable. The overall goal of the current protocol is to describe a previously reported18 preparation of AuNCs with a functionalizable carboxylic acid group on the surface by employing thioctic acid and polyethylene glycol (PEG) in an aqueous environment in detail and their conjugation with molecules bearing a primary amine following the acid-amine coupling method. Because of the ease of synthesis and high reproducibility, this protocol can be used and adapted by researchers from non-chemistry backgrounds.

One of the key requisites for applications of AuNCs in biomedical research is the ability to observe and measure AuNCs inside cells. Among the methods available to monitor nanoparticle uptake by cells, flow cytometry (FCM) and confocal laser scanning microscopy (CLSM) offer robust, high-throughput methods which allow fast measurements of internalization of fluorescent nanomaterials in large number of cells19. Here, FCM and CLSM method for direct measurement and analysis of PL AuNCs inside cells, without the need for additional dyes, have also been presented.

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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 the same conditions overnight.
    NOTE: Freshly prepare the NaBH4 solution in ice-cold ultrapure water and add to the reaction mixture immediately after preparation.
    NOTE: The synthesis of AuNCs is easily scalable. Up to 2 L of AuNCs was synthesized in a single batch, without any change in the optical properties of the particles.
  4. (Critical) The next day, purify the solution by applying three cycles of centrifugation/filtration using a membrane filtration device with a molecular weight cut-off of 3 kDa. Without this purification procedure, the following step does not work properly.
  5. Add thiol-terminated polyethylene glycol (MW 2,000; 15.6 mg; 7.8 μmol) to the solution, adjust the pH to 7-7.5 and stir the mixture overnight to obtain 1. Purify the dispersion by applying three cycles of centrifugation/filtration using a membrane filtration device with a molecular weight cut-off of 3 kDa.
    NOTE: Adjustment of the pH to 7-7.5 is extremely important. Higher pH can result in a blue shift of emission maxima.

2. Conjugation of 3-(aminopropyl)triphenylphosphonium bromide (TPP) on the surface of 1

  1. Mix the 1 solution (24 mL) prepared in the previous step and 3-(aminopropyl)triphenylphosphonium bromide (12 mg, ~30 μmol). Adjust the pH to 4.5 with 1 M HCl.
    NOTE: 3-(Aminopropyl)triphenylphosphonium (TPP) bromide salt was prepared as described in the literature20.
  2. Start the reaction by adding an excess of N-(3-dimethyl-aminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC·HCl) (60 mg, 312 μmol). The pH of the solution will increase and should not be allowed to go beyond 6. Monitor the pH of the reaction mixture for the first hour. If the pH increases above 6, reduce it to 4.5–6 by adding 1 M HCl.
  3. Stir the reaction mixture overnight at room temperature.
  4. Purify the dispersion by applying three cycles of centrifugation/filtration using a membrane filtration device with a molecular weight cut-off of 3 kDa to obtain 2. Dilute 2 obtained here with ultrapure water to the initial volume of 24 mL. The concentration of Au in the solution is 200 μg/mL.

3. Cell culture

  1. Culture HeLa cells (HPA culture collection) in Dulbecco’s modified Eagle’s Medium supplemented with 10% fetal bovine serum in 5% CO2 at 37 °C.
  2. Split and passage the cells when they reach ~80% confluence. To minimize acquisition of new mutants, the number of cell propagations should not exceed 30.

