Biosensing with conically shaped nanopores and nanotubes

In this review we consider recent results from our group that are directed towards developing "smart" synthetic nanopores that can mimic the functions of biological nanopores (transmembrane proteins). We first discuss the preparation and characterization of conical nanopores synthesized using the track-etch process. We then consider the design and function of conical nanopores that can rectify the ionic current that flows through these pores under an applied transmembrane potential. Finally, two types of sensors that we have developed with these conical nanopores are described. The first sensor makes use of molecular recognition elements that are bound to the nanopore mouth to selectively block the nanopore tip, thus detecting the presence of the analyte. The second sensor makes use of conical nanopores in a resistive-pulse type experiment, detecting the analyte via transient blockages in ionic current.     

Solid-state nanopores for ion and small molecule analysis

Solid-state nanopore in analytical chemistry has developed rapidly in the 1990s and it is proved to be a versatile new tool for bioanalytical chemistry. The research field of solid-state nanopore starts from mimicking the biological nanopore in living cells. Understanding the transport mechanism of biological nanopore in vivo is a big challenge because of the experimental difficulty, so it is essential to establish the basic research of artificial nanopores in vitro especially for the analysis of ions and small molecules. The performance of solid-state nanopores could be evaluated by monitoring currents when ions and molecules passed through. The comparison of the two types of nanopores based on current-derived information can reveal the principle of biological nanopores, while the solid-state nanopores are applied into practical bioanalysis. In this review, we focus on the researches of the solid-state nanopores in the fabrication process and in the analysis of ions and small molecules. Fabrication methods of nanopores, ion transport mechanism, small molecule analysis and theoretical studies are discussed in detail.     

Solid-state nanopores for ion and small molecule analysis

Solid-state nanopore in analytical chemistry has developed rapidly in the 1990s and it is proved to be a versatile new tool for bioanalytical chemistry. This review focuses on the analysis of ions and small molecules with nanopores including nanopipettes, polymer film nanopores, Si₃N₄ nanopores, graphene nanopores, MoS₂ nanopores and MOFs.     

Electrokinetic transport through the nanopores in cell membrane during electroporation

In electroporation, applied electric field creates hydrophilic nanopores in a cell membrane that can serve as a pathway for inserting biological samples to the cell. It is highly desirable to understand the ionic transfer and fluid flow through the nanopores in order to control and improve the cell transfection. Because of submicron dimensions, conventional theories of electrokinetics may lose their applicability in such nanopores. In the current study, the Poisson-Nernst-Planck equations along with modified Navier-Stokes equations and the continuity equation are solved in order to find electric potential, fluid flow, and ionic concentration through the nanopores. The results show that the electric potential, velocity field, and ionic concentration vary with the size of the nanopores and are different through the nanopores located at the front and backside of the cell membrane. However, on a given side of the cell membrane, angular position of nanopores has fewer influences on liquid flow and ionic transfer. By increasing the radius of the nanopores, the averaged velocity and ionic concentration through the nanopores are increased. It is also shown that, in the presence of electric pulse, electrokinetic effects (electroosmosis and electrophoresis) have significant influences on ionic mass transfer through the nanopores, while the effect of diffusion on ionic mass flux is negligible in comparison with electrokinetics. Increasing the radius of the nanopores intensifies the effect of convection (electroosmosis) in comparison with electrophoresis on ionic flux.     

Nucleobase recognition at alkaline pH and apparent pKa of single DNA bases immobilised within a biological nanopore

The four DNA bases are recognized in immobilized DNA strands at high alkaline pH by nanopore current recordings. Ionic currents through the biological nanopores are also employed to measure the apparent pK(a) values of single nucleobases within the immobilised DNA strands.     

平行四面体纳米孔道中DNA超结构的组装和离子输运特性

<正>受生物膜离子通道结构和功能的启发,人工制备固体纳米孔道门控开关器件一直备受关注[1,2].基于仿生纳米孔道的非对称离子传输性质制备的离子二极管和场效应管装置对于构建离子电路和能量转换的纳米器件至关重要[3,4].然而,仿生制备的固体纳米孔道在离子传输过程中有漏电流的存在,严重影响了固体纳米孔道应用的灵敏度和信噪比[5].针对这一问题,研究者利用DNA分子的特殊识别和自组装的功能特性,相继构筑了基于DNA和纳米孔道的智能响应体系[6,7].但在之前的研究工作中,分[8]     

Modulating ion current rectification generating high energy output in a single glass conical nanopore channel by concentration gradient

Inspired by biological systems that have the inherent skill to generate considerable bioelectricity from the salt content in fluids with highly selective ion channels and pumps on cell membranes,herein,a fully abiotic,single glass conical nanopores energy-harvesting is demonstrated.Ion current rectification(ICR)in negatively charged glass conical nanopores is shown to be controlled by the electrolyte concentration gradient depending on the direction of ion diffusion.The degree of ICR is enhanced with the increasing forward concentration difference.An unusual rectification inversion is observed when the concentration gradient is reversely applied.The maximum power output with the individual nanopore approaches10~4pW.This facile and cost-efficient energy-harvesting system has the potential to power tiny biomedical devices or construct future clean-energy recovery plants.     

