Zeng-Qiang Wu

Anomalous diffusion of electrically neutral molecules in charged nanochannels.

Diffusion is the principal means of passive transport whereby ions or molecules driven by thermal motion move along their concentration gradients. This spontaneous process is widespread in nature. For example, as the main form of transport for vital materials through cell membranes, diffusion plays a fundamental role in living cells. Recently, self-supporting membranes that contain single or arrays of nanochannels are attracting increasing attention in nanotechnology, chemistry, physics, and biology. Inspired by biological membrane channel systems, most notably the a-hemolysin channel, artificial analogues of such nanoscopically sized pores have been developed for potential application in controlled growth of nanostructures, nanofiltration devices, bioseparation, and biosensing. Compared to biologically based nanochannels, artificial inorganic nanochannels have potential advantages of being less fragile, more stable, easily tailored, and flexible with regard to surface modification. Anodization of aluminum under appropriate electrochemical conditions yields extended membranes containing self-assembled, uniform, parallel pores with diameters of a few tens to a few hundreds of nanometers, high pore density, and well-defined morphology. Such porous anodic alumina (PAA) membranes could find applications in chemical and biological separations as well as for analytical purposes. Motion of molecules across these charged membranes is the basis of numerous systems of technological and biological interest. Detailed knowledge of such mass-transport behavior is therefore crucial for understanding and optimizing prospective device structures. Although many application models have been successfully established on the basis of the widely studied migration mechanism of ions or charged biomolecules in nanochannels, little is known about the motion of electrically neutral molecules on this size scale. Herein we show a distinct diffusion phenomenon of electrically neutral molecules in charged alumina nanochannels. Mass transfer across a porous membrane is governed by the Nernst–Planck equation. For a one-dimensional system, the mass transfer J along the x axis can be written as Equation (1) where D, C, and z are the diffusion coefficient,

Real‐Time Monitoring of Mass‐Transport‐Related Enzymatic Reaction Kinetics in a Nanochannel‐Array Reactor

To understand the fundamentals of enzymatic reactions confined in micro-/nanosystems, the construction of a small enzyme reactor coupled with an integrated real-time detection system for monitoring the kinetic information is a significant challenge. Nano-enzyme array reactors were fabricated by covalently linking enzymes to the inner channels of a porous anodic alumina (PAA) membrane. The mechanical stability of this nanodevice enables us to integrate an electrochemical detector for the real-time monitoring of the formation of the enzyme reaction product by sputtering a thin Pt film on one side of the PAA membrane. Because the enzymatic reaction is confined in a limited nanospace, the mass transport of the substrate would influence the reaction kinetics considerably. Therefore, the oxidation of glucose by dissolved oxygen catalyzed by immobilized glucose oxidase was used as a model to investigate the mass-transport-related enzymatic reaction kinetics in confined nanospaces. The activity and stability of the enzyme immobilized in the nanochannels was enhanced. In this nano-enzyme reactor, the enzymatic reaction was controlled by mass transport if the flux was low. With an increase in the flux (e.g., >50 microL min(-1)), the enzymatic reaction kinetics became the rate-determining step. This change resulted in the decrease in the conversion efficiency of the nano-enzyme reactor and the apparent Michaelis-Menten constant with an increase in substrate flux. This nanodevice integrated with an electrochemical detector could help to understand the fundamentals of enzymatic reactions confined in nanospaces and provide a platform for the design of highly efficient enzyme reactors. In addition, we believe that such nanodevices will find widespread applications in biosensing, drug screening, and biochemical synthesis.

Morpholino-Functionalized Nanochannel Array for Label-Free Single Nucleotide Polymorphisms Detection

The sensitive identification of single nucleotide polymorphisms becomes increasingly important for disease diagnosis, prevention, and practical applicability of pharmacogenomics. Herein, we propose a simple, highly selective, label-free single nucleotide polymorphisms (SNPs) sensing device by electrochemically monitoring the diffusion flux of ferricyanide probe across probe DNA/morpholino duplex functionalized nanochannels of porous anodic alumina. When perfectly matched or mismatched target DNA flows through the nanochannels modified with probe DNA/morpholino duplex, it competes for the probe DNA from morpholino, resulting in a change of the surface charges. Thus, the diffusion flux of negatively charged electroactive probe ferricyanide is modulated since it is sensitive to the surface charge due to the electrostatic interactions in electric double layer-merged nanochannels. Monitoring of the change in diffusion flux of probe enables us to detect not only a single base or two base mismatched sequence but also the specific location of the mismatched base. As is demonstrated, SNPs in the PML/RARα fusion gene, known as a biomarker of acute promyelocytic leukemia (APL), have been successfully detected.

