A solid-state nanopore-based single-molecule approach for label-free characterization of plant polysaccharides

Abstract

Polysaccharides are important biomacromolecules existing in all plants, most of which are integrated into a fibrillar structure called the cell wall. In the absence of an effective methodology for polysaccharide analysis that arises from compositional heterogeneity and structural flexibility, our knowledge of cell wall architecture and function is greatly constrained. Here, we develop a single-molecule approach for identifying plant polysaccharides with acetylated modification levels. We designed a solid-state nanopore sensor supported by a free-standing SiN _x membrane in fluidic cells. This device was able to detect cell wall polysaccharide xylans at concentrations as low as 5 ng/μL and discriminate xylans with hyperacetylated and unacetylated modifications. We further demonstrated the capability of this method in distinguishing arabinoxylan and glucuronoxylan in monocot and dicot plants. Combining the data for categorizing polysaccharide mixtures, our study establishes a single-molecule platform for polysaccharide analysis, opening a new avenue for understanding cell wall structures, and expanding polysaccharide applications.

Keywords: acetylation; ion current; polysaccharides; single-molecule; solid-state nanopore.

Figure 1

Solid-state nanopore setup for plant polysaccharide xylan detection. (A) Schematic graph showing nanopore setup for plant polysaccharide detection. The red dashed arrow is the force direction and single xylan molecule translocation event. (B) The representative open pore currents generated in the experiments. The insert at the top left corner shows the transmission electron micrograph of a 4-nm nanopore. Scale bar, 2 nm. (C) Representative raw nanopore current traces and events at the indicated voltages. (D) Molecular formula of polysaccharide samples. The region between R1 and R2 is composed of repeating units that represent the extension of repeating units at the reducing and nonreducing ends. The red parts represent the acetyl group.

Figure 2

Discriminating native xylans from wild-type and hyperacetylated mutant plants. (A) Determination of acetate content in native xylans from wild-type Nipponbare (NP) and the hyperacetylated mutant brittle leaf sheath1 (bs1). ∗∗Statistical significance calculated using Student's t test at P < 0.01. (B) Partial proton NMR spectra showing increased acetyl signals of native xylans from bs1 than that from NP. (C) Partial HSQC spectra of native xylans from NP and bs1, showing purity and acetylation levels of native xylans. Arrows and arrowheads indicate the signals derived from xylosyl and acetylated xylosyl residues on xylan backbones, respectively. (D) The representative current traces of NP and bs1 xylans recorded at a voltage of 150 mV. (E) Scatterplots showing event distribution of NP and bs1 xylans probed at a voltage of 150 mV. (F) Comparison of the normalized current amplitude I_b/I₀ and dwell time values of NP and bs1 xylans at the indicated positive bias voltage. Data represent the mean ± SD of three biological replicates.

Figure 3

Analysis of deacetylated xylans from wild-type and bs1 plants. (A and B) (A) The representative current traces of deacetylated xylans from wild-type (NP-ac) and bs1 (bs1-ac) at a positive bias voltage of 150 mV; (B) scatterplots showing event distribution of NP-ac and the corresponding native NP xylan. (C) Scatterplots showing event distribution of bs1-ac and the corresponding native xylan of bs1. (D) Scatterplots showing event distribution of NP-ac and bs1-ac xylan. (E and F) Line charts of normalized current amplitude I_b/I₀ and dwell time between NP and NP-ac (E) and between bs1 and bs1-ac (F) at a series of positive bias voltages. Data represent the mean ± SD of three biological replicates. The data for native xylan in (E and F) are the same as in Figure 2F.

Figure 4

Perforation performance of xylans from the type I cell wall. (A) Diagrams of xylans from type I and II cell walls, which are variable in the side chain substitution with acidic (methyl) glucuronic acid (GX) or neutral arabinose, respectively. (B) Scatterplots showing event distribution of NP-ac and meGX xylan under 150 mV. (C) The normalized current amplitude I_b/I₀ histogram of meGX and NP-ac xylan under 150 mV. (D) The dwell time histograms of meGX and NP-ac xylan under 150 mV.