This is a simple protocol for the synthesis of functionalizable, near-infrared emitting photoluminescent gold nanoclusters and their detection using a commercial setup. One of the major advantages of this technique is that the attachment of thiol-functionalized ligand and the coupling of amine-functionalized ligand on the surface of gold nanoclusters does not adversely affect the photoluminescent properties and coil stability. It can be envisaged that the combination of intense photoluminescence properties and conjugation with biomolecules will allow for the in vitro detection of low-concentration anilides, biosensing, solubling, and bio-imaging.
Demonstrating the procedure will be Klaudia Kvakova, my PhD student. The flow cytometry will be demonstrated by research assistant Alzbeta Magdolenova. The microscopic part will be demonstrated by Vaclav Bocan, a grad student from Lenka Libusova Laboratory.
Start by adding 7.8 milligrams of thioctic acid in 60 microliters of two-molar sodium hydroxide to 23.4 milliliters of ultrapure water and stirring the mixture until completely dissolved. Next add 10.2 microliters of hydrogen tetrachloraurate to the solution and, after 15 minutes, add 480 microliters of freshly prepared sodium borohydride while stirring vigorously. Continue stirring the reaction mixture overnight.
On the next day, purify the solution using three cycles of ultrafiltration with a molecular weight cut-off of three kilodaltons, then add 15.6 milligrams of thiol-terminated polyethylene glycol to the solution, adjust the pH to between 7 and 7.5, and stir the mixture overnight to obtain nanocluster 1. On the following day, repeat the three cycles of centrifugation and filtration to purify the dispersion. Mix the nanocluster 1 solution with TPP, then adjust the pH to 4.5 with one-molar hydrogen chloride.
Start the reaction by adding an excess of EDC HCL and monitor the pH of the mixture for the first hour. If the pH increases above six, reduce it by adding hydrogen chloride. Stir the reaction mixture overnight at room temperature.
On the next day, perform three cycles of centrifugation and filtration as previously described to obtain nanocluster 2. Dilute 2 with ultrapure water to a volume of 24 milliliters. Seed the cells in a 12-well plate at a density of 20, 000 cells per well.
Incubate them for 48 hours, then aspirate the medium and add 400 microliters of complete culture medium with or without 500 micrograms of nanoparticles per well. Return the cells to the 37-degrees Celsius incubator for nanocluster internalization. After two hours, detach the cells with standard trypsinization.
Collect them in polypropylene microcentrifuge tubes and centrifuge for five minutes at 350 times g and four degrees Celsius. Prepare FCM buffer according to manuscript directions and use one milliliter of the buffer to wash the cell pellets. Centrifuge the cells for another five minutes, then resuspend them in 500 microliters of the FCM buffer and store them at four degrees Celsius.
Prior to acquisition of the sample, make sure that the instrument has the corresponding optical configuration. Place the 780 over 60 bandpass filter in front of the 405A detector. Filter all samples using a five-milliliter polystyrene round-bottomed tube with a cell strainer cap.
Specify the cytometer configuration. Plot a two-parameter dot plot of the FSC-A and SSC-A to show distribution of cells. To exclude doublets, create a two-parameter dot plot of FSC-H versus FSC-A and plot a single-parameter histogram for the fluorescent channel area to monitor the relative fluorescence intensity in the sample.
Acquire untreated sample at a low flow rate to minimize coincident events. During acquisition, adjust the PMT voltages to get the untreated population on scale on the FSC versus SSC plot. If necessary, adjust the PMT voltages for the FL channel to place the unstained population on the left corner of the histogram.
Then select the specific Gate tab in the software and draw an appropriate gate around the desired population. The cells inside the gate will move to the next checkpoint. Set up the experiment and record the data.
At 24 hours post seeding, add 100 micrograms of 2 to each dish chamber containing 0.5 milliliters of medium with HeLa cells. Return the dish to the incubator and allow the cells to internalize the nanoclusters, then discard the medium and wash the cells with prewarmed, fresh medium. Fill each chamber with 800 microliters of fresh medium and proceed with imaging.
To image the cells, use a confocal microscope with Plan-APOCHROMAT and a 63x oil-objective lens. Mount the dish on the inverted stage with the chamber warmed up to 37 degrees Celsius and supplied with a humidified, 5%carbon dioxide atmosphere. Detect internalized gold nanoclusters by using a 405-nanometer laser set to 2%power with an appropriate beam splitter, setting the range of detection wavelengths between 650 and 760 nanometers.
Set the resolution of the image to 2048 by 2048 pixels. In the acquisition speed setting, aim for a pixel dwell time around four microseconds and acquire the image with two times averaging, then set the pinhole to one area unit and, for higher sensitivity, use the photon counting mode. For correct illumination and transmitted light with DIC use Kohler's setting of the condenser and field stop.
To acquire transmitted light, use a 488-nanometer laser at 0.7%power without any fluorescence detector assigned, making sure to set an appropriate beam splitter for the laser wavelength. Absorption spectra indicated that gold nanoclusters 1 and 2 do not have a characteristic surface plasmon band and show broad emission from 550 to 850 nanometers. The photoluminescence strongly increased after attachment of TPP to the surface of 1.
Emission from the nanoclusters was also visible under a 365-nanometer UV light. The emission was stable and its wavelength was independent of the excitation wavelength but the intensity was maximal when excited with UV light. Flow cytometry was used to confirm nanocluster 2 uptake by HeLa cells.
Near fluorescence was dependent on both incubation time and concentration of nanocluster 2. The gold nanoclusters within cells were imaged noninvasively with a standard confocal laser scanning microscope. The cells were stained with nanocluster 2 and, after 24 hours of incubation, bright red photoluminescence was observed inside the cells.
When attempting this protocol, it is important to remember that the removal of unreacted and hydrolyzed sodium borohydride is very important, otherwise thiolated polyethylene glycol will not bind to the surface of gold nanocluster. Additionally, the photoluminescence of gold nanoclusters starts from 560 nanometers. To avoid the background autofluorescent in confocal microscopy experiments, the photons should be collected above 650 nanometers.
