Optimising the signal-to-noise ratio is essential for resolving the entire population of your particle sample. If the blockades are too small (i.e. within the RMS noise range), they may become lost in the noise signal and not be accounted for by the software. This is especially important for the recording of the calibration particles.
The Capture Assistant (Custom Planner) allows for easy optimisation of the conditions with the help of the "Green Zone." This green zone guides the user to correctly adjust measurement settings (such as stretch, pressure, and voltage) to maximise resolution. If you are using the programmable Custom Assistant, and your data quality is not ideal, try switching to the Classic Capture mode instead. Classic Capture allows you to optimise each parameter yourself, so you can get the best possible data out of every nanopore.
To improve your overall signal-to-noise ratio, ensure you have optimised the following parameters:
- Stretch: Within the range of 44.5 - 48 mm (50 mm if you want to sacrifice the pore), adjust the stretch slowly until your average blockade magnitude (for your calibration and sample particles) sits between 0.15 and 0.5 nA at a background current of approximately 100 nA. If you cannot get sufficiently large blockades even at 44.5 mm, use a smaller nanopore.
- Voltage: Once you have optimised the stretch, it is recommended adjust the voltage until the baseline current sits between 120 and 140 nA (or 150 nA if you can maintain the stability of the system). Using the Classic Capture mode will allow you to set the voltage higher than the default setting programmed into the Custom Assistant, often greatly improving the signal-to-noise ratio.
- RMS noise: After optimising the voltage and before you pipette in your particles, check that the RMS noise is smaller than 15 pA. If it is higher, use the steps below to minimise the noise.
- Pressure: Adjust the dilution (of the calibration particles) until you get a particle rate between 200 - 600 particles per minute at a pressure of 5 mbar. For concentration measurements requiring 2 - 3 pressures, aim for a minimum particle rate of 500 particles per minute at the lowest pressure, and 1500 particles per minute at the higher pressure. The difference between each different pressure should be at least 2 mbar.
- Reagent quality and system stability: Ensure that you filter your electrolyte immediately before using it to make up dilutions. If working with biological samples, you should use the Izon Reagent Kit components to coat and protect your nanopore from being modified and blocked by proteins and other biological matter present in samples.
There are three key sources that contribute to noise and signal interference:
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Short-circuits:
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Electrolyte can creep in between metal connection points inside or around the fluid cell, causing significant noise fluctuations.
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Remove the upper fluid cell, then wash and dry it, making sure all of the metal parts of the fluid cell are dry. Then re-establish the baseline current.
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Always ensure that you are using the reverse-pipetting technique when introducing fluid to the upper or lower fluid cells, and do not pipette more than 35 µL into the upper fluid cell, or more than 75 µL into the lower well, or the fluid may leak between connection points and cause the noise to creep up.
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Partial nanopore blockage:
- Particle or bubble build up on either side of the aperture can cause partial blocking of the nanopore (indicated by a sudden >5% drop in baseline current or particle rate).
- To prevent these blockages, replace the electrolyte in the lower fluid cell every half hour.
- If the current becomes unstable and the noise is high, replace the fluid in the lower fluid cell with filtered deionised water as follows:
- Remove applied pressure.
- Stretch the nanopore to 47mm.
- Remove the upper fluid cell. A droplet of fluid should remain on top of the nanopore. Immediately pipette 35 µL of deionised water to the centre of the nanopore surface to maintain pore hydration.
- Rinse the upper fluid well with deionized water and dry with compressed gas or lint-free tissues.
- Replace the content in the lower fluid well with deionised water.
- Gently wipe the top of nanopore with lint-free tissue.
- Immediately fit the upper fluid cell and pipette 35 μL of deionised water into the upper fluid well ensuring no bubbles present.
- Apply maximum pressure for approximately 5 minutes.
- Once completed, check if the baseline current is now stable with measurement electrolyte, if not repeat Step 3-8
- External/environmental noise:
- 50/60 kHz noise from nearby laboratory equipment and ungrounded power supplies can interfere with the baseline current signal.
- To reduce influence from external noise, do not operate the qNano in close proximity to large laboratory appliances that draw a lot of power, and ensure the fluid cell metal cap is used during data recording to minimise noise on the baseline current and obtain better blockade resolution.