In Situ Monitoring of Protein Adsorption on a Nanoparticulated Gold Film by Attenuated Total Reflection Surface-Enhanced Infrared Absorption Spectroscopy

In situ surface enhanced infrared absorption spectroscopy (SEIRAS) with an attenuated total reflection (ATR) configuration has been used to monitor the adsorption kinetics of bovine hemoglobin (BHb) on a Au nanoparticle (NP) film. The IR absorbance for BHb molecules on a gold nanoparticle film deposited on a Si hemispherical optical window is about 58 times higher than that on a bare Si optical window and the detection sensitivity has been improved by 3 orders of magnitude. From the IR signal as a function of adsorption time, the adsorption kinetics and thermodynamics can be explored in situ. It is found that both the electrostatic interaction and the coordination bonds between BHb residues and Au NP film surface affect the adsorption kinetics. The maximum adsorption can be obtained in solution pH 7.0 (close to the isoelectric point of the protein) due to the electrostatic interaction among proteins. In addition, the isotherm of BHb adsorption follows well the Freundlich adsorption model.

Exploration of two-enzyme coupled catalysis system using scanning electrochemical microscopy.

In biological metabolism, a given metabolic process usually occurs via a group of enzymes working together in sequential pathways. To explore the metabolism mechanism requires the understanding of the multienzyme coupled catalysis systems. In this paper, an approach has been proposed to study the kinetics of a two-enzyme coupled reaction using SECM combining numerical simulations. Acetylcholine esterase and choline oxidase are immobilized on cysteamine self-assembled monolayers on tip and substrate gold electrodes of SECM via electrostatic interactions, respectively. The reaction kinetics of this two-enzyme coupled system upon various separation distance precisely regulated by SECM are measured. An overall apparent Michaelis-Menten constant of this enzyme cascade is thus measured as 2.97 mM at an optimal tip-substrate gap distance of 18 μm. Then, a kinetic model of this enzyme cascade is established for evaluating the kinetic parameters of individual enzyme by using the finite element method. The simulated results demonstrate the choline oxidase catalytic reaction is the rate determining step of this enzyme cascade. The Michaelis-Menten constant of acetylcholine esterase is evaluated as 1.8 mM. This study offers a promising approach to exploring mechanism of other two-enzyme coupled reactions in biological system and would promote the development of biosensors and enzyme-based logic systems.

Propagation of concentration polarization affecting ions transport in branching nanochannel array.

A new ionic current rectification device responsive to a broad range of pH stimuli has been fabricated using porous anodic alumina membrane with tailor-made branching nanochannel array. The asymmetric geometry is achieved by changing oxidation voltage using a simple two-step anodization process. Due to the protonation/deprotonation of the intrinsic hydroxyl groups upon solution pH variation, the nanochannels array-based device is able to regulate ionic current rectification properties. The ion rectification ratio of the device is mainly determined by the branching size and surface charges, which is also confirmed by theoretical simulations. In addition, theoretical simulations show that the slight difference in ion rectification ratio for the nanochannel devices with different branching numbers is due to the propagation of concentration polarization. Three dimensional simulations clearly show the nonuniform concentration profiles in stem nanochannel near the junction interface due to the presence of branching nanochannels. The present ionic device is promising for biosensing, molecular transport and separation, and drug delivery in confined environments.

Study on the influence of cross-sectional area and zeta potential on separation for hybrid-chip-based capillary electrophoresis using 3-D simulations.

