In 1880, Pyotr Ilyich Tchaikovsky was commissioned to create an overture to commemorate the defeat of Napoleon’s grand armée outside of Moscow in 1812. His use of 16 real cannons as part of the percussion section has definitely made this piece stand out in terms of impact. Unfortunately, he personally hated it, he thought it was loud and noisy but without artistic merit. Fortunately in diagnostics we are more concerned with loud signals than we are with artistic merit. In this issue in our series on Signal versus Noise, I am will focus on the amount of signal (the loudness) of the signal provided per binding event.
All diagnostic assays come down to the measurement of binding events between the aptamer and the target. When I say “the amount of signal per binding event” what I really mean is the number of signal molecules activated or present per molecule of target bound. In most aptamer diagnostic applications the constraint has been that only a single detection molecule is attached per aptamer. It is routine to synthesize aptamers with a wide variety of signal molecule moieties attached. The problem is that aptamers do not have Fc regions, the whole aptamer is involved in binding, thus the signal is only incorporated on one terminus or the other of the aptamer.
Why is this a problem? With one signal molecule per aptamer the maximum signal per binding event cannot be higher than 1:1. It does not matter if we have an excess of aptamers to target or an excess of target to aptamer, our maximum signal to binding event ratio will be 1:1. With antibodies it is routine to attach many signal molecules per antibody molecule thus resulting in a much higher signal/binding event ratio. A higher signal to binding event ratio means that it is possible to obtain greater sensitivity with an antibody than with an aptamer, independently of binding affinity.
At NeoVentures we have overcome this constraint with our proprietary hybrid gold nanoparticle (GNP) signal amplification system. We are able to routinely conjugate an average of 2500 oligonucleotides to an individual 40 nm GNP. We have incorporated an 80:20 ratio of signal oligo (labeled with biotin, and then strep/HRP) to detection aptamer. With this approach we can detect down to 100 fM nucleoprotein from SARS-CoV-2 in a mL of saliva.
This represents a true breakthrough for the application of aptamers in diagnostics. Now, we can actually design aptamer based detection systems with a higher signal per binding event than is currently possible with antibodies. This increases the strength of the signal without increasing the background noise. A clear signal with very low amounts of target protein is beautiful music to my ears. It is possible to overcome a lot of background noise if you use a cannon to play your notes rather than a flute.