Synthesis of germanium nanospheres as trappable optical probes (GeNTOPs)

Materials for germanium nanosphere synthesis

Chemicals

• Germanium oxide (GeO₂) 99.99 % trace metal basis (Sigma Aldrich).
• Quercetin (Sigma Aldrich).
• Sodium hydroxide (NaOH,Sigma Aldrich).
• 37 % Hydrochloric acid (HCl, Sigma Aldrich).
• Sodium borohydride (NaBH₄, Merck).
• Deionized water Type I (MilliQ, see Equipment).

Equipment

• Magnetic stirrer with heating function C-MAG HS7 with ES-D5 thermometer (IKA).
• Laboratory dish Boro 3.3 with 140 mm diameter (VWR).
• Silicone oil (Sigma Aldrich).
• Magnetic stir bars 8-35 and 4-10.
• Vortex mixer RS-VA 10 (Phoenix Instruments).
• Centrifuge 5430R with rotor F-35-6-30 (Eppendorf).
• Ultrasonic cleaner with heating function USC-THD (VWR).
• Digital pH-meter646 (Knick).
• Nanopure system MilliQ with Q-POD and Biopak filter.
• Refrigerator (4^◦C)
• Conical centrifuge tubes 15 mL.
• Protein LoBind tubes 1.5 mL (Eppendorf).
• Duran laboratory glass bottle 50 mL with. standard screw cap PP/green (Schott).

Materials for lipid bilayer coating of germanium nanospheres

Chemicals

• 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC); Avanti Polar Lipids, Inc.
• 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)- 2000](DSPE-COOH, Avanti Polar Lipids, Inc.).
• Calcium chloride.
• Chloroform.
• Deionized water type I (MilliQ, see Equipment).
• Buffer 1: 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES),150 mM NaCl, pH 7.4.
• Buffer 2: 5 mM HEPES, 200 mM NaCl, 1 mM tris(2-carboxyethyl) phosphine  (TCEP), 5 mM EDTA, pH 7.4.
• Buffer 3: 25 mM HEPES, 100 mM NaCl, 0.25 mM CaCl₂, pH 7.4.
• Buffer 4: 25 mM HEPES, 25 mM NaCl, 1 mM TCEP, 0.25 mM CaCl₂, pH 7.4.

Equipment

• Ultrasonic cleaner with heating function USC-THD (VWR).
• Centrifuge 5430 R with rotor F-45-30-11 (Eppendorf).
• Centrifuge MiniSpin plus with rotor F-45-12-11 (Eppendorf).
• Lyophilizer Alpha 2-4 LSC (Christ).
• Vortex mixer RS-VA10 (Phoenix Instruments).
• Thermomixer C (Eppendorf).
• Desiccator
• Nanopure system MilliQ with Q-POD and Biopak filter.
• Vacuum pump PC 3004 Vario with CVC 3000 display (Vacuumbrand).
• Conical centrifuge tubes 15 mL.
• Protein LoBind tubes 1.5 mL and 2 mL (Eppendorf).
• Brown 1.5 mL screwed glas flasks (Fischerbrand).

Methods

Synthesis of germanium nanospheres

Germanium nanospheres were synthesized by a wet chemical reduction method. The germanium nanosphere synthesis is a one-step synthesis. Germanium oxide is suspended with the stabilizing agent quercetin in an aqueous sodium hydroxide solution (Solution A) and reduced by adding an aqueous sodium borohydride solution (Solution B). Upon reduction, crystalline germanium nuclei form that will further undergo growth. This growth process can be stopped at different time points to obtain the desired size of nanospheres. Termination is achieved by separating the quercetin-coated germanium nanospheres from the synthesis solution by centrifugation and redispersion in deionized water. After 3 h the synthesis resulted in roughly30-nm-diameter germanium nanospheres as trappable optical probes (GeNTOPs). Subsequently, the size increased nearly linearly with time with about 30 nm/h up to 8 h. The 70-nm-diameter GeNTOPs used were obtained by stopping the synthesis after 5 h. Smaller nanospheres can be obtained by stopping the reaction earlier.

Solution A: solution of germanium oxide with stabilizing agent

1. Mix 17.0 mg germanium oxide and 96.0 mg quercetin in 10 mL of 0.15 M sodium hydroxide in a 50 mL Schott bottle (Tip 1) and adjust the pH with 37 % HCl to pH = 8.8.
2. Mix the solution with a vortex device.
3. The resulting solution should be orange and will show small phase separated droplets. It is recommended to use the solution within a few hours.

Solution B: sodium borohydride solution

1. Dissolve quickly 29.5 mg of sodium borohydride (reducing agent) in 3 mL of ice cold deionized water in a 15 mL falcon tube.
2. Vortex the solution3x and store it in the fridge (Tip 2).
3. After mixing,the solution should be stored in the fridge for 20 min before usage (Tip 3).

