Yi-Lun Ying

Nanopore-Based Sequencing and Detection of Nucleic Acids

Nanopore-based techniques, which mimic the functions of natural ion channels, have attracted increasing attention as unique methods for single-molecule detection. The technology allows the real-time, selective, high-throughput analysis of nucleic acids through both biological and solid-state nanopores. In this Minireview, the background and latest progress in nanopore-based sequencing and detection of nucleic acids are summarized, and light is shed on a novel platform for nanopore-based detection.

Nanopore Analysis of β-Amyloid Peptide Aggregation Transition Induced by Small Molecules

β-Amyloid 42 (Aβ42) is the predominant form of the amyloid peptide, which is found in the plaques of the brains of Alzheimers (AD) patients and is one of the most abundant components in amyloid aggregates. Information of the Aβ42 aggregation states is essential for developing an understanding of the pathologic process of amyloidoses. Here, we used α-hemolysin (α-HL) pores to probe the different aggregation transition of Aβ42 in the presence of β-cyclodextrin (β-CD), a promoter of Aβ42 aggregations, and in the presence of Congo red (CR), an inhibitor of aggregations. Analyzing the characteristic transit duration times and blockade currents showed that β-CD and CR have opposite effects on the aggregation of Aβ42. Translocation events of the monomeric Aβ42 peptide were significantly lower in amplitude currents than protofilaments, and protofilaments were captured in the α-HL nanopore with a longer duration time. CR binds to Aβ42 and its peptide fibrils by reducing the aggregated fibrils formation. In this process it is assumed CR interferes with intermolecular hydrogen bonding present in the aggregates. In contrast to CR, β-CD promotes the aggregation of Aβ42. These differences can readily be analyzed by monitoring the corresponding characteristic blockade events using a biological α-HL nanopore.

Chrominance to Dimension: A Real-Time Method for Measuring the Size of Single Gold Nanoparticles

Noble metal nanoparticles have excellent optical and chemical properties and are widely used in optics, sensors, and biomedicines. The inherent characteristics of metal nanoparticles, particularly their size, play important roles in their applications. The ability to readily measure the size of single nanomaterials on-site is crucial to the rapid development of single-particle sensors. In this study, we developed a facile and real-time method for estimating the diameter of single gold nanoparticles (GNPs) that range from 35 to 110 nm in diameter; this technique uses the chrominance of the GNPs plasmon resonance scattering light that is captured by a dark-field microscope (DFM). The RGB (three primary colors, red, green, and blue) chrominance information from the dark-field image can be directly converted into the diameters of the GNPs using the relationship between the particle size and the scattering light peak wavelength; this conversion was carried out using Matlab program based on an RGB-To-Wavelength (RTW) process. This approach is more convenient, less time-consuming, and enables observation under arbitrary conditions compared to the scanning electron microscopy (SEM) technique. The differences between the diameters of the GNPs that were calculated using this method and those that were measured using SEM were less than 5 nm. The RTW method has also been applied in the monitoring of the refractive index of the media surrounding the GNPs, and their dynamic acting within cells in real-time.

A Stimuli-Responsive Nanopore Based on a Photoresponsive Host-Guest System

The open-close states of the ion channels in a living system are regulated by multiple stimuli such as ligand, pH, potential and light. Functionalizing natural channels by using synthetic chemistry would provide biological nanopores with novel properties and applications. Here we use para-sulfonato-calix[4]arene-based host-guest supramolecular system to develop artificial gating mechanisms aiming at regulating wild-type α-HL commanded by both ligand and light stimuli. Using the gating property of α-hemolysin, we studied the host-guest interactions between para-sulfonato-calix[4]arene and 4, 4′-dipyridinium-azobenzene at the single-molecule level. Subsequently, we have extended the application of this gating system to the real-time study of light-induced molecular shuttle based on para-sulfonato-calix[4]arene and 4, 4′-dipyridinium-azobenzene at the single-molecule level. These experiments provide a more efficient method to develop a general tool to analyze the individual motions of supramolecular systems by using commercially available α-HL nanopores.