Protein biosensors based on biofunctionalized conical gold nanotubes

There is increasing interest in the concept of using nanopores as the sensing elements in biosensors. The nanopore most often used is the alpha-hemolysin protein channel, and the sensor consists of a single channel embedded within a lipid bilayer membrane. An ionic current is passed through the channel, and analyte species are detected as transient blocks in this current associated with translocation of the analyte through the channel-stochastic sensing. While this is an extremely promising sensing paradigm, it would be advantageous to eliminate the very fragile lipid bilayer membrane and perhaps to replace the biological nanopore with an abiotic equivalent. We describe here a new family of protein biosensors that are based on conically shaped gold nanotubes embedded within a mechanical and chemically robust polymeric membrane. While these sensors also function by passing an ion current through the nanotube, the sensing paradigm is different from the previous devices in that a transient change in the current is not observed. Instead, the protein analyte binds to a biochemical molecular-recognition agent at the mouth of the conical nanotube, resulting in complete blockage of the ion current. Three different molecular-recognition agents, and correspondingly three different protein analytes, were investigated: (i) biotin/streptavidin, (ii) protein-G/immunoglobulin, and (iii) an antibody to the protein ricin with ricin as the analyte.     

Concentration-gradient-dependent ion current rectification in charged conical nanopores

Ion current rectification (ICR) in negatively charged conical nanopores is shown to be controlled by the electrolyte concentration gradient depending on the direction of ion diffusion. The degree of ICR is enhanced with the increasing forward concentration difference. An unusual rectification inversion is observed when the concentration gradient is reversely applied. A numerical simulation based on the coupled Poisson and Nernst-Planck (PNP) equations is proposed to solve the ion distribution and ionic flux in the charged and structurally asymmetric nanofluidic channel with diffusive ion flow. Simulation results qualitatively describe the diffusion-induced ICR behavior in conical nanopores suggested by the experimental data. The concentration-gradient-dependent ICR enhancement and inversion is attributed to the cooperation and competition between geometry-induced asymmetric ion transport and the diffusive ion flow. The present study improves our understanding of the ICR in asymmetric nanofluidic channels associated with the ion concentration difference and provides insight into the rectifying biological ion channels.     

Chemically-modified nanopores for sensing

Sensing with chemically-modified nanopores is an emerging field that is expected to have major impact on bioanalysis and fundamental understanding of nanoscale chemical interactions down to the single-molecule level. The main strength of nanopore sensing is that it implies the prospect of label-free single-molecule detection by taking advantage of the built-in transport-modulation-based amplification mechanism. At present, fabrication and application of solid-state nanopores are becoming the focus of attention because, compared with their biological counterparts, they offer greater flexibility in terms of shape, size, and surface properties, as well as superior robustness. A breakthrough in label-free nanopore sensing for real-world applications is therefore expected from implementing solid-state nanopores, an area that is still developing. Without claiming comprehensiveness, the focus of this review comprises recent results and trends in nanopore-based sensing (i.e. emerging technologies for fabricating solid-state nanopores, their chemical functionalization, and detection methods for quantitative analysis).     

基于仿生膜的功能化单纳米通道在分析化学中的应用

灵感来源于蛋白质离子通道的仿生功能化单纳米通道,已逐渐成为一种成熟的单分子检测技术和离子整流器。功能化纳米通道包括两种:基因改造的蛋白质纳米通道和固体加工的纳米通道。常用的固体纳米通道有三种:在纳米氮化硅或石墨烯上用聚焦离子束(FIB)或电子束(FEB)轰击得到的纳米通道,化学腐蚀聚合物薄膜中的重金属离子轨迹得到的锥形纳米通道和拉制毛细管或激光刻蚀得到的玻璃纳米孔。相对于蛋白质纳米通道,固态的人工纳米通道具有更优越的机械稳定性,并可用于各种功能基团的修饰。经过近十年的发展,包括蛋白质纳米通道在内的各种仿生的纳米通道已广泛用于对小分子、蛋白质和聚合物等其他一些对象的定性和定量检测。本综述详细介绍了近年来国内外该领域的发展,并对未来的发展方向作了简要的展望。     

