Table 2 |.
Troubleshooting table
| Step | Problem | Possible reason | Solution |
|---|---|---|---|
| 5 | Low concentration of COOH-SWNT suspension | Not enough dry COOH-SWNT added | It is hard to accurately measure small amounts of dry CNTs; use more COOH-SWNT powder for making the suspension |
| Probe tip was touching the sides of the conical tube or was not optimally placed | Make sure the probe tip is not touching the conical tube and is submerged fully in the solution, resting at a distance halfway from the fluid meniscus | ||
| The solution overheated during the probe-tip sonication | Change the ice bath every 10 min to prevent overheating. If none of these solutions work, the resulting COOH-SWNT suspension can be concentrated using 100,000-MWCO centrifugal filters | ||
| 11 | Excessive agglomeration or pelleting of activated COOH-SWNTs during washes with PBS (some degree of SWNT destabilization can happen, and it will not affect the next steps, if the SWNTs can be suspended again with bath sonication) | PBS salt concentration is too high | Make sure to use 0.1× PBS for washes, as 1x PBS will affect the colloidal stability of SWNTs adversely because of the high salt concentration |
| The starting COOH-SWNT solution was not well suspended or was too old | Suspend the COOH-SWNTs well in water and use only the supernatant of the centrifuged COOH-SWNTs. If using COOH-SWNTs from a previously suspended batch, bath-sonicate for at least 30 min or make a new COOH-SWNT suspension | ||
| Too much EDC and/or NHS was used | EDC-NHS is used in excess for this reaction, but too much of it can agglomerate the SWNTs. Decrease the amount of EDC-NHS until no agglomeration is observed | ||
| Too much solution was passed through the filtration membrane during washing | Always keep the membranes submerged in the reaction solution to prevent pelleting of SWNTs on membranes. If more liquid than desired is passing through and the membrane surface is exposed, decrease the centrifugation speed and/or time | ||
| 19 | PEI-SWNT reaction product is not suspending in water or has a low final concentration (<20 mg L−1) | The PEI was not well-dissolved in solution before addition to the activated COOH-SWNTs | After making the PEI suspension, place it on an orbital shaker at maximum speed overnight |
| Overheating of the solution during probe-tip sonication | Make sure to change the ice bath at least once or twice during the probe-tip sonication. Alternatively, use the pulse feature of the ultrasonic homogenizer with a ‘1 s on and 2 s off’ setting | ||
| Addition of not enough or too much PEI polymer for the reaction (not enough PEI may cause multiple SWNTs to be covalently bonded together by one PEI polymer, and too much PEI may cause formation of larger particles through non-specific adsorption, both of which will prevent the successful suspension of individual SWNTs) | Use the optimized amount suggested in this paper and, if scaling the reaction up or down, also scale the amount of PEI accordingly. Here, we suggest using 40 mg of PEI (25,000 MW, branched) for 2 mg of COOH-SWNTs | ||
| 25 | PEI-SWNT agglomeration upon plasmid DNA addition | PEI-SWNT positive charge density is not enough to carry added plasmid negative charge | Either increase the PEI-SWNT amount or decrease the plasmid DNA amount. Alternatively, redo PEI-SWNT synthesis to achieve particles with higher zeta potential values |
| 29 | Observed toxicity or damage in the infiltrated leaves | Too much PEI-SWNT was used for infiltration | For the reaction detailed here, we suggest using 500–1,000 ng of PEI-SWNTs per infiltration, because more may cause damage. Use less PEI-SWNTs if damage is observed with 500 ng |
| PEI-SWNT particles contain too much free PEI | If the washing is not performed efficiently, there may be too much free PEI left in the solution. Perform more wash cycles after the PEI reaction | ||
| SWNTs were functionalized with too much PEI | Instead of using 40 mg of PEI for 2 mg of COOH-SWNTs, repeat the reaction with less PEI. We suggest testing PEI amounts between 20 and 40 mg per 2 mg of COOH-SWNTs to troubleshoot | ||
| 30A(iv), B(iii), C(iv) | No gene expression was observed with any method | Plants or leaves used for delivery were too old or unhealthy | Use 1-month-old healthy plants and young leaves to test the constructs |
| Need to optimize the DNA-loading protocol | For DNA-loading optimization, we suggest testing between 500 and 1,000 ng of PEI-SWNTs in a total of 100 μL of solution per leaf infiltration, with the following SWNT/DNA mass ratios: 6:1, 3:1,1:1, 1:3, and 1:6 | ||
| PEI-SWNTs may be toxic to cells even though no visible leaf damage is observed | Follow the troubleshooting suggestions for Step 29 | ||
| Plasmid DNA construct is not working | Make sure the plasmid DNA is fully sequenced and induces expression when delivered via Agrobacterium to leaves or via PEG transfection in protoplasts | ||
| 30B(iii) | No fluorescent protein expression is detected with confocal microscopy | Low laser power | Use high laser power (80–100% of maximum) and a 20× objective. Scan the entire area carefully for cells that are expressing the fluorescent protein |
| Gene expression was not strong and uniform as with Agrobacterium-mediated delivery | Unlike Agrobacterium-mediated expression, the expression via SWNTs is less strong, and not all cells in the infiltration area will express the protein of interest |