Reaction: germanium nanospheres

1. Pre-heat the silicone oil bath to about 60 ^◦C on a magnetic stirrer using a stir bar.
2. Stir the substrate and stabilizing agent containing Solution A continuously in the preheated oil bath for 10 min (Tip 4).
3. Add the reducing agent (Solution B) drop wise. The orange solution will turn yellowish (Tip 5).
4. Let the solution react in the closed flask for up to 8 h under constant stirring (Tip 6 and 7).  The solution will turn dark orange over time.
5. Redisperse the sedimented particles (dark red deposit) by gentle shaking in a rotating, swirling fashion.
6. Centrifuge the solution for 5 min at 7,830 rpm (Centrifuge 5430 R).
7. Collect the nanosphere pellet and resuspend it in 10 mL deionized water (Tip 8).
8. Vortex the solution for redispersion.
9. Repeat steps 6–8.
10. Repeat step 6 and resuspend the nanosphere pellet in 2 mL deionized water.
11. Sonicate the resuspension for 5 min to redisperse the nanospheres.
12. Store the nanospheres in the fridge.
13. Depending on the synthesis time, the resulting germanium nanospheres will have a diameter of about 30–200 nm.

Lipid bilayer coatingof germanium spheres

To optimize germanium nanospheres for optical trapping and functionalization with bio-molecules, we coated the GeNTOPs with a lipid bilayer following established procedures. Carboxylated and uncarboxylated lipids are mixed, dried and redissolved in HEPES buffer. Multilamellar vesicles (MLVs) are produced by vortexing of the solution and subsequently small unilamellar vesicles (SUVs) are generated by sonication. SUVs are collected by centrifugation and mixed with calcium chloride and GeNTOPs for fusion. Lipid-bilayer-coated GeNTOPs are washed, collected and lyophilized for long-term storage.

Solution C: lipid mixtures

1. Dissolve each lipid type (DMPC and DSPE-COOH) in chloroform (10 mg/mL).
2. Mix the chloroform solutions of the lipids in a 1:4 ratio of DMPC:DSPE-COOH.
3. Prepare 1 mL lipid solutions in brown 2 mL flasks.
4. Dry the lipid solutions in the desiccator under a 50 mbar vacuum overnight.
5. Store the dry lipids in the flasks in the freezer at -20 ^◦C.

Solution D: SUV emulsions

1. Hydrate two flasks of the lipid mixtures (Solution C) with 2 mL of 80 ^◦C warm Buffer 1 to achieve a final total lipid concentration of about 0.5 mM.
2. Pipette and vortex the hydrated lipid mixture for 2 min to form MLVs. The presence of MLVs turns the emulsion turbid.
3. Transfer the MLV emulsion into a 2 mL protein low binding tube and sonicate for 30 min at 80 ^◦C to form SUVs (Tip 9).
4. Separate the MLVs from the SUVs by centrifugation for 30 min at 13,000 rpm (MiniSpin plus). After centrifugation, the supernatant should be clear and a colorless pellet should be visible.
5. Collect the supernatant containing the SUVs.

Solution E: GeNTOP solution

1. Vortex the GeNTOPs obtained from the synthesis protocol until they are suspended.
2. Sonicate the particles for 5 min for dispersion.

Reaction: lipid-bilayer coating of GeNTOPs

1. Add 2 mL of SUV emulsion (Solution D) to 2 mL of germanium nanospheres (Solution E).
2. Add 50 µL of 3 mM calcium chloride(CaCl₂).
3. Vortex quickly and distribute the volume to four protein low binding 1.5 mL Eppendorf tubes.
4. Incubate all four tubes for 45 min at 80 ^◦C and 600 rpm in the thermomixer (Tip 9).
5. Keep the samples overnight (12 h) at 4 ^◦C.
6. Centrifuge the samples for 15 min at 13,000 rpm (Centrifuge 5430 R).
7. Collect the nanosphere pellet and resuspend it in 1 mL Buffer 2.
8. Vortex the solution for redispersion.
9. Repeat steps 6-8 but use Buffer 3.
10. Repeat steps 6-8 but use Buffer 4.
11. Repeat steps 6-8 but use deionized water.
12. Lyophilize the samples and store at 4 ^◦C or use them freshly for your experiments (Tip 10).

Tips

1. Weigh the chemicals inside of e.g. Eppendorf tubes and spin the tubes down shortly before opening them to reduce any loss of spilled powder. Furthermore, pour the powder into the Schott bottle and use all of the 10 mL sodium hydroxide solution needed for the reaction to wash the residues out of the tubes. We washed the germanium oxide and the quercetin by successively pipetting 3x and 7x, respectively, 1 mL of the NaOH solution into the powder residues and pouring the solution with the dissolved powder residues into the Schott bottle.
2. Instead of the fridge, an ice bath possibly can be an alternative.
3. Based on observations, a time between solution preparation and usage of 20 min lead to the best results. Similar observations have been made by Choi et al. (Catal. Commun. 84, 80-84 (2016))  for its catalytic activation by metal nanoparticles.
4. 50 % of the reaction solution should be submerged in the oil bath.
5. The solution was added drop wise with a 1 mL pipette.There is no need to do this step with smaller drop sizes.
6. Remember that the synthesis time determines the final particle size.
7. The stirring speed of the magnetic stirrer (see Equipment) was around 25 %.
8. Alternatively, distribute the solution volume equally to two falcon tubes to avoid a centrifugation balance.
9. The temperature needs to be above the melting temperature of the lipids to achieve a good mixture
10. Samples were snap frozen with liquid nitrogen and placed in the precooled (-80^◦C) lyophilizer. A vacuum of 0.018 mbar was applied while the temperatures where slowly adjusted to room temperature over roughly 1 day (standard lyophilization procedure of the device).

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