Selective enrichment of endogenous peptides by chemically modified porous nanoparticles for peptidome analysis

We report the development of a combined strategy for high capacity, comprehensive enrichment of endogenous peptide from complex biological samples at natural pH condition. MCM-41 nanoparticles with highly ordered nanoscale pores (i.e. 4.8nm) and high-surface area (i.e. 751m(2)/g) were synthesized and modified with strong cation-exchange (SCX-MCM-41) and strong anion-exchange (SAX-MCM-41) groups. The modified nanoparticles demonstrated good size-exclusion effect for the adsorption of standard protein lysozyme with molecular weight (MW) of ca. 15kDa; and the peptides with MW lower than this value can be well adsorbed. Step elution of the enriched peptides with five salt concentrations presented that both modified nanoparticles have high capacity and complementarity for peptides enrichment, and the SAX-MCM-41 nanoparticles has obviously high selectivity for acidic peptides with pI (isoelectric point) lower than 4. Large-scale enrichment of endogenous peptides in 2mg mouse liver extract was achieved by further combination of SCX-MCM-41 and SAX-MCM-41 with unmodified MCM-41 nanoparticles. On-line 2D nano-LC/MS/MS was applied to analyze the enriched samples, and 2721 unique peptides were identified in total. Two-dimensional analysis of MW versus pI distribution combined with abundance of the identified peptides demonstrated that the three types of nanoparticles have comprehensive complementarity for peptidome enrichment.     

Electrophoretic size separation of particles in a periodically constricted microchannel

The size separation of Brownian particles with the same free mobility in an electrophoretic microchannel with alternating thick regions and narrow constrictions is studied theoretically. The electrophoretic mobility is field dependent and generally increases with field strength. In weak fields, Brownian diffusion dominates and the migration is controlled by the entrance effect. Therefore, smaller particles migrate faster than larger ones. In strong fields, however, the particle tends to follow electric field lines. Smaller particles are susceptible to Brownian motion and thus influenced by the nonuniform electric field in the well significantly. As a result, larger particles possess higher mobilities. Our simulation results agree with the experimental observations and provide guidance for efficient nanofluidic separation.     

Effect of electric field on liquid infiltration into hydrophobic nanopores

Understanding the variation of nanofluidic behavior in the presence of an external electric field is critical for controlling and designing nanofluidic devices. By studying the critical infiltration pressure of liquids into hydrophobic nanopores using molecular dynamics (MD) simulations and experiments, important insights can be gained on the variation of the effective liquid-solid interfacial tension with the magnitude and sign of electric field, as well as its coupling with the pore size and the solid and liquid species. It is found that the effective hydrophobicity reduces with the increase of electric intensity and/or pore size, and the behavior is asymmetric with respect to the direction of the electric field. The underlying molecular mechanisms are revealed via the study of the density profile, contact angle, and surface tension of confined liquid molecules.     

Specific enrichment of prokaryotic DNA using a recombinant DNA-binding protein

Targeted enrichment of DNA is often necessary for its detection and characterization in complex samples. We describe the development and application of the novel molecular tool for the specific enrichment of prokaryotic DNA. A fused protein comprising the DNA-binding subunit of the bacterial topoisomerase II, gyrase, was expressed, purified, and immobilized on magnetic particles. We demonstrated the specific affinity of the immobilized protein towards bacterial DNA and investigated its efficiency in the samples with high background of eukaryotic DNA. The reported approach allowed for the selective isolation and further detection of as few as 5 pg Staphylococcus aureus DNA from the sample with 4?×?10⁶-fold surplus of human DNA. This method is a promising approach for the preparation of such type of samples, for example in molecular diagnostics of sepsis.     

