A German research team has developed a method for using solid-state nanopores for stochastic sensing of proteins.
The technique, which they detailed in a paper published this week in the online edition of Nature Nanotechnology, uses solid-state nanopores functionalized with recombinant his-tagged proteins to sense target analytes, and could prove a versatile approach for high-sensitivity protein detection and quantification, Ulrich Rant, a researcher at the Technical University of Munich and an author on the paper, told ProteoMonitor.
When voltage is applied across a membrane containing a nanopore, molecules passing through the nanopore will cause changes in conductivity that can be measured, enabling detection of those molecules. Thus far, the technology has been applied primarily to the study of nucleic acids, and a number of commercial firms, such as Oxford Nanopore, are developing it for high-throughput DNA sequencing.
Nanopore technology is applicable to protein detection, as well, although, Rant noted, certain modifications are required to adapt it for this purpose.
For instance, much nanopore work has revolved around use of biological nanopores like staphylococcal α-hemolysin. While these nanopores are large enough for nucleic acid work, they are typically too small to study larger molecules like intact proteins.
Given that limitation, Rant said, the researchers turned to solid-state nanopores, which they could build via electron beam lithography and reactive ion etching to have pore sizes that would accommodate the proteins of interest.
The problem then, Rant said, "was how to incorporate molecular recognition into the pore" in order to functionalize it for detection of specific proteins.
Past attempts to incorporate receptors for target proteins into nanopores have had trouble limiting the number of receptors that attach to a nanopore, he said. "So you have many, many receptors sticking in the pores, and when you exposed these pores to protein solutions the proteins would go into the pore and then they would irreversibly bind [to the receptors], and this would clog the pores to some extent. So the pore diameter would effectively get narrower, and in the end you would have a reduced pore conductance that was irreversible."
Rant and his colleagues solved this problem by incorporating a small number of multivalent nitrilotriacetic acid, or NTA, groups into a molecular monolayer of alkane-thiols it assembled over the pore interior. These NTA groups bind His-tagged proteins, allowing the researchers to immobilize these proteins within the pore and giving it the biochemical functionality of the His-tagged proteins. With this arrangement, the researchers could then detect analytes of interest based on their interaction with the immobilized His-tagged proteins within the pore.
"We managed to place a single receptor site in the pore, so if one protein goes in there, it will bind to the receptor and then it will unbind again and you will see not only a decrease in the ionic current of the pore, but you will also see an increase again when the protein leaves the pore," Rant said.
Using this technique, the researchers built a nanopore capable of detecting various IgG antibody subclasses by functionalizing the nanopore with His-tagged protein A – a bacterial cell wall protein. They analyzed rat and hamster IgG samples, identifying relative concentrations of IgG1 and IgG2. They also detected residual amounts of IgG1 in a commercially purified rat IgG2 sample, demonstrating the high sensitivity of the technique.
In principle, any His-tagged protein should work with the system, Rant said. "Our aim was to make this kind of approach immediately applicable to many different protein systems, and His-tagged proteins are probably the most prevalent recombinant protein tags out there, so you could easily apply it to thousands of different proteins."
Nanopore-based protein detection will likely prove most useful for low-abundance protein detection, he said, noting that "even if you only have a hundred or a thousand [binding] events, you can get very good data from those events."
There are, however, drawbacks inherent to nanopore technology, Rant noted. While well-suited to measuring moderately strong protein binding, it is poorly suited to measuring either very weak or very strong binding events.
"Because you are doing a single-molecule experiment, if you want to detect many proteins you have to detect them one after the other," he said. "And if they bind very strongly to a receptor in the pore, then you have a problem because you have to wait a long time until the pore is free again and you can start to detect the next binding event. So, if you have a high-affinity reaction I would say the nanopore is not very well-suited."
"Also," he added, "if you have a very weak interaction the nanopore might not be well-suited because if the binding is so short that it occurs on a microsecond time scale, then the electronics are just not fast enough to capture the event."
For normal affinities – within nanomolar to millimolar kD values – though, the technology could prove a powerful tool for high-sensitivity protein detection, Rant said. He and his colleagues have not patented their work and currently have no commercialization plans, but, he said, a commercial version of the tool could probably be brought to market within five to ten years were someone interested in doing so.
"From a technological point of view it's not very difficult to reproduce," he said. "We used electron beam lithography and reactive ion etching to pattern the pores. These are very standard processes in nanotechnology. So with the technology that we used, it's easy to make many of these pores in parallel if a semiconductor plant desired to do so."
Rant added that work currently being done by companies like Oxford Nanopore on DNA sequencing could also be applied to protein detection, further speeding development of the technology.
"I think the technology that has been developed for DNA sequencing could be directly applied to the problems here," he said. "Like parallel readout, the electronics that are now being developed, and so on."
Indeed, Oxford Nanopore has said that it is pursuing nanopore-based protein detection in addition to its DNA work. While the company has not released any commercial nanopore protein detection devices, in January, a team including Oxford Nanopore researchers and company founder Hagan Bayley published a study in the Journal of the American Chemical Society on using nanopores linked to aptamers for protein detection.
Their approach differed from that of Rant and his colleagues in that they used an αHL protein pore with an aptamer attached to a cysteine residue near the entrance of the pore. Because the αHL pore, unlike the solid state pores used by the German team, is too small for proteins to enter, Bayley and colleagues attempted to detect binding by measuring changes in current caused by aptamer-protein binding outside the pore.
In experiments using thrombin-binding aptamers, the researchers wrote that they were able to quantify nanomolar concentrations of thrombin, suggesting that "aptamer-based nanopores have the potential to be integrated into arrays for the parallel detection of multiple analytes."
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