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Analytical Chemistry

A Bouncing, Swinging New Detection Method

 

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.

10 comments on “A Bouncing, Swinging New Detection Method”

  1. ezra abrams says:

    1 pg/mL in blood is very impressive
    I mean, really impressive – blood is a horrible sample format.

    and even more impressive if it is a homogeneous no wash assay – just mix and go (sometimes called an emergent property assay)

    Quanterix and T2biosystems are to successful companies based on ultra sensitive assays.

    But it isn’t clear that for the larger markets we need more sensitive assays; I think the big guys – say Roche – can already do this but don’t bother cause their is no reliable diagnostic need ; it isn’t really clear that you need more sensitivity for PSA or TSH, and the sensitivity for infectious isn’t good enough by logs (I think the desired spec is one gram negative/mL of blood)

  2. Aaron says:

    Genmark, formerly known as Osmetech, has an on-market ferrocene based electrochemical DNA detection chip and the amount of capital it took to get that absolutely wonderful product to market was staggering, perhaps $500M. The same seems to be the case for up-and-coming electrochemical biosensor startups. One reason for this, it is easy to blow through millions of dollars getting the bare electrode material to behave properly.

    The senior author on this paper founded a company called Xagenic and raised > $23M before selling to General Atomics. The commercialization of electrochemical biosensors is very capital intensive, and when they got started there wasn’t a COVID pandemic to drive the development of new technologies.

    https://www.genomeweb.com/diagnostics/xagenic-closes-series-b-round-raising-238m#.YGIGeS1h39A

    https://www.ga.com/general-atomics-acquires-assets-of-xagenic-inc

  3. cynical1 says:

    Could this technology be used to attach an antibody with unknown specificity to the DNA linker thereby allowing one to determine what those antibodies are directed against? I’m thinking something like the antibodies found in the CSF of MS patients whose targets are mostly unknown and correlate with disease activity.

    1. morecynical says:

      How does that enable you to determine what protein those antibodies are directed against? I suppose you could flow known proteins over the assay one-at-time, but that seems like a complicated way to answer the question (as opposed to just using the antibodies to immunoprecipitate the target)

      1. sort_of_knowledgeable says:

        Since the target can be released from the antibody,

        ” incubation with liquid blanks restores their activity as the target protein diffuses off,”

        It might be possible to conduct analysis of targets after release

  4. Param says:

    I am somewhat surprised they are still publishing on this. “Somewhat” because I understand the science can continue to progress incrementally, so no surprise there. The underlying technology started over two decades ago when Shana Kelley was in Barton’s lab at Caltech, with patent applications from 1999 and 2003. Other early patent applications from Kelley followed from 2005 to 2007 from the Trustees of Boston College. The relevant patent applications were published in 2010 from the University of Toronto.

    “You can imagine all sorts of monitors and sensor applications for something like this, with many diagnostic and clinical applications.” They thought so too. A company was formed to commercialize this technology. It was called Xagenic founded in 2010. Over the next 5 years, Xagenic raised over $60 million. It was eventually acquired by General Atomics in 2018.

    Interesting science is one thing. Creating commercial products are another thing altogether. Issues about real world applications, business model, market size, company leadership, and operational management all matter more. It didn’t work out in this case. If anyone thinks they can do better, you know where to go to get the sub-license, while some background IP has reverted back to University of Toronto.

  5. Gene says:

    I assume there’s a missing word?

    “Making the DNA linker [longer] led to a slower response”

    1. steve says:

      In that case maybe add some Viagra?

      1. steve says:

        Sorry, guess the headline got to me.

  6. Kaleberg says:

    Wow! This may turn endocrinology into a real science. (I know, it already is a real science, but data on blood levels is, at best, spotty. There’s just so much unknown land that being able to make sensors like this is bound to lead to some interesting places. Even better, a lot of the patents are running out soon, and technology tends to advance one patent expiration at a time.)

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