How the asymmetry of internal potential influences the shape of I-V characteristic of nanochannels

Ion transport in biological and synthetic nanochannels is characterized by such phenomena as ion current fluctuations, rectification, and pumping. Recently, it has been shown that the nanofabricated synthetic pores could be considered as analogous to biological channels with respect to their transport characteristics [P. Yu. Apel et al., Nucl. Instrum. Methods Phys. Res. B 184, 337 (2001); Z. Siwy et al., Europhys. Lett. 60, 349 (2002)]. The ion current rectification is analyzed. Ion transport through cylindrical nanopores is described by the Smoluchowski equation. The model is considering the symmetric nanopore with asymmetric charge distribution. In this model, the current rectification in asymmetrically charged nanochannels shows a diodelike shape of I-V characteristic. It is shown that this feature may be induced by the coupling between the degree of asymmetry and the depth of internal electric potential well. The role of concentration gradient is discussed.     

Translocation of biomolecules through solid‐state nanopores: Theory meets experiments

The interest in polynucleotide translocation through nanopores has moved from purely biological to the need of realizing nanobiotechnological applications related to personalized genome sequencing. Polynucleotide translocation is a process in which biomolecules, like DNA or RNA, are electrophoretically driven through a narrow pore and their passage can be monitored by the change in the ionic current through the pore. Such a translocation process, which will be described here offers a very promising technology aiming at ultra‐fast low‐cost sequencing of DNA, though its realization is still confronted with challenges and drawbacks. In this review, we present the main aspects involved in the polynucleotide translocation through solid‐state nanopores by discussing the most relevant experimental, theoretical, and computational approaches and the way these can supplement each other. The discussion will expose the goals that have been reached so far, the open questions, and contains an outlook to the future of nanopore sequencing. © 2011 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 49: 985–1011, 2011     

Ion current rectification and rectification inversion in conical nanopores: a perm-selective view

Ionic transport in charged conical nanopores is known to give rise to ion current rectification. The present study shows that the rectification direction can be inverted when using electrolyte solutions at very low ionic strengths. To elucidate these phenomena, electroneutral conical nanopores containing a perm-selective region at the tip have been investigated and shown to behave like classical charged nanopores. An analytical model is proposed to account for these rectification processes.     

Protein Detection Through Single Molecule Nanopore

A single nanopore represents a versatile single-molecule probe that can be employed to reveal several important features of proteins, such as physical structure, backbone flexibility, mechanical stability, their folding state, binding affinity to other interacting ligands and enzymatic activity. In this review, we summarize the development and current research related to the field of protein detection by nanopore, as well as a few examples of the pioneer work on protein detection. We first discuss the principle of electrical detection with nanopores and how this technique provides information from current traces. Then the development from peptide detection with biological nanopore to protein detection through solid-state nanopore is described. Finally, we prospect the measurement of protein shape and construction using nanopore technology for the applications in life research area.     

Surface chemical functionalization of cylindrical nanopores derived from a polystyrene-poly(methylmethacrylate) diblock copolymer via amidation

This paper demonstrated covalent functionalization of surface -COOH groups on cylindrical nanopores derived from a polystyrene-poly(methylmethacrylate) diblock copolymer (PS- b-PMMA) via amidation mediated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). The surface functionalization led to conversion of the surface charge of the nanopores and also to the shrinkage of effective pore radius, as verified using cyclic voltammetry for PS- b-PMMA-derived nanoporous films immobilized on gold substrates. For native PS- b-PMMA-derived nanoporous films, the redox current of anionic Fe(CN) 6 (3-) decreased with increasing solution pH due to the deprotonation of the surface -COOH groups, whereas those of cationic Ru(NH 3) 6 3+ and uncharged 1,1'-ferrocenedimethanol (Fc(CH 2OH) 2) were similar regardless of pH. In contrast, upon EDC-mediated amidation of the nanopore surface with ethylenediamine, the redox current of Ru(NH 3) 6 3+ decreased with decreasing pH and those of Fe(CN) 6 (3-) and Fc(CH 2OH) 2 were independent of pH. The decrease in redox current of Ru(NH 3) 6 3+ at acidic pH was consistent with the presence of -NH 2 groups on the nanopore surface as a result of the covalent immobilization of ethylenediamine. Furthermore, the redox current of Fc(CH 2OH) 2 decreased upon amidation of the nanopores with tetraethyleneglycol monoamine ((PEO) 4NH 2), reflecting the shrinkage of the effective pore radius. The control of the surface charge and effective radius of the nanopores via EDC-mediated amidation will provide a simple means for controlling the selectivity of molecular mass transport through PS- b-PMMA-derived nanopores.     

