Peering Inside Nanopores

“We make money from invisible holes.  But we can’t afford to be invisible.”

Hans van der Voorn, Executive Chairman of nanotechnology start-up firm Izon, spreads out a series of brochures highlighting his company’s brightly-coloured, cheerful-looking devices, each housing its trademark nanopore technology.

“We made a conscious decision not to have any grey instruments. It’s a little unusual, I suppose, for science, which can be a bit serious. [Our competitors] are all beige or grey boxes. They look like little microwaves. Boring as hell. We wanted these to be the centre of attention in someone’s lab.”

From an initial research idea five years ago, Izon’s devices have emerged on the international market as the self-proclaimed “world’s most comprehensive nanoparticle analysis system”. Inside each coffee-grinder-sized machine is a plastic membrane containing a single flexible nanopore — a tiny hole which can be “tuned” by stretching and relaxing the plastic to allow particles of different sizes to pass through one-at-a-time.

As they cross, the particles trigger a characteristic dip in the electric current that runs across the membrane, allowing precise measurements of the quantities and characteristics of the particles in transit. Under the right conditions, the technology can record particles as small as an individual molecule.

One of Izon’s product lines has been customized to count viruses — an application particularly suited to vaccine research laboratories. Another major target for Izon is the emerging field of nanomedicine, which uses nanoparticles for targeted drug delivery. Here, reliable measurements of the number and attributes of the nanoparticles involved are critically important.

Bringing their initial concept out of the lab into working prototypes and fully-fledged commercial-scale production has taken a great deal of hard work and investigation, but Hans says they still face many unknowns. “We’re still learning. I don’t think we’ll ever stop doing research.”

Over the past four years, that research has involved MacDiarmid partner organization Industrial Research Limited (IRL), and has more recently grown into a multi-faceted collaboration with the Institute itself. Out of this  collaboration, a new understanding of the basic science underpinning Izon’s technology is emerging, with direct applications for the company’s commercial goals.

In 2008, MacDiarmid, IRL and Izon (then known as Australo) launched a joint project entitled, “Nanotechnologies for DNA sequencing”. It aimed to combine two very different technologies, each with the capacity to measure objects on the scale of a single molecule, and apply them to the complex task of decoding genes.

The idea behind the project was IRL senior scientist Jeff Tallon’s brainchild: if Izon’s nanopores could be adapted to thread a single nucleotide of DNA through the pore, the genetic sequence could be read off base-by-base as it passed through the hole.

The second technology, MacDiarmid’s surface enhanced Raman spectroscopy (SERS) system, had demonstrated the ability to detect individual molecules with its laser light-scattering technique.

Perhaps this feat could be replicated at the site of the nanopore, and together the two technologies would form the basis for a world-leading, novel solution for rapid DNA sequencing.

That was the theory, at any rate.

As the project has progressed, it’s become clear that its real value will have a lot more to do with fundamental science than with DNA.

“Since we first started, lots of others have come to the party. When people start talking about a $30 genome, that’s not a market we’re realistically going to chase,” says Hans.

“The original purpose [of the project] may never be met, but you find out a lot in the meantime”.

Currently, although single molecule-scale resolution is attainable using the nanopores, it can’t be achieved consistently. IRL’s contribution to the project has focused on extending the pores’ capacity.

Virus-sized particles, which Izon’s current devices target, are around 50 nanometers wide.

To refine this down to the width of a single strand of DNA — only two nanometers across — more work was clearly needed.

To that end, MacDiarmid principal investigator Geoff Willmott — based in IRL’s micro- and nano-fluidics lab – has been running a series of experiments examining which conditions give the clearest signal when a particle crosses the pore. Does a surface electrical charge on the pore make a difference? How do different nanoparticles and different solutions behave? What is the strength and duration of the signal that results? Nailing down these basic parameters will help Izon generate more reliable outcomes for smaller particles.

Another useful development came to light when the project team realised the nanopores did not appear to be stretching as much as they expected. Images from the University of Canterbury’s microscopes showed that this came down to faulty assumptions about the geometry of the holes.

Instead of the regular, cone-shape punctures the researchers had expected, they found more complicated three dimensional structures when they peered inside the tiny pores. So working out how much the pores open as the plastic is stretched turns out to be a tricky problem — something not factored into Izon’s original mathematical models.

Thus far in the company’s product development, Hans says, they’ve relied extensively on calibration to work around the gaps in their understanding of the fundamental science. Currently, with each product sold they supply standard-sized particles and guidance on calibrating the instrument for different uses.

“We can get a very adequate answer by sending through a set of known nanoparticles. But we still don’t have a model that’s good enough to give someone a measurement from first principles. When we do, that will be a big advantage.”

Refining the model with data produced by the collaboration will be a major help as the company targets new markets and refines its products, he says. Izon sells primarily to research laboratories at present, but envisions additional applications for its technology. These include medical testing and diagnostics, and eventually hand-held devices for air and water quality testing, as well as point-of-care diagnostics in the GP’s office. A better understanding of the fundamental science will improve accuracy and effectiveness, laying the groundwork for these future applications.

Another of the unexpected outcomes of the collaboration has seen Izon’s flexible plastic film, or elastomer, adapted for research into the inner workings of the SERS technique in Pablo Etchegoin’s plasmonics laboratory.

Single-molecule resolution, the starting point behind the DNA sequencing project, can only be achieved where very high SERS resonance develops. Typically, SERS works when a resonant “hotspot” develops on a surface that’s been coated with a random array of gold or silver nanoparticles. These hotspots are dependent on very closely-spaced particles landing just the right distance apart, so there is little control over where and how they form.

In an effort to introduce that element of control, post-doctoral researcher Kamal Hossain has been coating the membrane inside Izon’s device with gold nanoparticles. As he stretches and relaxes the plastic, he can make direct observations of how the changing spacing between particles affects the SERS signal.

If successful, these will be the first experimental investigations of their kind, providing new fundamental insights into plasmonic resonance. It is hoped the precise observations of stretching and deformation will feed back into Izon’s product development as well.

At Izon, Hans van der Voorn says this type of fundamental research isn’t something he would have predicted, but that’s the nature of collaboration. Other “children” of the original collaborative project with MacDiarmid include spin off projects with Massey, Auckland, Canterbury and Victoria Universities, and the addition of MacDiarmid PhD graduate Sam Yu to Izon’s science staff.

According to Hans, this kind of basic research wouldn’t happen without the initiative of collaborators and partner organisations. As the company has grown, its internal research priorities have inevitably shifted to focus exclusively on product development and marketing.

Well-targeted incentives for research and development early in a company’s growth go a long way, he says. Izon received TechNZ funding from 2005 to 2008 that enabled them to reduce the size of their devices. They’re eager to apply again, with the aim of fully automating the particle analysis process.

However, he believes restricting new R&D incentives to large companies that should be able to fund themselves is a waste of money.

“When we’re a 100 million dollar company, I’m not going to have my hand out.” At that stage, he says, Izon looks forward to being able to fund the MacDiarmid Institute and sponsor research in universities and CRIs.

He hopes there will be a continued recognition of the advantages of a diverse range of research funding opportunities. Izon’s experience has shown that there are substantial – and often unpredictable — benefits from collaborations between research institutions and the commercial sector.

“It’s not MacDiarmid or us. It’s MacDiarmid and us. Because what they do helps us, and what we do helps them. We become symbiotic.”