Single-Molecule Tools for Bioanalysis

Shuo Huang

• Publisher: Jenny Stanford Publishing
• Year: 2022
• eBook ISBN: 9781000409215
• Topic: Médecine
• Subtopic: Biotechnologie en médecine

In the last three decades, the fast development of single-molecule techniques has revolutionized the way we observe and understand biological processes. Some of these techniques have been further adapted as tools for bioanalysis. This book summarizes and details the frontiers of the development of these tools as well as their applications. The contributors are young and established researchers in their respective fields. The main content originates from the lecture notes of a chemistry graduate course taught by the book editor at Nanjing University. This book is suitable to be used as a textbook for a high-level undergraduate or an entry-level graduate course. The systematically written content provides a thorough illustration of the mechanisms of each methodology presented.

Chapter 1 Single-Molecule Analysis by Biological Nanopores

Yuqin Wang and Shuo Huang

School of Chemistry and Chemical Engineering, Nanjing University, China

State Key Laboratory of Analytical Chemistry for Life Science, Nanjing, China

Chemistry and Biomedicine Innovation Center (ChemBIC), Nanjing, China

[email protected]

Biological nanopores are a type of proteins which form pores and were developed for in vitro single-molecule sensing. In view of their ease of use, consistency and precision of preparation, biological nanopores can be further engineered or modified for highly specialized sensing applications. Using single-channel recording, the identity of an analyte is reported from its interaction with the pore restriction during its translocation. Being geometrically compatible with single-stranded DNA or single-stranded RNA, biological nanopores, such as α-hemolysin (α-HL) or Mycobacterium smegmatis porin A (MspA), have long been considered the most promising candidates for third-generation sequencing. After research of ~3 decades, the prototype of a nanopore sequencer was first demonstrated in 2012 and is now widely used in a variety of genomics research programs. Sustained research of nanopore sequencing has also stimulated its other applications, such as sensing of single ions, small molecules, macromolecules, biomacromolecules, or their combinations. In this chapter, we introduce the mechanism and the methodology of the biological nanopore techniques along with a tutorial protocol. We hope the reader will benefit from reading this chapter by successfully carrying out a highly simplified nanopore measurement or becoming inspired for their own research.

1.1 Introduction

1.1.1 Single-Molecule Biophysics and Nanopore

The field of single-molecule biophysics lies at the interface of physics, biology, and chemistry; its aim is to understand the mechanism of biological phenomena on a single-molecule scale. Propelled by the dramatic improvement of modern technologies such as patch-clamp technology [1], electron microscopy (EM) [2], scanning probe microscopy (SPM) [3], optical tweezers [4], magnetic tweezers [5], single-molecule fluorescence [6], super resolution microscopy [7], Förster resonance energy transfer [8], and total internal reflection fluorescence microscopy [9], researchers can now easily sense or even visualize unprecedented insights into enzyme kinetics [10], conformational dynamics [11], protein folding kinetics [12], breaking of chemical bonds [13], and ligand-binding activities [14] in a single molecule.

Each of the single-molecule methods mentioned above is irreplaceable, but not universal. For particular single-molecule applications, the proper selection of the methodology becomes critical for success. Nanopore technology is a unique method which is particularly suitable as a sensor for chain-shaped and electrically charged polymers such as nucleic acids [15]. The predecessor of the nanopore method could be traced back to the invention of the patch-clamp technology in the 1970s, which described the first instrument enabling human beings to monitor single ion channel activities on cell membranes [1]. This invention gained Neher and Sakmann the Nobel Prize in physiology or medicine in 1991 and is now widely used as a tool for electrophysiology studies of transmembrane porins or ion channels. The nanopore method, which conceptually originated from patch-clamp measurements, determines molecular identities by probing pore blockage events caused by molecular interactions of an analyte with a nanopore sensor (Figure 1.1A) [16]. In a typical nanopore measurement, a strand of a single-molecule analyte (e.g. a piece of single-stranded DNA [ssDNA]) electrophoreses through the nanoscopic aperture, generating a transient resistive pulse signal containing the molecular identity information (Figure 1.1B,C). This molecular transport process through a nanopore is termed a “translocation” event. Molecular identities are recognized by analyzing the trace fluctuations caused by the molecular translocation. The nanopore method is so sensitive that detection of subtle differences between analyte molecules is possible, making it an efficient single-molecule sensor like a miniaturized Coulter counter [17].

