Single Molecule Detection Of Proteins In Single Cells
An interview with Professor David Walt, Tufts University, conducted by April Cashin-Garbutt, MA
Can You Please Give An Introduction To The Single Molecule Array (Simoa) You Have Developed And That You Will Be Discussing In Your Talk At Pittcon 2017?
We developed this technology almost 10 years ago. It was developed after a key experiment was carried out by David Rissin, who was a PhD candidate in my laboratory at the time.
At the time, we were working with microwell arrays for a different project. At a lab meeting I posed the question: “How many molecules of a fluorescent molecule would it take to bring the concentration in one of these very tiny microwells to a level that would be easily detectable?” We performed the calculation and it turned out to be about 100 molecules of a fluorescent dye.
David carried out an experiment using a fiber optic microwell array, which had approximately 60,000 microwells on the end of an optical fiber. We trapped a very dilute solution of an enzyme in these microwells–each microwell had a volume of the order of 40 femtoliters (10-15 liters)—a very tiny volume.
The experiment was designed to trap a very dilute solution of an enzyme containing a high concentration of a substrate. Substrates are the starting materials for enzymes, such that if there were an enzyme present, the enzyme would catalyze the conversion of that substrate into many molecules of a fluorescent product. This process would occur quite rapidly with β-galactosidase, which is a fairly active enzyme.
The idea was that we would run this reaction at such a dilute concentration of enzyme that only a fraction of the wells would contain an enzyme molecule and only those wells that did would fluoresce. After David Rissin worked out the technical details required to carry out the experiment, he demonstrated that one could clearly see which wells contained an enzyme molecule because they turned very bright red in contrast to the wells that contained no enzyme molecule, which remained black.
After this demonstration, we observed that as we decreased the enzyme concentration, we observed fewer fluorescent wells because there were fewer enzyme molecules in the array. We published a paper in Nano Letters and claimed that this was the first time that anyone had measured concentration by counting molecules.
So this experiment was the first demonstration that you could take a solution, confine it into very tiny volumes and literally count the number of molecules present in that solution. In this case, the β-galactosidase concentration could be measured by simply counting the number of bright wells relative to the number of dark wells.
Read the rest of the Q&A article at news-medical.net
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