I In situ biosynthesized gold nanoclusters for self-imaging of cancer cells and in vivo self-bio-marking of tumors
| Preparation of in situ biosynthesized gold nanoclusters ● TIMING 75 h
1 | Culture HepG2 cells in 7 × 9 culture flasks in 15mL medium at 37 °C, 5% CO2. Prepare the cells of a whole culture flask to be tested.
2 | Incubate HepG2 cells of a whole culture flask with 10 μmol/L HAuCl4 solution for 24 or 48 h at 37 °C, 5% CO2.
3 | Trypsinize the incubated HepG2 cells and harvest the cells in eppendorf tubes.
4 | Disperse the cells in PBS, centrifuge the mixture at 1000 rpm for 5 min and collect the cells.
5 | Repeat this procedure three times.
6 | Re-disperse the collected cells in 2 mL Milli-Q water and use the cells for extracting the gold nanoclusters.
7 | Extract the gold nanoclusters from incubated HepG2 cells by a frequently repetitive freeze-thaw method from -20 °C to 37 °C for ~5 h.
▲CRITICAL STEP Avoid any biochemical reagent to decompose the structure of gold nanoclusters during this procedure.
8 | Centrifuge the mixture at 1500 rpm for 3 min. Discard the precipitated cell debris and collect the supernatant with gold nanoclusters.
PAUSE POINT The above supernatant is stable at 4 ˚C up to about 2 weeks.
| UV/Vis absorbance spectra measurement for gold nanoclusters ● TIMING 0.5 h
9 | Dilute the above supernatant 5 times.
10 | Add 2 mL of the above diluted mixture to a cuvette.
11 | Place the cuvette at the sample cell position.
12 | Record UV/Vis absorbance spectra (700-200 nm) for the obtained gold nanoclusters with scan speed 120 nm/min.
| Fluorescence spectra measurement for gold nanoclusters ● TIMING 0.5 h
13 | Dilute the above supernatant 5 times.
14 | Add 2 mL of the above diluted mixture to a cuvette.
15 | Place the cuvette at the sample cell position.
16 |Record fluorescence spectra (500-700 nm) for the obtained gold nanoclusters with a scan speed of 60 nm/min at excitation wavelength 470 nm (Excitation Slit 3 nm; Emission Slit 3 nm).
| Transmission electron microscope (TEM) of gold nanoclusters ● TIMING 16 h
17 |Further sonicate the above supernatant for ~3 h.
▲CRITICAL STEP Keep gold nanoclusters in good monodispersion.
18 | Disperse 5 μL the above supernatant with decuple dilution on copper grids, and dry overnight.
19 | Acquire the TEM image of gold nanoclusters.
| X-ray photoelectron spectra ● TIMING 15 h
20 | Freeze-dry the above supernatant overnight and collect the pale yellow powder.
PAUSE POINT The above collected powder is stable at 4 ˚C up to about 2 months.
21 | Record the X-ray photoelectron spectra of the gold nanoclusters from the mixture.
22 | Set the typical operating pressure at around 5 × 10 -10 Torr in the analysis chamber.
23 | Take survey spectra (0–1000 eV) at a constant analyzer pass energy of 160 eV.
24 | Acquire all high-resolution spectra for Au4f, with a pass energy of 60 eV, a step of 0.1 eV and a dwell time of 1000 ms.
25 | Fit the XPS peaks by using the publicly available software XPSPEAK v. 4.1. Use the Shirley function as a background and fit the individual peaks by a Gaussian-Lorentzian cross-product.
| SEM images of the cells interspersed by in situ biosynthesized gold nanoclusters ● TIMING 80 h
26 | Seed HepG2 cells on ITO glass substrate in a cell culture dish containing DMEM with 10% (vol/vol) FBS and 1% (vol/vol) penicillin-streptomycin, respectively. Incubate the cells at 37 °C in a humidified 5% CO2 atmosphere overnight.
27 | Add 2 μL of HAuCl4 stock solution (10 mmol/L) to a final concentration of 10 μmol/L. Shake the dish gently to ensure good mixing.
28 | Incubate the dish at 37 °C in a humidified 5% CO2 atmosphere for 24 or 48 h depending on the cell proliferation state.
29 | Rinse the incubated cells three times with PBS.