Investigating electron-transfer processes using a biomimetic hybrid bilayer membrane system

Here we report a protocol to investigate the electron-transfer processes of redox-active biomolecules in biological membranes by electrochemistry using biomimetic hybrid bilayer membranes (HBMs) assembled on gold electrodes. Redox-active head groups, such as the ubiquinone moiety, are embedded in HBMs that contain target molecules, e.g., nicotinamide adenine dinucleotide (NADH). By using this approach, the electron-transfer processes between redox molecules and target biomolecules are mediated by mimicking the redox cycling processes in a natural membrane. Also included is a procedure for in situ surface-enhanced Raman scattering (SERS) to confirm the electrochemically induced conformational changes of the target biomolecules in the HBMs. In addition, each step in constructing the HBMs is characterized by electrochemical impedance spectroscopy (EIS), high-resolution X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). The time required for the entire protocol is ∼12 h, whereas the electrochemical measurement of electron-transfer processes takes less than 1 h to complete.

Accurate Data Process for Nanopore Analysis

Data analysis for nanopore experiments remains a fundamental and technological challenge because of the large data volume, the presence of unavoidable noise, and the filtering effect. Here, we present an accurate and robust data process that recognizes the current blockades and enables evaluation of the dwell time and current amplitude through a novel second-order-differential-based calibration method and an integration method, respectively. We applied the developed data process to analyze both generated blockages and experimental data. Compared to the results obtained using the conventional method, those obtained using the new method provided a significant increase in the accuracy of nanopore measurements.

In situ high throughput scattering light analysis of single plasmonic nanoparticles in living cells.

Plasmonic nanoparticles have been widely applied in cell imaging, disease diagnosis, and photothermal therapy owing to their unique scattering and absorption spectra based on localized surface plasmon resonance (LSPR) property. Recently, it is still a big challenge to study the detailed scattering properties of single plasmonic nanoparticles in living cells and tissues, which have dynamic and complicated environment. The conventional approach for measuring the scattering light is based on a spectrograph coupled to dark-field microscopy (DFM), which is time-consuming and limited by the small sample capacity. Alternatively, RGB-based method is promising in high-throughput analysis of single plasmonic nanoparticles in dark-field images, but the limitation in recognition of nanoparticles hinders its application for intracellular analysis. In this paper, we developed an automatic and robust method for recognizing the plasmonic nanoparticles in dark-field image for RGB-based analysis. The method involves a bias-modified fuzzy C-means algorithm, through which biased illumination in the image could be eliminated. Thus, nearly all of the gold nanoparticles in the recorded image were recognized both on glass slide and in living cells. As confirmed, the distribution of peak wavelength obtained by our method is well agreed to the result measured by conventional method. Furthermore, we demonstrated that our method is profound in cell imaging studies, where its advantages in fast and high-throughput analysis of the plasmonic nanoparticles could be applied to confirm the presence and location of important biological molecules and provide efficiency information for cancer drug selection.

Single molecule analysis of light-regulated RNA:spiropyran interactions

The photo-regulated interactions between an RNA aptamer and photochromic spiropyran were investigated at a single-molecule level via an α-hemolysin nanopore. Upon irradiation of alternating UV/visible light, the translocation process of the RNA aptamer could be optically tuned on account of its different binding affinity with two photoisomers, spiropyran and merocyanine. This provides a general analytic model for understanding the mechanism of a photo-regulated biomolecule conformational change at a single-molecule level.

Single-molecule analysis in an electrochemical confined space

Electrochemical analysis of single molecules is a method with the strong ability of the enhanced efficiency and ultra-sensitivity. Here, we demonstrate that the electrochemical confined space could efficiently convert single molecule characteristics into measurable electrochemical signatures with high temporal resolution. The human telomere repeat sequence T8 was used as a probe to determine the electrochemical confined effect in a nanopore. Our results show that the nanopore with comparable confined space of the telomere repeat sequence exhibits the most distinguishable single-molecule signals which suggest the folded conformation of T8. This method will greatly extend the lifetime of a metastable conformation for a single biomolecule by strong analyte-nanopore interactions, which brings the new insight into the understanding of the biomolecule’s function at single-molecule level.

Driven Translocation of Polynucleotides Through an Aerolysin Nanopore

Aerolysin has been used as a biological nanopore for studying peptides, proteins, and oligosaccharides in the past two decades. Here, we report that wild-type aerolysin could be utilized for polynucleotide analysis. Driven a short polynucleotide of four nucleotides length through aerolysin occludes nearly 50% amplitude of the open pore current. Furthermore, the result of total internal reflection fluorescence measurement provides direct evidence for the driven translocation of single polynucleotide through aerolysin.