Orientation and lateral mobility of cytochrome c on the surface of ultrathin lipid multilayer films.

We have previously shown that cytochrome c can be electrostatically bound to an ultrathin multilayer film having a negatively charged hydrophilic surface; furthermore, x-ray diffraction and absorption spectroscopy techniques indicated that the cytochrome c was bound to the surface of these ultrathin multilayer films as a molecular monolayer. The ultrathin fatty acid multilayers were formed on alkylated glass, using the Langmuir-Blodgett method. In this study, optical linear dichroism was used to determine the average orientation of the heme group within cytochrome c relative to the multilayer surface plane. The cytochrome c was either electrostatically or covalently bound to the surface of an ultrathin multilayer film. Horse heart cytochrome c was electrostatically bound to the hydrophilic surface of fatty acid multilayer films having an odd number of monolayers. Ultrathin multilayer films having an even number of monolayers would not bind cytochrome c, as expected for such hydrophobic surfaces. Yeast cytochrome c was covalently bound to the surface of a multilayer film having an even number of fatty acid monolayers plus a surface monolayer of thioethyl stearate. After washing extensively with buffer, the multilayer films with either electrostatically or covalently bound cytochrome c were analyzed for bound protein by optical absorption spectroscopy; the orientation of the cytochrome c heme was then investigated via optical linear dichroism. Polarized optical absorption spectra were measured from 450 to 600 nm at angles of 0 degrees, 30 degrees, and 45 degrees between the incident light beam and the normal to the surface plane of the multilayer. The dichroic ratio for the heme alpha-band at 550 nm as a function of incidence angle indicated that the heme of the electrostatically-bound monolayer of cytochrome c lies, on average, nearly parallel to the surface plane of the ultrathin multilayer. Similar results were obtained for the covalently-bound yeast cytochrome c. Furthermore, fluorescence recovery after photobleaching (FRAP) was used to characterize the lateral mobility of the electrostatically bound cytochrome c over the monolayer plane. The optical linear dichroism and these initial FRAP studies have indicated that cytochrome c electrostatically bound to a lipid surface maintains a well-defined orientation relative to the membrane surface while exhibiting measurable, but highly restricted, lateral motion in the plane of the surface.     

Vectorially oriented membrane protein monolayers: profile structures via x-ray interferometry/holography.

X-ray interferometry/holography was applied to meridional x-ray diffraction data to determine uniquely the profile structures of a single monolayer of an integral membrane protein and a peripheral membrane protein, each tethered to the surface of a solid inorganic substrate. Bifunctional, organic self-assembled monolayers (SAMs) were utilized to tether the proteins to the surface of Ge/Si multilayer substrates, fabricated by molecular beam epitaxy, to facilitate the interferometric/holographic x-ray structure determination. The peripheral membrane protein yeast cytochrome c was covalently tethered to the surface of a sulfhydryl-terminated 11-siloxyundecanethiol SAM via a disulfide linkage with residue 102. The detergent-solubilized, photosynthetic reaction center integral membrane protein was electrostatically tethered to the surface of an analogous amine-terminated SAM. Optical absorption measurements performed on these two tethered protein monolayer systems were consistent with the x-ray diffraction results indicating the reversible formation of densely packed single monolayers of each fully functional membrane protein on the surface of the respective SAM. The importance of utilizing the organic self-assembled monolayers (as opposed to Langmuir-Blodgett) lies in their ability to tether specifically both soluble peripheral membrane proteins and detergent-solubilized integral membrane proteins. The vectorial orientations of the cytochrome c and the reaction center molecules were readily distinguishable in the profile structure of each monolayer at a spatial resolution of 7 A.     

The location of cytochrome c on the surface of ultrathin lipid multilayer films using x-ray diffraction.