4. AuNC internalization into HeLa cells

  1. Seed the cells in a 12-well plate at a density of 20,000 cells/mL (1 mL/well). The goal is to achieve ~50% confluence after 48 h.
  2. At 48 h post-seeding, aspirate the culture medium and add 400 µL of complete culture medium (for untreated controls) or 500 µg of nanoparticles in 400 µL of complete cultured medium (for treated samples) to each well. Return the cultures to a 37 °C incubator.
    NOTE: Addition of high volumes of AuNC solution adversely affects the cell viability. AuNC solutions need to be concentrated. Thus 2 obtained in step 2.4 is concentrated 100 times. 40 mL AuNC was concentrated to 400 μL. A 25 μL aliquot of this concentrated solution was added to 400 µL cell culture media to obtain the desired AuNC concentration.
  3. After 2 h of internalization, detach the cells by standard trypsinization according to the manufacturer’s protocol.
  4. Collect the samples in polypropylene microcentrifuge tubes and centrifuge for 5 min at 350 x g at 4 °C.
  5. Prepare the following FCM buffer: pre-chilled phosphate buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.47 mM NaH2PO4, pH 7.4) supplemented with 2% bovine serum albumin at 4 °C.
  6. Wash the pellets with 1 mL of FCM buffer and centrifuge for 5 min at 350 x g at 4 °C.
  7. Resuspend the pellet in 500 µL of FCM buffer and store samples at 4 °C prior to analysis.

5. Flow cytometry analysis

  1. Filter all samples using a 5 mL polystyrene round-bottom tube with a cell-strainer cap.
  2. Before acquiring data using the instrument software, specify the cytometer configuration.
  3. Format all dot-plots and histograms for ‘Acquisitions’.
  4. Plot a two-parameter dot plot of the forward scatter area (FSC-A) and side scatter area (SSC-A) to show distribution of cells. To exclude doublets, create a two-parameter dot plot of FSC height (FSC-H) vs. FSC-A. To monitor the relative fluorescence intensity in the sample, plot a single-parameter histogram for the fluorescent channel area (FL-A). Use a linear scale to depict FSC and SSC data, and a logarithmic scale for all fluorescent parameters.
  5. Acquire untreated sample (without nanoparticles) at a low flow rate to minimize coincident events (if allowed by the instrument). During acquisition, adjust the photomultiplier tube (PMT) voltages to get the untreated population on scale on the FSC vs SSC plot. If necessary, adjust PMT voltages for the FL channel to place the unstained population on the left corner of the histogram.
  6. Select the specific ‘gate tab’ in the software and draw an appropriate gate around the desired population. Cells inside the gate will move to the next checkpoint.
  7. Record 10,000 events per sample.
  8. Record all samples under the same instrument settings.
  9. Use an appropriate program to analyze the flow cytometry data.
    NOTE: Some applications might require customized setup of filters. For filter exchange always follow the manufacture’s recommendations in the user guide.
    NOTE: The experiment can be saved and reloaded to preserve the instrument settings and gating strategy.
    NOTE: A side scatter height (SSC-H) vs. side scatter area (SSC-A) plot can be also used for doublet exclusion. This type of gating can be more sensitive as the FSC detector is not usually a PMT.

6. Internalization of 2 into HeLa cells for confocal laser scanning microscopy (CLSM)

  1. Seed the cells onto a 4-chamber glass bottom 35 mm dish at a density of 250,000 cells/mL (0.5 mL/chamber). Keep the chamber in a 37 °C incubator with 5% CO2 atmosphere. The goal is to achieve ~50% confluence after 24 h.
  2. At 24 h post-seeding, add 100 µg of 2 (or 10 µL from the stock solution of 10 mg/mL) to each dish chamber containing 0.5 mL of medium with the cells (for treated samples).
  3. Return the dish to the incubator. Let the cells internalize the AuNCs for 24 h prior to using them for CLSM.
  4. After the internalization period, discard the medium and wash the cells with pre-warmed fresh medium for 5 min. Repeat the washing step once more. Then fill each chamber with 800 µL of fresh medium.

