May 23, 2018
by Carol Lynn Alpert
All hands in attendance today.
Science is unforgiving. Before you can even get to the main event you’re training for, you must contend with miles of equipment, logistics, protocol, and supply hurdles along the way. This week Peter and Abhijit finally resolved the problem of the “clicking” Smaract microscope stage, and Kil Ho and Abby made progress winnowing through some of the seemingly unending set of variables found in vendor-supplied QDots, buffer solutions, quantum yields, and processing steps.
The Smaract stage arrived back from Germany with the diagnosis that the clicking is due to the “stick-slip” behavior of the piezoelectric material, and Abhijit’s new blog post contains a link to a YouTube video that demonstrates the mechanics of this. Basically the plan is to avoid imaging at the moment the stick-slip is occurring. Abhijit also replaced the aluminum plate beneath the stage with a new steel plate. Its increased stiffness will further reduce drift. He then set up benchmarking tests using 100 nm fluorescent microspheres, and determined a full-width half-maximum point spread function (PSF) of 305 nm. However, a spherical aberration was noted along the vertical axis in the axial direction and the fluorescent beads drifted 140 nm over a 10-minute period. The team suspects that ambient air current and temperature deviations may be at fault, and so they intend to enclose the microscope stage. Abhijit also used one-micrometer-diameter beads to begin to test incoherent holography capacity of the system. To benchmark the system he will have to perform this same test using 100 nm beads.
Kil Ho and Abby are still plowing through the weeds of dozens of variables affecting their QDot – DNA conjugation efforts. Kil Ho had initially thought that switching to commercially-available QDots would help him work out a set of optimal protocols before switching back to lab-made QDs, but now it seems the opposite may be true. What he’s discovered so far in testing CdSe QDots supplied from four different vendors is that not only is there variability between vendors, there is also variability between different batches from the same vendor. Perhaps more significantly, there seem to be considerable discrepancies between the advertised quantum yield of each product and what they actually yield on testing in the lab. Not only that, but once any chemistry is performed with the QDots, their yield tends to plunge. The QDots are typically delivered in a minimal amount of buffer solution; some of them lose stability during buffer or ligand exchange chemistry, essential steps in the process of DNA conjugation. In his May 13 blog post, Kil Ho reports data obtained from taking QDot products from two different vendors through some of the necessary process steps. This revealed considerable variability and reduced utility. Kil Ho is actually gathering data on products from four different vendors. The plan is to publish the data, as a heads-up to the field that differences in advertised yield and practical-use yield will vary significantly, and that buffering solutions and exchanges are key factors for success in any conjugation efforts. Certainly for our goal of DNA conjugation, we must double down on the quest to determine an optimal buffer solution, and a significant factor is the percentage of magnesium in that solution.
Meanwhile, Abby was tackling the very high ratio (1:1500) required for the QDot-Dye conjugation technique. She’s currently using dye as a substitute for the desired DNA, trying to figure out how to use a smaller ratio since DNA is very expensive. Abby’s hunch was that she could substitute cheaper PEG amine-terminated “filler” molecules for some of the single-strand DNA. While waiting for the new DNA and filler molecules to arrive, she worked on other parts of the QDot-Dye conjugation process and found that she could reduce the ratio of dye required (as a DNA stand-in) by significantly increasing the EDC to QD ratio. She was able to get the QDot:Dye ratio down to 1:15. And yet, when the new DNA and PEG amine filler molecules arrived, QDot-DNA conjugation failed. Now Abby is asking “Does the DNA sequence matter during QD-DNA conjugation?” which is the title of her latest blog post. There is some suggestion in the literature that the “T” nucleotide thymine is the least reactive, so she will try a DNA sequence consisting solely of Ts, terminated with a dye molecule on one end so that she can do a quick check for binding. The size of the DNA string may also matter. Abby is also checking techniques for gold nanoparticle conjugation that may be relevant as well as a review paper on QDot conjugation from Dubertret. In other news, Abby was able to design and build new separation columns that should work with the larger QDot-DNA conjugates, although so far she has only able to confirm that they successfully separating QD-Dye conjugates from non-conjugated dye molecules.
Carol Lynn reported the sad news that Jeanne Antill passed away this week after a prolonged struggle with late-stage cancer. Jeanne, an artist, graphic designer and video producer, was a much-loved member of QSTORM team and the MOS Strategic Projects Group. She designed our QSTORM Legacy site (qstorm-legacy.org) and peopled it with wonderfully whimsical illustrations that conveyed ideas and themes well beyond the capacity of the camera.
We will meet again on July 5th at 11 am. Conflicting travel schedules preclude a June meeting.