30 | Dehydrate through the following series of 15 min ethanol washes: 30%, 50%, 70%, 90%, 95% and 100%, respectively.
▲CRITICAL STEP Make sure the cytoskeleton maintain good during the above gradient dehydration and fixation procedure.
31 | Transfer to critical point drying apparatus containing ethanol as intermediate reagent. Use a suitable holding device.
32 | Smaller-area contamination spots can be examined at electron accelerating voltages compatible with Energy Dispersive X-ray Spectroscopy.
33 | Reduce penetration of low kinetic energy electrons probes closer to the immediate material surface.
34 | Visualize by high-resolution FE-SEM at electron accelerating voltage 2.00 kV and with the work distance 4.4 mm from low kinetic energy electrons probes to the immediate material surface.
35 | Record the energy dispersive X-ray spectroscopy of the gold nanoclusters interspersed on cytomembrane from a random region of 2 μm × 2 μm with electron accelerating voltage 2.00 kV.
| Preparation of cell optical imaging ● TIMING 3 d
36 | Seed HepG2, K562, or L02 cells on quartz substrate in a cell culture dish containing DMEM with 10% (vol/vol) FBS and 1% (vol/vol) penicillin-streptomycin, respectively. Incubate the cells at 37 °C in a humidified 5% CO2 atmosphere overnight.
37 | Prepare the fluorescence or Raman imaging sample by following Steps 24-26 of the PROCEDURE.
A | Cell fluorescence imaging ● TIMING ~1.5 h
i | Place the prepared cell sample on CLSM objective stage.
▲CRITICAL STEP Make sure the cells maintain normal physiological status during fluorescence signal record process.
ii | Record cell imaging for the incubated cells by a confocal laser scanning microscopy with a 488-nm excitation laser beam focused using a 20 × IR coated objective.
B | Cell Raman spectroscopy ● TIMING ~1.0 h
i | Gently remove PBS and transfer the prepared cell sample on Raman microscope objective stage.
▲CRITICAL STEP Make sure the cells maintain good status during Raman signal record process.
ii | Record cell Raman spectroscopy by confocal Raman microscopy with an excitation wavelength at 532 nm, 50% laser intensity, and overlaying twice with 100 s integration time.
| Preparation of injected mice for imaging ● TIMING 3 d
38 | Inject HAuCl4 solution prepared in PBS (100 μL of 10 mmol/L for each mouse) into BALB/c mice bearing HepG2 or K562 tumors near the tumor location through the subcutaneous injection.
39 | Normally maintain the injected mice in a specific pathogen free (SPF) house at 24 ± 2 °C with a standard 12-hour light/12-hour dark cycle for 24, 48 or 72 hours.
| In vivo bio-imaging ● TIMING ~1.0 h
40 | Anesthetize the experimental mouse with 5% isoflurane (21% oxygen, balance nitrogen) in an induction chamber until breathing is slow and deep, at different time points post injection (e.g. 24, 48, or 72 h).
41 | Position the mouse on its back on a microscope stage for imaging, during the imaging procedures under isoflurane anesthesia.
▲CRITICAL STEP Perform all imaging procedures under isoflurane anesthesia for experimental reproducibility and to minimize the torment of experimental anmimals.
42 | Select the appropriate excitation wavelength (488 nm) and adjust the mouse’s posture as needed while monitoring the live image.
43 | Record in vivo image of the mice under Maestro EX in vivo fluorescence imaging system (CRI, Inc.) at different time points post injection (e.g. 24, 48, or 72 h).
44 | Euthanatize those mice after 1 week post injection.
! CAUTION The animal bodies should be collected in a designated freezer for radioactive contaminated biohazardous waste.
II Gold nanoclusters and graphene nanocomposites for cancer cell imaging
| Preparation of gold nanoclusters ● TIMING 2d
Dissolve 1.0 g HAuCl4•4H2O with Millipore water in a 100 mL volumetric flask to make 1% (w/v) HAuCl4•4H2O solution.
Load 169.2 g TOAB into 16mL chloroform in a 50 mL three-neck flask.
Add 6 mL 1% (w/v) HAuCl4•4H2O solution to the flask, and place the stoppers in all the necks.