Ion separation in nanofluidics

Ionic species with a constant charge-to-size ratio (i.e. electrophoretic mobility) cannot be separated in electroosmotic or pressure-driven flow along microscale channels. In nanoscale channels, however, the enormous electric fields inside electrical double layers cause transverse ion distributions yielding charge-dependent mean ion speeds in the flow. Those ions with a constant charge-to-size ratio can thus be separated solely by charge (or equivalently, size) in nanofluidics. Here we develop an analytical model to optimize and compare the separation of such ions in nanochannel chromatography and nanochannel electrophoresis in terms of selectivity, plate height and resolution. Both planar and cylindrical geometries are considered. It is found that in nanoscale channels chromatography yields a larger selectivity and a larger minimum reduced plate height than electrophoresis does. The maximum resolution is, however, comparable between these two nanofluidic approaches, where the optimal channel half-height or tube radius is within the range of 1-10 times the Debye length. Our results also suggest that cations can be better separated in nanofluidics than can anions.     

Pre-binding dynamic range and sensitivity enhancement for immuno-sensors using nanofluidic preconcentrator

Almost all immuno-biosensors are inherently limited by the quality of antibodies available for the target molecule, and obtaining a highly sensitive antibody for a given target molecule is a challenge. We describe a highly efficient and flexible way to enhance immunoassay detection sensitivity and binding kinetics using a nanofluidic based electrokinetic preconcentrator. The device is a microfluidic integration of charge-based biomolecule concentrator and a bead-based immunoassay. Because the preconcentrator can increase the local biomolecule concentration by many orders of magnitude, it gives the immuno-sensor better sensitivity and faster binding kinetics. With a 30 min preconcentration, we were able to enhance the immunoassay sensitivity (with molecular background) by more than 500 fold from higher 50 pM to the sub 100 fM range. Moreover, by adjusting the preconcentration time, we can switch the detection range of the given bead-based assay (from 10-10 000 ng ml(-1) to 0.01-10 000 ng ml(-1)) to have a broader dynamic range of detection. As the system can enhance both detection sensitivity and dynamic range, it can be used to address the most critical detection issues in the detection of common disease biomarkers.     

Proteocubosomes: nanoporous vehicles with tertiary organized fluid interfaces

Proteocubosomes are nanostructured open-nanochannel hierarchical fluid vehicles characterized by a cubic lattice periodicity of the lipid/protein supramolecular assembly (protein-loaded cubosomes). They are obtained here at very high hydration levels by a three-dimensional (3D) self-assembly process, which exploits a protein-directed 3D patterning and fragmentation to create a new, tertiary-level structural order of fluid lipid/water interfaces. Our freeze-fracture electron microscopy study reveals that the proteocubosome structures are built up by patterned assemblies of nanocubosomes, which comprise 3D nanoporous fracture surfaces throughout. Complex cubosomic architectures, involving arrays of nanodroplets (larger than 20 nm) inside the proteocubosome particles, are established at high resolution. The soft-matter hierarchical nanocompartment formations display internal aqueous pores belonging to the D-type lipid cubic lattice nanochannel system that is proven by synchrotron X-ray diffraction. The reported nanostructured fluid may give rise to novel applications in nanofluidic biomimetic devices, porous protein drug delivery vehicles, nanoscale enzymatic bioreactors, and protein-encapsulating fluid nanomaterials.     

Autoionization of Excitons in Organic Solar Cells

It has been shown that in addition to the conventional mechanism of photovoltaic effect in organic solar cells (OSCs), which is the ionization of molecular excitons on impurities, a new mechanism is possible in nanoscale cells, namely, tunneling autoionization in a strong electric field of the pn junction. Its quantum yield for molecular excitons and charge-transfer excitons becomes higher than for ionization on impurities with a cell length of less than 20–25 or 60–80 lattice periods (~10 and ~25 nm), respectively. The possibility of creating cascades of series-connected OSCs with a quantum yield close to 1 on the basis of the new mechanism is discussed.     