X-ray diffraction and spectroscopic techniques were used to characterize ultrathin fatty acid multilayers having a bound surface layer of cytochrome c. Three to six monolayers of arachidic acid were deposited onto an alkylated glass surface, using the Langmuir-Blodgett method. These fatty acid multilayer films were stored either in a 1 mM NaHCO3 pH 7.5 solution or a buffered 10 microM cytochrome c solution, pH 7.5. After washing extensively with buffer, these multilayer films were assayed for bound cytochrome c by optical spectroscopy. It was found that the cytochrome c bound only to the odd-numbered monolayer films (which have hydrophilic surfaces). The theoretical number of cytochrome c molecules bound to the ultrathin multilayer films having three or five monolayers was calculated as N = 1.2 x 10(13)/cm2 (assuming a hexagonally close-packed monolayer of protein), which would produce an optical density of 0.002 at a wavelength of 550 nm; for a three or five monolayer ultrathin film that was incubated with cytochrome c, OD550 approximately equal to 0.002. The protein was released from the film when treated with greater than 100 mM KCl solution, as would be expected for an electrostatic interaction. Meridional x-ray diffraction data were collected from the arachidic acid films with and without a bound cytochrome c layer. A box refinement technique, previously shown to be effective in deriving the profile structures of nonperiodic ultrathin films, was used to determine the multilayer electron density profiles. The electron density profiles and their autocorrelation functions showed that bound cytochrome c resulted in an additional electron dense feature on the multilayer surface, consistent with a bound cytochrome c monolayer. The position of the bound protein relative to the multilayer surface was independent of the number of fatty acid monolayers in the multilayer. Future studies will use these methods to investigate the structures of membrane protein complexes bound directly to the surface of multilayer films.     

Comparison of the binding specificity of two bacterial metalloproteases, LasB of Pseudomonas aeruginosa and ZapA of Proteus mirabilis, using N-alpha mercaptoamide template-based inhibitor analogues

Nanospheres lithographic (NSL) method has been used to fabricate nano-structured arrays (NAs) of hexagonally close-packed gold (Au) using polystyrene beads [PS, diameter ～300 nm] as mask. The developed NA was incorporated with a customized and cheap microfluidics system to demonstrate its applicability as an alternative easy and efficient platform for multiplex analysis and Lab-on-a-Chip applications. The chip functionality was demonstrated with horseradish peroxidase (HRP) and anti-HRP antibody as model for recognition system. The enzyme-linked immunosorbent assay (ELISA) performed on fabricated protein biochip had a detection limit 100 pg/mL for HRP. The antibody chip was also checked for the shelf-life and it was found that these chips could be stored for 50 days when stored at 4°C without any significant loss of activity. Therefore, NAs based protein biochip with the correct microfluidics could find huge potential application in diagnostics and biosensing technology.     

Ultrasensitive electrical detection of protein using nanogap electrodes and nanoparticle-based DNA amplification

The present study describes an ultrasensitive protein biochip that employs nanogap electrodes and self-assembled nanoparticles to electrically detect protein. A bio-barcode DNA technique amplifies the concentration of target antigen at least 100-fold. This technique requires the establishment of conjugate magnetic nanoparticles (MNPs) and gold nanoparticles (AuNPs) through binding between monoclonal antibodies (2B2), the target antigen, and polyclonal antibodies (GP). Both GP and capture ssDNA (single-strand DNA) bonds to bio-barcode ssDNA are immobilized on the surface of AuNPs. A denature process releases the bio-barcode ssDNAs into the solution, and a hybridization process establishes multilayer AuNPs over the gap surface between electrodes. Electric current through double-layer self-assembled AuNPs is much greater than that through self-assembled monolayer AuNPs. This significant increase in electric current provides evidence that the solution contains the target antigen. Results show that the protein biochip attains a sensitivity of up to 1 pg/μL.     

Nanopore sensors: From hybrid to abiotic systems

This paper provides an overview of different nanostructured architectures utilised in electrochemical devices and their application in biosensing and bioelectronics. Emphasis is placed on the fabrication of nanostructured films based on a layer-by-layer (LBL) films approach. We discuss the theory and the mechanism of charge transfer in polyelectrolyte multilayer films (PEM), as well as between biomolecules and redox centres, for the development of more sensitive and selective biosensors. Further, this paper presents an overview of topics involving the interaction between nanostructured materials, including metallic nanoparticles and carbon materials, and their effects on the preservation of the activity of biological molecules immobilised on electrode surfaces. This paper also presents examples of biological molecules utilised in film fabrication, such as DNA, several kinds of proteins, and oligonucleotides, and of the role of molecular interaction in biosensing performance. Towards the utilisation of LBL films, examples of several architectures and different electrochemical approaches demonstrate the potential of nanostructured LBL films for several applications that include the diagnosis and monitoring of diseases. Our main aim in this review is to survey what can assist researchers by presenting various approaches currently used in the field of bioelectrochemistry utilising supramolecular architectures based on an LBL approach for application in electrochemical biosensing.     

