I am looking for advice on what kind of plate-reader we would benefit most from and what system is generally preferred from a user perspective.
Today, we are mostly running absorbance based assays using tartrazine dyed reagents (we produce our own reagents) to verify pipetting performance. We are measuring in the volume span of 0.1µL to 50µL.
I know there are a lot of different method much more likely to show us the true pipetting performance - would anyone have a specific method to recommend? Quant-iT kits or qubit seems promising. Any advice on designing a dual-dye assay?
Right now Varioskan lux looks like an alternative as it might cover some functions requested by other departments, main drawback is that it is rather large and bulky. Does anyone have experience with this system?
Best regards,
Wictor
Edit: Are there any plate reader that can do the fluorescence and absorbance measurement for each well subsequently? I.e., for each well do both measurements and then proceed to next well.
I have a SpectaMax M5 in my lab and it does the job well enough. I found a linear range for tartrazine between 1-7% (w/v) final concentration in 100 uL sample well (standard 96PCR plate), with OD between 0.5-2.5 before the R2 began to break down. I ran all my volume verification with on-plate std curve, but also found very little variation in the regression over 20 different plates so an external curve might have been acceptable. This method and well volume would be specific enough for your volume range down to ~0.5 uL unless you reduced the well volume and confirmed the linear range at that lower well volume. (or a better read plate with higher RFU, or better plates like A/2 chimney well, etc…)
The M5 is bulky, and would need multiple “experiments” set up within the same protocol to read Flu and Abs, but you’d need to switch between top-read and bottom read regardless to get better OD, which requires an adapter on the M5. Not using the adapter would risk tightening the linear range and subsequently the volume range you could measure - at least with the M5.
Just wanted to come back and specify that this was AFTER background correction. Without a plate blank, it would likely be ~1.5-3.5, so be sure to consider that in your calibration curve; whether or not plate blank will be available. Your calculated curve intercept will adjust accordingly.
I love this topic and wanted to chime in with a device I saw at SLAS. While it may be a bit expensive it does have amazing applications for volume verification during the running of an assay/experiment rather than relying on verification tests done seperately. The plate reader is called a Liquimetrix from Matrix Fluidix. It uses reflection of white light to calculate volume in a well regardless of plate type (once a plate type is properly configured). The total read time was marketed at 30 seconds/plate with reporting for both accuracy and precision.
I believe it was around the 120k mark, which is pricy but if it works as advertised can be justified as I don’t see these kind of capabilities from other platforms: “on demand” volume verification at run time for assays, in a possible integrated scenario.
This is definitely a liquid property and not a proprietary property, so it could also be programmed for other readers. For example, Softmax Pro (For equipment by Molecular Devices) includes functions PathCheck and Pipettor Check which can perform volume verification using NIR absorbance of water. It depends on (I believe) calculating the path length of the water column as opposed to the concentration which can then be used to calculate column height and volume depending on the well geometry. It’s all relative so you use a filled-well blank instead of plate blank to start with; kinda like a standard curve of 100-200µL water to calculate pathlength. Swap water with your liquid and there would be some useful dynamic range.
Since this tech in particular is NIR absorbance, using optically clear labware would definitely lead to better results but having higher quality imaging and specificity would for sure allow this to be done even with suboptimal well clarity. There are also other non-spectrophotometric options as well - I was actually looking into a project with ultrasonics for a while but other projects took priority. I think there’s a thread floating around somewhere in this forum where someone set up an in-house ultrasonic volume measurement for their plates.