Enrichment and separation of acidic and basic proteins using the centrifugal ultrafiltration followed by nanoparticle-filled capillary electrophoresis

This report describes a method for enrichment and separation of acidic and basic proteins using the centrifugal ultrafiltration followed by nanoparticle-filled capillary electrophoresis. To improve stacking and separation efficiencies of proteins, the separation buffer containing 1.6% poly(diallyldimethylammonium chloride) was added with gold nanoparticles (AuNP), poly(ethylene oxide), cetyltrimethylammonium bromide, and poly(vinyl alcohol). As a result, the use of AuNP as additives exhibited better efficiency in separation, stacking, and analysis time. Even for large-volume samples (110 nL), the separation efficiencies of acidic and basic proteins remained greater than 10(4) and 10(5) plates/m, respectively. To further enhance detection sensitivity, protein samples were enriched using the centrifugal ultrafiltration, followed by our proposed stacking method. The detection sensitivity was improved up to 314-fold compared to normal hydrodynamic injection. Additionally, the limits of detection at a signal-to-noise of 3 for most proteins were down to nanomolar range. We have validated the application of our method by means of analyses of 50 nM lysozyme in saliva samples. The proposed method was also successfully applied to the analyses of egg-white proteins, which have large differences in molecular weight and pI.     

Phosphate Diester Cleavage Promoted by the Metallomicelles of Ce(III) Complexes of Aza-Crown Ether with Different Numbers of Nitrogen Atoms

The design of artificial hydrolase has attracted extensive attention due to their scientific significance and potential application in the field of gene medicine and molecular biology. This work reports the catalytic activation of two aza-crown ether Ce(III) complexes and their metallomicelles as artificial hydrolase in bis(4-nitrophenyl) phosphate ester (BNPP) hydrolysis. The chemical composition of two complexes was determined by the fluorescence spectra and the mole ratio method for electronic absorption spectra. The bonding effect of BNPP and solubilizing effect of the complexes were proved by a method of fluorescence spectroscopy. The catalytic activity of different catalytic systems in BNPP hydrolysis was measured with UV-vis spectrophotometric method. These catalytic systems showed high catalytic activity for promoting BNPP hydrolysis at the almost physiological conditions. BNPP hydrolysis rate in these catalytic system is about 10⁷- to 10⁹-fold faster than that of the BNPP spontaneous hydrolysis in aqueous solution at the same conditions. The metallomicelle systems exhibited higher catalytic activity compared with the complex solution systems in BNPP hydrolysis, and hexadecyltrimethyl ammonium bromide micelle provides a useful catalytic environment for reaction. The acid effect of the catalytic system is ascribed to the formation of metal-bound hydroxide serving as a better kind of nucleophile.     

Highly rectified ion transport through 2D WSe2/MoS2 bi-layered membranes

Through a two-step vacuum-filtration process, WSe₂ and MoS₂ nanosheets were sequentially deposited onto a polymeric nanoporous support, forming WSe₂/MoS₂ bi-layered heterostructure. Highly rectified ion transport phenomenon is observed through the heterogeneous 2D layered membranes.     

Hydrolysis of glycosides with microgel catalysts

A dormant macromolecular catalyst was prepared by polymerization of an aqueous styrene-butyl acrylate miniemulsion in the presence of a new polymerizable pentadentate ligand. The catalyst was activated by binding Cu(II) ions to the ligand site and then explored for its ability to hydrolyze glycosidic bonds in alkaline solution. The performance was correlated to the catalytic activity shown by low molecular weight analogs. A turnover rate of up to 43 × 10(-4) min(-1) was previously observed for cleavage of the glycosidic bond in selected p-nitrophenylglycosides with a binuclear, low molecular weight catalyst; by contrast, the same reaction is more than 1 order of magnitude faster and has a turnover rate of up to 380 × 10(-4) min(-1) when using the prepared macromolecular catalyst. The catalyzed hydrolysis is about 10(5)-fold accelerated over the uncatalyzed background reaction under the provided conditions, while a significant discrimination of the α- and β-glycosidic bond or of the galacto- and gluco-configuration in the sugar moiety in the glycoside substrates is not observed.     