Multivalent protein binding in carbohydrate-functionalized monolayers through protein-directed rearrangement and reorientation of glycolipids at the air-water interface

Multivalent protein binding plays an important role not only in biological recognition but also in biosensor preparation. Infrared reflection absorption spectroscopy and surface plasmon resonance techniques have been used to investigate concanavalin A (Con A) binding to binary monolayers composed of 1,2-di-O-hexadecyl-sn-glycerol and derived glycolipids with the mannose moieties. The glycolipids in the binary monolayers at the air-water interface underwent both lateral rearrangement and molecular reorientation directed by Con A in the subphase favorable to access of the carbohydrate ligands to protein binding pockets for the formation of multivalent binding sites and the minimization of steric crowding of neighboring ligands for enhanced binding. The amounts of specifically bound proteins in the binary monolayers at the air-water interface were accordingly increased in comparison with those in the initially immobilized monolayers at the air-water interface. The directed rearranged binary monolayers with multivalent protein binding were preserved for the preparation of biosensors.     

DNA-directed capture of primary cells from a complex mixture and controlled orthogonal release monitored by SPR imaging

Many biological samples are composed of several cell types. Qualitative and quantitative analysis of these complex mixtures is of major interest for both diagnostic and biomedical applications. Because large amounts of biological material are often challenging to collect, tremendous efforts have been made for a decade to design miniaturized platforms-such as lab-on-a-chip or microarrays-to run sensitive and reliable analysis from tiny quantities of starting material. Although barely explored so far, the release of resolved cellular samples constitutes an exciting strategy for further cell analysis. Herein, we propose a DNA-based biochip suitable for cell-type analysis in a label-free manner. The DNA-array is firstly converted into antibody-array using antibody-DNA conjugates. These protein-DNA hybrid molecules are chemically synthesized by covalent coupling of short oligonucleotides to antibodies directed against cell-type specific markers. We show not only specific capture of primary spleen cells on protein-DNA microarray spots but also their fast and specific orthogonal release according to the antibody-DNA combinations by incorporating restriction sites in DNA. Both molecular and cellular interactions occurring on the biochip are monitored by surface plasmon resonance (SPR) imaging. This optical technique turns out to be a powerful way to monitor, in real-time, biological interactions occurring on the microarrayed features.     

Surface-Enhanced Raman Scattering in Tunable Bimetallic Core-Shell

In this paper, optical properties of multilayer spherical core-shell nanoparticles based on quasi-static approach and plasmon hybridization theory are investigated. Calculations show that light absorption spectrum of bimetallic multilayer core-shell has three intense plasmon resonance peaks, which are more suitable for multiplex biosensing based on surface-enhanced Raman scattering (SERS) and localized surface plasmon resonance (LSPR). The plasmon resonance peaks in bimetal nanshells are optimized by tuning the geometrical parameters. In addition, the optimal geometry is discussed to obtain the Raman enhancement factor in bimetallic multilayer nanoshell. SERS enhancement factor is calculated with consideration of dampings due to both the electron scattering and the radiation at the boundary and modified Drude model in dielectric function of bimetallic nanoshell. It is shown that bimetallic nanoshell with the small size exhibits strong SERS enhancement factor (~6.63 × 10⁵) with additional collision dampings and ~2.9 × 10⁹ with modified Drude model which are suitable for biosensing applications. In addition, any variation in blood concentration and oxygen level can be detected by this bimetallic core-shell nanoparticle with sensitivity of Δλ/Δn = 264.91 nm/RIU.     

金结合多肽及其在生物传感领域的应用

金结合多肽是近年来通过生物展示技术或人工设计所获得的一类可以特异性与金结合的多肽,因其良好的生物相容性及易修饰性,针对此类生物大分子的研究和应用成为包括生物传感在内的众多领域的研究热点。金结合多肽多用于生物传感器的敏感膜制备,具有识别分子有序定位、反应步骤少、条件温和、高灵敏度的优点。我们在简要总结金结合多肽的代表性序列及其与金的结合机理的前提下,评述了金结合多肽在生物传感领域的应用,着重论述了利用基因工程技术表达含有金结合多肽的融合蛋白这一敏感膜关键器件的方法途径。     

Interferometric Backward Third Harmonic Generation Microscopy for Axial Imaging with Accuracy Beyond the Diffraction Limit

A new nonlinear microscopy technique based on interference of backward-reflected third harmonic generation (I-THG) from multiple interfaces is presented. The technique is used to measure height variations or changes of a layer thickness with an accuracy of up to 5 nm. Height variations of a patterned glass surface and thickness variations of fibroblasts are visualized with the interferometric epi-THG microscope with an accuracy at least two orders of magnitude better than diffraction limit. The microscopy technique can be broadly applied for measuring distance variations between membranes or multilayer structures inside biological tissue and for surface height variation imaging.     