Figure 1.1 The origin of nanopore and its DNA-sensing applications. (A) The crystal structure of a heptameric α-HL nanopore. The heptameric pore appears with a mushroom shape, consisted of a wider cap (vestibule) and a narrower stem (β-barrel). The stem, which is composed of 14 antiparallel β strands, forms a cylindrical channel with a 2.6 nm diameter, permitting translocation of only ssDNA instead of dsDNA. (B) Traditional nanopore apparatus. A single α-HL nanopore can spontaneously insert into a freestanding lipid bilayer forming the only conducting path across the membrane. Analytes such as ssDNA are electrophoretically driven to pass through the pore, giving rise to the appearance of resistive pulses caused by the analyte. Reproduced with permission from ref. [18], Copyright (1999) The Biophysical Society. (C) Characteristic blockades of poly(U) translocation through an α-HL nanopore. Reproduced with permission from ref. [19], Copyright (1996) National Academy of Sciences, USA.

The nanopore measurement has advantages over other optics-based single-molecule methods because it monitors ionic current instead of photon counts. Due to limited photon emissions from fluorophores, single-molecule methods based on fluorescent microscopy such as single-molecule fluorescence resonance energy transfer (smFRET), normally produce noisy data which may limit its sensing resolution [8]. For a nanopore device based on natural ion channels, the measurement range is between 1 pA and 200 pA, which is equivalent to acquisition of 6.25–1250 million ions per second.

$\left(1\times {10}^{12}A\right)\times \frac{1s}{1.60\times {10}^{-19}C}=6.25\times {10}^{6}ions$

This amount of charge transport can be reliably amplified and measured by a patch-clamp amplifier with a satisfactory signal-to-noise ratio, but prolonged excitation causes severe photo bleaching of the fluorophore, limiting the duration of the measurement. On the other hand, the nanopore device can withstand hours, or days of continuous measurement.

1.1.2 Nanopore Methods

A nanopore sensor could be generally defined as a nanoscale aperture in an impermeable membrane connecting two chambers containing electrolyte solution. A wide range of materials and methods can be utilized to make nanopore devices with different geometries and properties. An ideal nanopore sensor has to be structurally stable and geometrically consistent. To fit the cross-sectional area of a single biomacromolecule, the size of a useful nanopore sensor is normally between 1 nm and 10 nm in diameter [20]. However, nanofabrication techniques in the 1990s cannot yet reliably produce such delicate a structure over an artificial material. Until 1996, the structural determination of the Staphylococcus aureus α-HL by X-ray crystallography [16] suggested a biomimetic strategy to produce nanopores, and this later became an initiator of all subsequent nanopore researches.

In general, nanopore devices can be further classified into “biological nanopores” and “solid-state nanopores.” All biological nanopores originate from natural transmembrane porins or their mimics, which can spontaneously penetrate a natural biomembrane or an artificial lipid bilayer and generate ion or molecular passages across the insulating membrane for biological sensing. Biological nanopores could be massively prepared on a large scale by standard molecular biology protocols such as prokaryotic or in vitro protein expression followed with the appropriate purification steps. Though naturally composed of amino acids, biological nanopores when stored properly can stay active for a few years with no noticeable difference during measurements. It has also been experimentally verified that a biological nanopore such as an α-HL can survive an extreme of salt concentration [21], temperature [22], pH [23], and denaturants [24–26] during measurements. However, it is the fragile lipid bilayer or biomembrane which is unable to withstand harsh measurement conditions such as a high applied electrical bias, violent mechanical vibrations, or the presence of strong detergents.

Alternatively, solid-state nanopores, which are porin mimics artificially fabricated on solid-state thin materials, were developed later and aimed to provide a more durable, silicon industry-compatible solution with a complete freedom of design flexibility. Various methods such as focused ion beam [27], electron beam [28], track-guided chemical etching [29], and dielectric breakdown [30] could be used for pore drilling. Solid-state nanopore techniques offer advantages of a more flexible pore geometry and patterning along with a variety of surface property modifications but suffers from a poor biosensing performance due to the inconsistency of pore manufacturing at the nanome...

Table of contents

• Cover Page
• Half-Title Page
• Title Page
• Copyright Page
• Table of Contents
• Preface
• Acknowledgements
• 1. Single-Molecule Analysis by Biological Nanopores
• 2. Optical Tweezers for Manipulation of Single Molecules
• 3. Single-Molecule Biosensing by Fluorescence Resonance Energy Transfer
• 4. DNA Origami as Single-Molecule Biosensors
• 5. Single-Molecule Manipulation by Magnetic Tweezers
• 6. Long-Time Recording of Single-Molecule Dynamics in Solution by Anti-Brownian Trapping
• Index