Electropreconcentration diagrams to optimize molecular enrichment with low counter pressure in a nanofluidic device

Concentration polarization (CP)-based focusing electrokinetics nanofluidic devices have been developed in order to simultaneously detect and enrich highly diluted analytes on-a-chip. However, stabilization of focal points over long time under the application of the electric field remains as a technical bottleneck. If pressure-assisted preconcentration methods have been proposed to stabilize propagating modes at low inverse Dukhin number , these recent protocols remain laborious for optimizing experimental parameters. In this paper, “electric field E/counter-pressure P” diagrams have been established during pressure-assisted electro-preconcentration of fluorescein as a model molecule. Such E/P diagram allows direct observation of the region for which the optimal counter-pressure P leads to a stable focusing regime. This region of stable focusing is shown to vary depending of the nanoslit length (100 μm < L_nanoslit < 500 μm) and the nature of the background electrolyte (KCl and NaCl). Longer nanoslits (500 μm) produce stabilization at low counter-pressure P, whereas NaCl offers a narrower region of stable focusing in the E/P diagram compared to KCl. Finally, the ability of such pressure-assisted protocol to concentrate negatively charged proteins has been tested with a more applicative protein, i.e., ovalbumin. The corresponding E/P diagram confirms the existence of the stable focusing regime at both low electric field E (≤20 V) and counter-pressure P (≤0.4 bar). With an enrichment factor as high as 70 after 2 min for ovalbumin at a concentration of 10 μM, such pressure-assisted nanofluidic electro-preconcentration protocol appears very promising to concentrate and detect biomolecules.     

Microchip-based cellular biochemical systems for practical applications and fundamental research: from microfluidics to nanofluidics

By combining cell technology and microchip technology, innovative cellular biochemical tools can be created from the microscale to the nanoscale for both practical applications and fundamental research. On the microscale level, novel practical applications taking advantage of the unique capabilities of microfluidics have been accelerated in clinical diagnosis, food safety, environmental monitoring, and drug discovery. On the other hand, one important trend of this field is further downscaling of feature size to the 10¹–10³ nm scale, which we call extended-nano space. Extended-nano space technology is leading to the creation of innovative nanofluidic cellular and biochemical tools for analysis of single cells at the single-molecule level. As a pioneering group in this field, we focus not only on the development of practical applications of cellular microchip devices but also on fundamental research to initiate new possibilities in the field. In this paper, we review our recent progress on tissue reconstruction, routine cell-based assays on microchip systems, and preliminary fundamental method for single-cell analysis at the single-molecule level with integration of the burgeoning technologies of extended-nano space.     

A simple, disposable microfluidic device for rapid protein concentration and purification via direct-printing

A facile and disposable microfluidic device for rapid protein concentration was fabricated by using a direct printing process. Two printed V-shaped microchannels in mirror image orientation were separated by a 100 mum wide toner gap. When a high electric field was applied across the two channels, nanofissures were formed by electric breakdown at the junction toner gap. This microfluidic device with nanofissures was used as a concentrator for protein. Negatively charged proteins were observed to concentrate at the anode side of the nanofissures upon application of an electric field across this junction. Using this device, about 10(3)-10(5)-fold protein concentration was achieved within 10 min. Systematic investigation showed that the concentration mechanism could be explained by the ion exclusion-enrichment effect of the nanofissures. In addition, the present microchip device integrated both functions of concentration and purification were confirmed. This simple on chip protein preconcentration and purification device could be a disposable sample preparation component in printed microfluidic systems used for practical biochemical assays.     

纳流控-电化学技术在生化分析领域的研究进展

纳流控作为一个崭新的研究领域正受到越来越多的关注,并且已被成功应用到纳米尺度分离、生化传感、能量转化等诸多领域. 纳流控的发展与电化学紧密相连,一方面,电化学可以为纳米孔道中的物质传输特性的研究提供驱动力;另一方面,纳米孔道可以为限域电化学研究提供微环境. 纳流控和电化学技术相辅相成,催生了许多单分子、单粒子分析以及纳米流体操控的新理念与新技术. 本综述从纳米孔道与电极的结合方式出发,对纳流控-电化学相关研究进行了总结与展望.     