A Two-Step Procedure for Quantitative Isolation of Pure Double Strand DNA from Animal Tissues and Cell Cultures

A two step procedure for the quantitative isolation of protein- and RNA-free double-strand DNA from animal tissue and cell homogenates is described. In the first step proteins not complexed with DNA are hydrolyzed with an immobilized protease (Proteinase K) that is separated by filtration after the de-proteinization. Then the DNA is adsorbed to hydroxylapatite (HA) and desorbed from the adsorbent by stepwise elution with buffers of increasing ionic strength. The DNA content was determined directly from the absorption at 260 nm. The melting curve of the isolated DNA showed that it was double stranded. The protein content in the DNA was determined from the ratio of the adsorbance at 260 to 230 nm. Non-histone proteins complexed to DNA determined the rate of deproteinization that was found to be tissue specific. These proteins were found to have a larger influence on the ratio A₂₆₀/A₂₃₀ than histones, indicating that their absorption (at 230 nm) is markedly perturbed when they are bound to DNA.     

表面等离子体-拉曼散射双模式复合生物芯片

本文提出了复合表面等离子体(SPR)无标记检测及表面增强拉曼散射(SERS)的显微成像技术.证明了双模式SPR-SERS生物芯片的可实施性,即在同一芯片上实现了表面等离子共振和表面增强拉曼显微检测.鉴于双模芯片的高保真性,基于显微技术的高精准、多通道无标记检测技术有望在临床医学检测中得以广泛应用.     

Dynamics of complex formation between biological and luminescent conjugated polyelectrolytes--a surface plasmon resonance study

A water-soluble polythiophene, POWT, with zwitterionic peptide like side chains possess good characteristics for biosensor applications. The zwitterionic side chains of the polymer can couple to biomolecules via electrostatic and hydrogen bonding. This creates possibilities to imprint biomolecules to spin-coated polymer films with maintained functionality, and use the resulting matrix as a biosensor. Polymer-biomolecular interaction studies done with surface plasmon resonance (SPR) reveal a well performing sensor matrix with high affinity for DNA hybridizations as well as for protein detection. The responses are distinct and very specific. A directional dependence of antibodies binding to POWT layer has also been observed. The polymer films have also been characterized by optical methods. Emission and absorption measurements in different buffer systems confirm that the polymer matrix can undergo structural and conformational changes on surfaces. The dielectric function in the interval 300-800 nm of POWT is reported, based on variable angle spectroscopic ellipsometry. This modeling reveals that a considerable amount of water is included in the material. The polymer layer possesses the characteristics needed for biochip applications and micro array techniques.     

Effects of lung surfactant proteins, SP-B and SP-C, and palmitic acid on monolayer stability

Langmuir isotherms and fluorescence and atomic force microscopy images of synthetic model lung surfactants were used to determine the influence of palmitic acid and synthetic peptides based on the surfactant-specific proteins SP-B and SP-C on the morphology and function of surfactant monolayers. Lung surfactant-specific protein SP-C and peptides based on SP-C eliminate the loss to the subphase of unsaturated lipids necessary for good adsorption and respreading by inducing a transition between monolayers and multilayers within the fluid phase domains of the monolayer. The morphology and thickness of the multilayer phase depends on the lipid composition of the monolayer and the concentration of SP-C or SP-C peptide. Lung surfactant protein SP-B and peptides based on SP-B induce a reversible folding transition at monolayer collapse that allows all components of surfactant to be retained at the interface during respreading. Supplementing Survanta, a clinically used replacement lung surfactant, with a peptide based on the first 25 amino acids of SP-B also induces a similar folding transition at monolayer collapse. Palmitic acid makes the monolayer rigid at low surface tension and fluid at high surface tension and modifies SP-C function. Identifying the function of lung surfactant proteins and lipids is essential to the rational design of replacement surfactants for treatment of respiratory distress syndrome.     

Structural investigation of the covalent and electrostatic binding of yeast cytochrome c to the surface of various ultrathin lipid multilayers using x-ray diffraction.

X-Ray diffraction was used to characterize the profile structures of ultrathin lipid multilayers having a bound surface layer of cytochrome c. The lipid multilayers were formed on an alkylated glass surface, using the Langmuir-Blodgett method. The ultrathin lipid multilayers of this study were: five monolayers of arachidic acid, four monolayers of arachidic acid with a surface monolayer of dimyristoyl phosphatidylserine, and four monolayers of arachidic acid acid with a surface monolayer of thioethyl stearate. Both the phosphatidylserine and the thioethyl stearate surfaces were found previously to covalently bind yeast cytochrome c, while the arachidic acid surface electrostatically binds yeast cytochrome c. Meridional x-ray diffraction data were collected from these lipid multilayer films with and without a bound yeast cytochrome c surface layer. A box refinement technique, previously shown to be effective in deriving the profile structures of ultrathin multilayer lipid films with and without electrostatically bound cytochrome c, was used to determine the multilayer electron density profiles. The surface monolayer of bound cytochrome c was readily apparent upon comparison of the multilayer electron density profiles for the various pairs of ultrathin multilayer films plus/minus cytochrome c for all cases. In addition, cytochrome c binding to the multilayer surface significantly perturbs the underlying lipid monolayers.     

