Here’s an ingenious new detection technique for biomolecules that builds on a number of reports over the last few years. People have been working on electrochemical detectors using DNA constructs on electrode surfaces, but this would appear to be an improved way to do it.
A team from Toronto reports using a “molecular pendulum” attached to an electrode surface, as shown at right. An antibody is attached to a DNA linker along with a molecule of ferrocene, and the latter was chosen for its redox potential relative to the electrode. When a molecule binds to the antibody, that change in the weight and size of the end of the pendulum changes how it behaves in its interactions with the surface (and with the other attached pendulums within range). This can be monitored as a change in the current as the negatively charged linker chain gets closer to the positively charged surface and brings the ferrocene(s) within range for electron transfer, and the group checked how this was affected by several variables.
Making the DNA linker longer led to a slower response, which was especially noticeably at low voltages. At a constant linker length, higher applied electric fields led to a faster response (which could easily become too fast). Variations with molecular charge of the analyte could be detected, but were small compared to other factors, which is useful. Changes related to the diffusion coefficient of the analyte were also detectable, related to molecular weight and size, but were also smaller than the effects of linker length and electric field strength. The same went for viscosity of the bulk fluid around the sensor, and the relations of these last few variables showed that calibration curves could be used to model them in a real detection system.
What kinds of fluids are we talking about, then, and what kinds of molecules? The team showed that the technique works with saliva, blood, tears, sweat, and urine when using an antibody to the muscle protein troponin as the end of the pendulum. That’s a good choice – that protein is normally not seen in blood, but it’s a widely used marker of cardiac muscle damage after a heart attack. This system could detect troponin down to 1 picogram/mL (about 40 femtomolar concentration) in blood, with no signal from negative control proteins. Tests of different protein/antibody pairs showed that brain-derived neurotrophic factor, interleukin-6, thioredoxin, carcino-embryonic antigen, alpha-1 fetoprotein, P53, IgE, and BDNF could all be detected selectively as well. The sensitivity was better for the larger proteins, as you would predict from the physical properties of the system – IL-6 had the narrowest window (at 184 amino acid residues) and AFP (at about 600 amino acids) the highest, but all of the differences were highly statistically significant. These sensors turn out to be reusable – incubation with liquid blanks restores their activity as the target protein diffuses off, and the electrodes could be stored for days or weeks in fluids like saliva with no real loss of sensitivity.
The last part of the paper shows that this technique can be used for real-time monitoring in a live animal. A small sensor electrode using the troponin system was place in a mouse’s mouth, and this set off a strong signal when the animal was injected with troponin (the signal started heading up at the 20 minute mark and peaks at about an hour. This also worked with mice that had been treated for four days with doxorubicin (which is known to cause cardiac damage in mice), with a strong signal with a spot check at Day 5 and nothing but baseline in the controls.
Many of the existing DNA/electrode systems don’t accommodate antibodies very easily, and tapping into those interactions is certainly a big advantage. You can imagine all sorts of monitors and sensor applications for something like this, with many diagnostic and clinical applications. Back in the R&D chemical biology world, there are plenty of weirdo ideas that could be tried as well. Can you monitor or distinguish different varieties of cells (human or pathogenic bacteria) based on surface antigens? At the other end of the scale, can the sensitivity of the system be increased to pick up even smaller target molecules, thus giving competition for something like SPR assays? As it stands, it would seem that this method could already handle protein-protein interactions. Similar to SPR in such cases, can on- and off-rates be measured? How small can these electrodes be made – could they go down to the size of patch-clamp techniques for single-cell experiments? And so on. Our world can use all the real-time tagless detection methods it can get, and I’ll be glad to see how this one evolves.