Size-selective concentration and label-free characterization of protein aggregates using a Raman active nanofluidic device

Trace detection and physicochemical characterization of protein aggregates have a large impact in understanding and diagnosing many diseases, such as ageing-related neurodegeneration and systemic amyloidosis, for which the formation of protein aggregates is one of the pathological hallmarks. Here we demonstrate an innovative label-free method for detecting and characterizing small amounts of early stage protein aggregates using a Raman active nanofluidic device. Sub-micrometre channels formed by a novel elastomeric collapse technique enable the separation and concentration of matured protein aggregates from small protein molecules. The Raman enhancement by gold nanoparticle clusters fixed below a micro/nanofluidic junction allows characterization of intrinsic properties of protein aggregates at concentration levels (～fM) much lower than can be done with traditional analytical tools. With our device we show for the first time the concentration dependence of protein aggregation over these low concentration ranges. We expect that our method could facilitate definitive diagnosis and possible therapeutics of diseases at early stages.     

Syntheses of 2-chloro-4-nitrophenyl beta-D-maltopentaosides with bulky modification and their application to the differential assay of human alpha-amylases.

Four novel maltopentaosides, 2-chloro-4-nitrophenyl O-(6-O-p-toluenesulfonyl-alpha-D-glucopyranosyl)-(1-->4)-tris[O- alpha-D-glucopyranosyl-(1-->4)]-beta-D-glucopyranoside (4), 2-chloro-4-nitrophenyl O-[6-O-(tert-butyldimethyl)silyl-alpha-D- glucopyranosyl]-(1-->4)-tris[O-alpha-D-glucopyranosyl-(1-->4)]-beta-D- glucopyranoside (5), 2-chloro-4-nitrophenyl O-[6-deoxy-6-(phenyl)sulfonyl-alpha-D- glucopyranosyl]-(1-->4)-tris[O-alpha-D-glucopyranosyl-(1-->4)]-beta-D- glucopyranoside (10), and 2-chloro-4-nitrophenyl O-(6-deoxy-6-phthalimido-alpha-D-glucopyranosyl)- (1-->4)-tris[O-alpha-D-glucopyranosyl-(1-->4)]-beta-D-glucopyranoside (11) were synthesized. Substrates 4, 5, 10, and 11 were hydrolyzed by human pancreatic alpha-amylase (HPA) from 1.1 to 2.9-fold faster than by human salivary alpha-amylase (HSA). Taking advantage of the difference in the hydrolytic rate of 5 (2.9-fold faster), we developed a new method for the differential assay of these two human alpha-amylases.     

Low-temperature direct bonding of glass nanofluidic chips using a two-step plasma surface activation process

Owing to the well-established nanochannel fabrication technology in 2D nanoscales with high resolution, reproducibility, and flexibility, glass is the leading, ideal, and unsubstitutable material for the fabrication of nanofluidic chips. However, high temperature (~1,000 °C) and a vacuum condition are usually required in the conventional fusion bonding process, unfortunately impeding the nanofluidic applications and even the development of the whole field of nanofluidics. We present a direct bonding of fused silica glass nanofluidic chips at low temperature, around 200 °C in ambient air, through a two-step plasma surface activation process which consists of an O₂ reactive ion etching plasma treatment followed by a nitrogen microwave radical activation. The low-temperature bonded glass nanofluidic chips not only had high bonding strength but also could work continuously without leakage during liquid introduction driven by air pressure even at 450 kPa, a very high pressure which can meet the requirements of most nanofluidic operations. Owing to the mild conditions required in the bonding process, the method has the potential to allow the integration of a range of functional elements into nanofluidic chips during manufacture, which is nearly impossible in the conventional high-temperature fusion bonding process. Therefore, we believe that the developed low-temperature bonding would be very useful and contribute to the field of nanofluidics.