7. CLSM Imaging of live Hela cells labeled with 2

  1. For microscopic imaging, use 63x oil (n = 1.518) objective lens (NA = 1.4) in a confocal microscope with Plan-Apochromat.
  2. Mount the dish on the microscope inverted stage with the chamber warmed to 37 °C and supplied with humidified 5% CO2 atmosphere.
  3. To detect internalized AuNC, use a 405 nm laser set at 2% power with an appropriate beam splitter. Set the range of detection wavelengths between 650 and 760 nm.
  4. Set the resolution of the image to 2048 x 2048 pixels. In the acquisition speed setting, aim for a pixel dwell time around 4 µs. Acquire the image with 2x averaging (line mode, averaging method mean). Set the pinhole to 1 Airy unit (for 405 nm light). For higher sensitivity, use photon-counting mode.
  5. For correct illumination in transmitted light with differential interference contrast (DIC), use Köhler’s setting of the condenser and the field stop. For acquisition of transmitted light, use a 488 nm laser at 0.7% power without any fluorescence detector assigned. Set an appropriate beam splitter for the laser wavelength.
  6. Acquire two images for each track (red fluorescence and DIC). Track AuNCs by their red fluorescence; cell boundaries are easily determined in the transmitted light with DIC pictures.

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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...

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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...

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Disclosures

Some portions of the methods and results were previously presented in the article by Pramanik et al.18 Here, these methods have been converted into practical point-by-point protocols. The authors declare no competing financial interests.

Acknowledgements

The authors are grateful to Alzbeta Magdolenova for her help with flow cytometry. The authors acknowledge the financial support from GACR project Nr. 18-12533S. Microscopy was performed in the Laboratory of Confocal and Fluorescence Microscopy co-financed by the European Regional Development Fund and the state budget of the Czech Republic, projects no. CZ.1.05/4.1.00/16.0347 and CZ.2.16/3.1.00/21515, and supported by the Czech-BioImaging large RI project LM2015062.

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Materials

NameCompanyCatalog NumberComments
1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochlorideTCI ChemicalsD1601https://www.tcichemicals.com/eshop/en/eu/commodity/D1601/;jsessionid=3AD046E5389206AAE33C8AAB5036CDD6?gclid=CjwKCAjwiZnnBRBQEiwAcWKfYrO69K6Np3tYeSsAouqGndUvzzsy1hStBPuHG-X3cpTIsAqq9z0cDBoC76MQAvD_BwE
Bovine serum albuminSigma-AldrichA4161https://www.sigmaaldrich.com/catalog/product/sigma/a4161?lang=en&region=CZ
Disodium hydrogen phosphate dihydratePENTA s.r.o.15130-31000https://www.pentachemicals.eu/soubory/specifikace/specifikace_281.pdf
DL-Thioctic acid, 98%Alfa AesarL04711https://www.alfa.com/en/catalog/L04711/
Hydrochloric acid 35%PENTA s.r.o.19350-11000https://www.pentachemicals.eu/soubory/specifikace/specifikace_512.pdf
Hydrogen tetrachloroaurate(III) trihydrate, ACS, 99.99% (metals basis), Au 49.0% minAlfa Aesar36400https://www.alfa.com/en/catalog/036400/
O-(2-Mercaptoethyl)-O′-methylpolyethylene glycol 2000Sigma-Aldrich743127https://www.sigmaaldrich.com/catalog/product/aldrich/743127?lang=en&region=CZ
Potassium chloridePENTA s.r.o.16200-31000https://www.pentachemicals.eu/soubory/specifikace/specifikace_346.pdf
Sodium borohydrideSigma-Aldrich452882https://www.sigmaaldrich.com/catalog/product/aldrich/452882?lang=en&region=CZ&gclid=CjwKCAjwiZnnBRBQEiwAcWKfYuoZKvdK_fH24F1gGugG4pamF2FFZLd36YyZmRTdGgkbm5SbyGP0jBoCoo0QAvD_BwE
Sodium chloridePENTA s.r.o.16610-31000https://www.pentachemicals.eu/soubory/specifikace/specifikace_376.pdf
Sodium dihydrogenphosphate dihydratePENTA s.r.o.12330-31000https://www.pentachemicals.eu/soubory/specifikace/specifikace_124.pdf
Sodium hydroxide pelletsPENTA s.r.o.15740-31000https://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 ScientificX12223https://www.thermofisher.com/order/catalog/product/X12223#/X12223

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