生物芯片与肿瘤研究

生物芯片技术是指通过微加工和微电子技术,在芯片表面构建微型生物化学分析系统,对组织细胞中的蛋白质、DNA或者其他生物组分进行高通量检测。生物芯片广泛应用于生命科学、司法鉴定、食品及营养科学、环境科学、农林科学、军事科学等多种领域。本文重点对其在肿瘤研究和诊断治疗中的应用做一简要综述。     

Immobilized protein films for assessing surface proteolysis kinetics

Enzymatic cleavage of protein substrates at solid surfaces is important in the food and detergent industries, and in biomedical applications. Creation of a reproducible protein substrate to study surface proteolysis is difficult as protein monolayers may not necessarily provide complete coverage of the surface, and protein multilayer systems are often unstable and nonuniform. We present a method to form a reproducible, immobilized, multilayer protein substrate. A 100-nm ovalbumin protein film is spin-cast onto an amine-functionalized silicon wafer and chemically cross-linked using glutaraldehyde to create a multilayer film. This protein film is stable in the presence of non-protease components such as detergents, and can be tailored to include different proteins and their mixtures, and varying degrees of susceptibility to proteolysis. Ellipsometry was used to measure the protein-film thickness as the substrate is cleaved by the protease subtilisin Carlsberg. The decrease in film thickness over time was found to be linear, indicating the depth-homogeneity of the model substrates. Lateral-homogeneity of the substrates was corroborated by atomic force microscopy (AFM) and by the reproducibility of the ellipsometric film thickness measured across different spots on the sample substrates. AFM of the multilayer protein surface before and after exposure to enzyme suggests uniform areal surface cleavage by the protease.     

Optimization of Surface Plasmon Resonance Biosensor with Ag/Au Multilayer Structure and Fiber-Optic Miniaturization

In this paper, we report a novel wavelength interrogation-based surface plasmon resonance (SPR) system, in which a film of three Ag layers and three Au layers are alternately deposited on a Kretschmann configuration as sensing element. This multilayer film shows higher sensitivity for refractive index (RI) measurement by comparing with single Au layer structure, which is consistent with its theoretical calculation. A sensitivity range of 2056–5893 nm/RIU can be achieved, which is comparable to RI sensitivities of other wavelength-modulated SPR sensors. Compared with Ag film, this Ag/Au multilayer arrangement offers anti-oxidant protection. This SPR biosensor based on a cost-effective Ag/Au multilayer structure is applicable to the real-time detection of specific interactions and dissociation of low protein concentrations. To extend the application of this highly-sensitive metal film device, we integrated this concept on an optical fiber. The range of RI sensitivities with Ag/Au multilayer was 1847–3309 nm/RIU. This miniaturized Ag/Au multilayer-based fiber optic sensor has a broad application in chemical and biological sensing.     

Biochip as a potential platform of serological interferon alpha2b antibody assay

The formation of antibodies against cytokines may play a major role in the generation of the immune response and may affect treatment protocols with recombinant cytokines. Interferon (IFN) is one of the effective therapeutic agents with anti-viral and anti-tumor specific effects. The appearance of IFN antibodies in patients may limit the natural and the therapeutic effect by IFNs. In contrast to conventional ELISA techniques, we here report a simple biochip methodology that enables identification of antibodies against cytokines and peptides. The method takes advantage of a functionalized self-assembled monolayer modified by N-hydroxysuccinimide (NHS). To validate this surface, four human proteins: IFNalpha2b, leptin, growth hormone and human IgG, with molecular sizes ranging between 14 and 150 kDa, were used. A number of other parameters for protein assay conditions by array technology were evaluated concomitantly. Finally, 56 serum samples from patients treated with recombinant human IFNalpha2b were simultaneously tested on single chip. In these patients, 16.1% (9 of 56 cases) were positive for IFNalpha2b antibodies. All results were confirmed in an ELISA, specific for the identification of IFNalpha specific antibodies in human samples. The potential application of this protein biochip can be amplified rapidly and reliably to test not only IFNalpha2b, but also other cytokine specific antibodies. The clinical relevance of such assays for investigations in autoimmune disorders is expected.