Engineering nanopore sensors for proteomic and metabolomic analysis

Abstract

Single-molecule analysis is a new emerging field of science, allowing the detection of analytes on the smallest biologically-relevant scale. 28 Whereas most methods require an ensemble of molecules to interact with the detector, single-molecule techniques require theoretically only one.In recent years, single-molecule analysis has excelled in oligonucleotide sequencing, maturing into commercial products capable of sequencing long stretches of oligonucleotides. These techniques are collectively known as third-generation (DNA) sequencing, not to be confused with NGS which also includes second-generation sequencing techniques. At the time of writing, two third-generation methods have reached the stage of commercialisation, namely: zero-mode waveguided sequencing by synthesis, and nanopore based sequencing.The first technique, zero-mode waveguided sequencing by synthesis, utilises nanostructures smaller than the wavelength of light in order to reduce the detection volume to less than the diffraction limit of visible light. 29 A polymerase is immobilised in the nanostructure, which elongates single stranded DNA using fluorescently labelled nucleotides. 30 The nucleotides are added in 0.1 to 10 µM range, such that (on average) there is only a single nucleotide present in the sampling volume which is in the attoto zeptoliter range (10 -18 -10 -23 L). The nucleotides are excited using a laser, causing nucleotide emission which is optically detected. The time of emission is an indication of binding, as unbound nucleotides enter and exit the sampling volume more rapidly. The laser excitation is, however, the main limitation of the system, as this causes photo-oxidation of the polymerase, rendering it inactive over-time.Contrary to other sequencing techniques, nanopores provide a polymerase-and label-free platform.The principle working of nanopore based sequencing is similar to nanometer-sized Coulter counters.They allow the differentiation of nucleotides (and other molecules)-due to ion displacement-based on the size and interaction of the analyte with the nanopore and it's environment. 31 For the purpose of oligonucleotide sequencing, an ATP-dependent helicase (e.g. Phi29) is brought on top of a nanopore (e.g. an engineered α-hemolysin or Mycobacterium smegmatis porin A nanopore). 32 The nucleobases are fed through the pore using the helicase, controlling speed by ATP concentration, and the nucleobases are read as a decrease in ionic current. Nucleobases translocating through the nanopore are not measured one-by-one. At any given time, there will be several nucleobases excluding ionic current, such that a chain of nucleobases is measured. Sequencing is performed by re-constructing the most probable sequence of nucleotides causing the excluded current over time.Events•s -1 where β > 0 Events•s -1 where β > 1 Events•s -1 where β > 10