Ion current rectification at nanopores in glass membranes

The origin of ion current rectification observed at conical-shaped nanopores in glass membranes immersed in KCl solutions has been investigated using finite-element simulations. The ion concentrations and fluxes (due to diffusion, migration, and electroosmotic convection) were determined by the simultaneous solution of the Nernst-Planck, Poisson, and Navier-Stokes equations for the two-ion (K+ and Cl-) system. Fixed surface charge on both the internal and external glass surfaces that define the pore structure was included to account for electric fields and nonuniform ion conductivity within the nanopores and electric fields in the external solution near the pore mouth. We demonstrate that previous observations of ion current rectification in conical-shaped glass nanopores are a consequence of the voltage-dependent solution conductivity in the vicinity of the pore mouth, both inside and outside of the pore. The simulations also demonstrate that current rectification is maximized at intermediate bulk ion concentrations, a combination of (i) the electrical screening of surface charge at high concentrations and (ii) a fixed number of charge-carrying ions in the pore at lower concentration, which are physical conditions where the voltage dependence of the conductivity disappears. In addition, we have quantitatively shown that electroosmotic flow gives rise to a significant but small contribution to current rectification.     

Surface-charge induced ion depletion and sample stacking near single nanopores in microfluidic devices

We report integrated nanopore/microchannel devices in which single nanopores are isolated between two microfluidic channels. The devices were formed by sandwiching track-etched conical nanopores in a poly(ethylene terephthalate) membrane between two poly(dimethylsiloxane) microchannels. Integration of the nanopores into microfluidic devices improves mass transport to the nanopore and allows easy coupling of applied potentials. Electrical and optical characterization of these individual nanopores suggests double layer overlap is not required to form an ion depletion region adjacent to the nanopore in the microchannel; rather, excess surface charge in the nanopore contributes to the formation of this ion depletion region. We used fluorescent probes to optically map the ion depletion region and the stacking of fluorescein near the nanopore/microchannel junction, and current measurements confirmed formation of the ion depletion region.     

Characterization of nanoporous gold electrodes for bioelectrochemical applications

The high surface areas of nanostructured electrodes can provide for significantly enhanced surface loadings of electroactive materials. The fabrication and characterization of nanoporous gold (np-Au) substrates as electrodes for bioelectrochemical applications is described. Robust np-Au electrodes were prepared by sputtering a gold-silver alloy onto a glass support and subsequent dealloying of the silver component. Alloy layers were prepared with either a uniform or nonuniform distribution of silver and, post dealloying, showed clear differences in morphology on characterization with scanning electron microscopy. Redox reactions under kinetic control, in particular measurement of the charge required to strip a gold oxide layer, provided the most accurate measurements of the total electrochemically addressable electrode surface area, A(real). Values of A(real) up to 28 times that of the geometric electrode surface area, A(geo), were obtained. For diffusion-controlled reactions, overlapping diffusion zones between adjacent nanopores established limiting semi-infinite linear diffusion fields where the maximum current density was dependent on A(geo). The importance of measuring the surface area available for the immobilization was determined using the redox protein, cyt c. The area accessible to modification by a biological macromolecule, A(macro), such as cyt c was reduced by up to 40% compared to A(real), demonstrating that the confines of some nanopores were inaccessible to large macromolecules due to steric hindrances. Preliminary studies on the preparation of np-Au electrodes modified with osmium redox polymer hydrogels and Myrothecium verrucaria bilirubin oxidase (MvBOD) as a biocathode were performed; current densities of 500 μA cm(-2) were obtained in unstirred solutions.     

Chiral Selective Transport of Proteins by Cysteine‐Enantiomer‐Modified Nanopores

Chirality is an intriguing and intrinsic feature of life and is highly associated with many significant biological processes. However, whether it influences the translocation behavior of proteins remains unclear. Herein, based on biomimetic strategies, we made chiral nanopores modified with cysteine enantiomers, and studied the chirality gating effects on protein transport. The results show that protein is preferentially transported through nanopores modified with l ‐cysteine because of chiral interaction, indicating chirality strongly influences protein transport process. This study presents a new method for better understanding the role of chirality in selective protein transport processes and provides a convenient approach for studying protein chiral separation and targeted treatments.