Hybrid chips combing microchips with capillaries have displayed particular advantages in achieving UV‐vis and mass spectroscopic detection. In this work, systematic 3‐D numerical simulations have been carried out to explore the influence of junction interface cross‐sectional area and ζ‐potential distribution on sample band broadening in hybrid‐chip electrophoresis separation. In this case, the ratio of cross‐sectional area of chip to capillary channel (Sratio) is used as the parameter of the variation in junction interface cross‐sectional area. Theoretical simulations demonstrated that the decrease of the Sratio would increase the separation efficiency in the hybrid‐chip‐based CE with uniform ζ‐potential distribution. ζ‐potential distribution along the axial direction of the channel also affects mass transport in hybrid‐chip‐based CE. Therefore, the effect of ζ‐potential distribution has been considered in the 3‐D simulation. Theoretical simulation results reveal that ζ‐potential distribution rather than the interface cross‐sectional area variation (Sratio) controls the sample band broadening and manipulates sample separation efficiency in the hybrid‐chip‐based CE with non‐uniform ζ‐potential distribution. Both the theoretical simulations and experimental results show that optimal hybrid‐chip CE separation efficiency can be achieved at Sratio=1.

Exploring the temperature-dependent kinetics and thermodynamics of immobilized glucose oxidase in microchip

Herein, we report a method to investigate the thermodynamics and kinetics of immobilized enzyme (Glucose Oxidase, GOD) catalytic reaction on a microfluidics platform with precise temperature-control using a home-made plexiglass temperature controllable holder. This approach allows us to extract kinetic and thermodynamic parameters of the immobilized enzyme catalytic reactions easily, showing significant advantages over the traditional calorimetric and microcalorimetric methods which require complex fabrication of thermally isolated system. In our approach, the Arrhenius equation is introduced to establish the relationship between the kinetics and thermodynamics of the immobilized GOD. Results show that the obtained activation energy (Ea = 60.56 kJ mol−1) and the activation enthalpy (ΔHa = 53.08 kJ mol−1) are smaller than free enzymes, demonstrating that the immobilized GOD exhibits improved thermal stability compared with free enzymes. The present work offers an alternative approach to achieve the kinetics and thermodynamics of immobilized enzyme catalytic reactions on a microfluidics chip and promote our understanding of enzyme catalytic reactions.

Interconnected ordered nanoporous networks of colloidal crystals integrated on a microfluidic chip for highly efficient protein concentration

We report a controllable method to fabricate silica colloidal crystals at defined position in microchannel of microuidic devices using simple surface modification. The formed PCs (photonic crystals) in microfluidic channels were stabilized by chemical cross‐linking of Si‐O‐Si bond between neighboring silica beads. The voids among colloids in PCs integrated on microfluidic devices form interconnected nanoporous networks, which show special electroosmotic properties. Due to the “surface‐charge induced ion depletion effect” mechanism, FITC‐labeled proteins can be efficiently and selectively concentrated in the anodic boundary of the ion depletion zone. Using this device, about 103‐ to 105‐fold protein concentration was achieved within 10 min. The present simple on chip protein concentration device could be a potential sample preparation component in microfluidic systems for practical biochemical assays.

Effect of Nanoemitters on Suppressing the Formation of Metal Adduct Ions in Electrospray Ionization Mass Spectrometry

In the work, we showed that the use of nanoemitters (tip dimension <1 μm, typically ∼100 nm) could dramatically reduce the nonspecific metal adduction to peptide or protein ions as well as improve the matrix tolerance of electrospray ionization mass spectrometry (ESI-MS). The proton-enriched smaller initial droplets are supposed to have played a significant role in suppressing the formation of metal adduct ions in nanoemitters. The proton-enrichment effect in the nanoemitters is related to both the exclusion-enrichment effect (EEE) and the ion concentration polarization effect (ICP effect), which permit the molecular ions to be regulated to protonated ones. Smaller initial charged droplets generated from nanoemitters need less fission steps to release the gas-phase ions; thus, the enrichment effect of salt was not as significant as that of microemitters (tip dimension >1 μm), resulting in the disappearing of salt cluster peaks in high mass-to-charge (m/z) region. The use of nanoemitters demonstrates a novel method for tuning the distribution of the metal-adducted ions to be in a controlled manner. This method is also characterized by ease of use and high efficiency in eliminating the formation of adduct ions, and no pretreatment such as desalting is needed even in the presence of salt at millimole concentration.