RESUMO
The application of solid-state (SS) nanopore devices to single-molecule nucleic acid sequencing has been challenging. Thus, the early successes in applying SS nanopore devices to the more difficult class of biopolymer, glycosaminoglycans (GAGs), have been surprising, motivating us to examine the potential use of an SS nanopore to analyze synthetic heparan sulfate GAG chains of controlled composition and sequence prepared through a promising, recently developed chemoenzymatic route. A minimal representation of the nanopore data, using only signal magnitude and duration, revealed, by eye and image recognition algorithms, clear differences between the signals generated by four synthetic GAGs. By subsequent machine learning, it was possible to determine disaccharide and even monosaccharide composition of these four synthetic GAGs using as few as 500 events, corresponding to a zeptomole of sample. These data suggest that ultrasensitive GAG analysis may be possible using SS nanopore detection and well-characterized molecular training sets.
Assuntos
Heparitina Sulfato/química , Aprendizado de Máquina , Nanoporos , Sequência de Carboidratos , Dissacarídeos/química , Glicômica/métodos , Glicômica/normas , Heparitina Sulfato/síntese química , Monossacarídeos/químicaRESUMO
A nanopore can be fairly-but uncharitably-described as simply a nanofluidic channel through a thin membrane. Even this simple structural description holds utility and underpins a range of applications. Yet significant excitement for nanopore science is more readily ignited by the role of nanopores as enabling tools for biomedical science. Nanopore techniques offer single-molecule sensing without the need for chemical labelling, since in most nanopore implementations, matter is its own label through its size, charge, and chemical functionality. Nanopores have achieved considerable prominence for single-molecule DNA sequencing. The predominance of this application, though, can overshadow their established use for nanoparticle characterization and burgeoning use for protein analysis, among other application areas. Analyte scope continues to be expanded, and with increasing analyte complexity, success will increasingly hinge on control over nanopore surface chemistry to tune the nanopore, itself, and to moderate analyte transport. Carbohydrates are emerging as the latest high-profile target of nanopore science. Their tremendous chemical and structural complexity means that they challenge conventional chemical analysis methods and thus present a compelling target for unique nanopore characterization capabilities. Furthermore, they offer molecular diversity for probing nanopore operation and sensing mechanisms. This article thus focuses on two roles of chemistry in nanopore science: its use to provide exquisite control over nanopore performance, and how analyte properties can place stringent demands on nanopore chemistry. Expanding the horizons of nanopore science requires increasing consideration of the role of chemistry and increasing sophistication in the realm of chemical control over this nanoscale milieu.
Assuntos
Glicômica , Nanoporos , Imagem Individual de Molécula/métodos , Técnicas Biossensoriais/métodosRESUMO
We developed a flow cell apparatus and method for streamlined, real-time measurements of nanopore conductance (G) in response to pH changes. By time-resolving the measurements of interfacial kinetics, we were able to probe nanopore surface coating presence and properties more thoroughly than in our previous work. Nanopores have emerged as a prominent tool for single-molecule sensing, characterization, and sequencing of DNA, proteins, and carbohydrates. Nanopore surface chemistry affects analyte passage, signal characteristics, and sensor lifetime through a range of electrostatic, electrokinetic, and chemical phenomena, and optimizing nanopore surface chemistry has become increasingly important. Our work makes nanopore surface chemistry characterizations more accessible as a complement to routine single-pH conductance measurements used to infer nanopore size. We detail the design and operation of the apparatus and discuss the trends in G and capacitance. Characteristic G vs pH curves matching those obtained in previous work could be obtained with the addition of time-resolved interfacial kinetic information. We characterized native and chemically functionalized (carboxylated) silicon nitride (SiNx) nanopores, illustrating how the method can inform of thin film compositions, interfacial kinetics, and nanoscale chemical phenomena.