Add a magnetic stir bar and mix the bi-phase solution vigorously at room temperature (23℃ to 25℃) until HAuCl4 is transferred into the organic phase. The aqueous phase should be transparent while the organic phase should have a color of bright amber.
Add 40.2 μL 1-DT into the flask, and stir for 10 minutes.
Prepare 1.55 mol/L NaBH4 solution
▲CRITICAL STEP The NaBH4 solution should be freshly prepared.
Load 25 mL 1.55 mol/L NaBH4 solution drop wisely. With the addition of the reducing reagent, the color of organic phase should change from bright amber to deep purple within a few seconds.
The nucleation should be completed within 20 minutes. Keep stirring for 3 hours and stop.
Separate the organic phase (upper layer) with a separating funnel, and load it into a 50 mL flask.
Evaporate the separated organic solution with a rotary evaporator at 50℃ for 4 minutes.
Re-dissolve the product in 2 mL chloroform.
To remove the leftover organics, like TOAB and 1-DT, precipitate the product in 80 mL ethanol in -4℃ refrigerator overnight.
Get the dark brown precipitant by 15,000 rpm centrifugation at 4℃. Filter it out and wash it with ethanol for two to three times and re-dissolve it in 2mL chloroform. Repeat step 11.
The final solution should contain 1.46×10-4 mol gold nanocluster.
Mix 500 μL gold nanocluster solution (contains 3.64×10-5 mol Au) with 10mL CTAB solution (contains 6.86×10-2 g CTAB, according to molar ratio Au : CTAB = 1:5). Stir the mixture vigorously until not any phase boundary can be observed.
▲CRITICAL STEP From this step on, the reactions need to be conducted in the dark as much as possible.
Leave the solution to stand in 55℃ water bath for 3 hours to evaporate the chloroform and get the aqueous solution of gold nanoclusters.
Make 3.64×10-2 mol/L aqueous solution of goldnanocluster. The color of the solution should be light brown.
PAUSE POINT The final aqueous gold nanocluster solution can be stored at 4℃ in dark. After lyophilization, the sample can be stored at 4℃ for months.
The diameter of such prepared nanoclusters is 2 - 3 nm. It does not exhibit the surface plasmon resonance characteristic peak at around 520 nm, but around 490 nm.
| Functionalization of gold nanocluster with RGO. ● TIMING 1 d
Prepare pH 5.0 acetic acid/sodium acetate buffer. And make 4 mL 0.5 mg/mL chitosan solution with the buffer with 30 minutes sonication at room temperature.
Dissolve 0.8 mg RGO in 1.5 mL chitosan solution with about 1 hour sonication at room temperature until there is not any black precipitant.
Add the aqueous solution of RGO to the aqueous solution of GNCs (3.64×10-5 mol) in a 10:1 molar ratio of RGO/ GNCs and homogenized in an ultrasonic bath for 30 min.
Incubate the above reaction mixture in the dark for 1 hour to allow loading of the GNCs onto RGO.
The final mixture then needs to be separated by centrifugation at 15000 rpm for 10 minutes.
Remove the supernatant and wash the aggregates with Millipore water twice to remove the residual RGO.
The sample can be stored at 4℃ refrigerator after lyophilization.
| Drug loading on GNC-RGO nanocomposites. ● TIMING 1d
Prepare 1μg/mL aqueous solution of GNC-RGO nanocomposites.
Incubate the nanocomposites with 0.22μg/mL Doxorubicin (DOX) solution for 1 hour at 37℃ in the water bath.
Drug-loaded GNC-RGO samples were separated through centrifugation at 10000 rpm for 15 min and carefully washed three times with ultrapure water.
PAUSE POINT The sample can be stored at 4℃ in dark after lyophilization.
! CAUTION Do not freeze the aqueous solution of GNC-RGO nanocoposites or drug-loaded GNC-RGO nanocomposites.
| Acquisition of confocal fluorescent images of cells treated with GNC, GNC-RGO nanocomposites ● TIMING 3 d
- Seed 500,000 HepG2 cells in 35 mm petri dish with glass bottom, overnight.
- Load 0.25 μg/mL DOX or DOX-laoded GNC-RGO nanocoposites and incubate with cells for 24 h.
- Wash the cells twice with PBS and load fresh medium.
- Acquire images on confocal fluorescent microscope using a 490 nm excitation laser beam and 40 X objective lens.