生物素修饰纳米银探针的制备及在蛋白芯片可视化检测中的应用

采用寡核苷酸为连接分子成功制备了生物素修饰的纳米银探针, 并建立了纳米银催化同种金属离子的特异性还原显色反应. 实验采用蛋白质芯片为分析工具, 以微量人IgG为蛋白分析模式研究了纳米银探针/氢醌/硝酸银体系的显色分析性能. 实验结果表明, 上述检测体系可对160 fg~100 pg含量范围内的微量蛋白显示可视化结果, 蛋白点的灰度值与其浓度具有良好的相关性, 最小蛋白检测量可达160 fg. 同时还开展了与商品化链亲和素纳米金/银增强试剂显色方法的对比实验, 结果表明, 本法制备的探针对蛋白的检出限降低了约40倍, 且具有存储稳定、反应快速等优点.     

Using a silver-enhanced microarray sandwich structure to improve SERS sensitivity for protein detection

A simple and sensitive method, based on surface-enhanced Raman scattering (SERS), for immunoassay and label-free protein detection is reported. A series of bowl-shaped silver cavity arrays were fabricated by electrodeposition using a self-assembled polystyrene spheres template. The reflection spectra of these cavity arrays were recorded as a function of film thickness, and then correlated with SERS enhancement using sodium thiophenolate as the probe molecule. The results reveal that SERS enhancement can be maximized when the frequency of both the incident laser and the Raman scattering approach the frequency of the localized surface plasmon resonance. The optimized array was then used as the bottom layer of a silver nanoparticle–protein–bowl-shaped silver cavity array sandwich. The second layer of silver was introduced by the interactions between the proteins in the middle layer of the sandwich architecture and silver nanoparticles. Human IgG bound to the surface of this microcavity array can retain its recognition function. With the Raman reporter molecules labeled on the antibody, a detection limit down to 0.1 ng mL^?1 for human IgG is easily achieved. Furthermore, the SERS spectra of label-free proteins (catalase, cytochrome C, avidin and lysozyme) from the assembled sandwich have excellent reproducibility and high quality. The results reveal that the proposed approach has potential for use in qualitative and quantitative detection of biomolecules.

Fabrication and chemical surface modification of nanoporous silicon for biosensing applications

In this work, porous silicon (PS) films with varied porosity (68–82%) were formed on the p-type, boron-doped silicon wafer (100) by the electrochemical anodisation in an aqueous hydrofluoric acid and isopropyl alcohol solution at different current densities (I _d) ranging from 20–70 mA cm^?2, respectively. Biofunctionalisation of the PS surface was carried out by chemically modifying the surface of PS by the deposition of 3-aminopropyltriethoxysilane thermally leading to high density of amine groups covering the PS surface. This further promotes the immobilisation of immunoglobulin (human IgG and goat anti-human IgG binding) on to the PS surface. Formation of nanostructured PS and the attachment of antibody–antigen to its surface were characterised using photoluminescence (PL), Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy techniques, respectively. The possibility of using these structures as biosensors has been explored based on the significant changes in the PL spectra before and after exposing the PS optical structures to biomolecules. These experimental results open the possibility of developing optical biosensors based on the variation of the PL position of the PL spectra of PS-based devices.     

Capillary-Channeled Polymer (C-CP) Films as Processing Platforms for Protein Analysis by Matrix-Assisted Laser/Desorption Ionization Mass Spectrometry (MALDI-MS)

Polypropylene (PP) capillary-channeled polymer (C-CP) films have parallel, μm-sized channels that induce solution wicking via capillary action. Efficient mass transport from the solution phase to the channel surface leads to adsorption of hydrophobic protein solutes. The basic premise by which C-CP films can be used as media to manipulate analyte solutions (e.g., proteins in buffer), for the purpose of desalting or chromatographic separation prior to MALDI-MS analysis is presented here. Cytochrome c and myoglobin prepared in a Tris-HCl buffer, and ribonuclease A, lysozyme, and transferrin prepared in phosphate buffered saline (PBS), are used as the test solutions to demonstrate the desalting concept. Protein analysis is performed after deposition on a C-CP film with and without a water washing step, followed by spray deposition of a typical sinapinic acid matrix. Extracted MALDI mass spectra exhibit much improved signal-to-noise characteristics after water washing. A mixture of cytochrome c and myoglobin (2 μL of 2.5 μM each in Tris-HCl buffer) was applied, washed with water and spatially separated via simple capillary action (wicking) using a reversed-phase solvent composition of 0.1% trifluoroacetic acid (TFA) in 50:50 acetonitrile (ACN):H₂O. Subsequent application of sinapinic acid followed by imaging of the film using MALDI-MS reveals that as the protein solution is wicked down the film, separation occurs.     

Antibody-antigen interaction on polystyrene: an in situ ellipsometric study

The objective of this investigation was to monitor the adsorption of antibodies to polystyrene surfaces using ellipsometry. Commercial polystyrene slides used for solid state diagnostics were selected as substrates and the adsorption of three different antibodies (human IgG, bovine IgG and goat anti-human IgG) were evaluated. Based on theoretical models describing the ellipsometric data, it was concluded that the adsorption of antibodies should result in layers that are sufficiently thick to be able to monitor the adsorption in terms of adsorbed amount and thickness of the layer with a reasonable precision. The experimental results confirmed this assumption and values of 2.0-2.3 mg/m(2) were detected for the adsorbed amount with a corresponding thickness of 10-16 nm. It was furthermore found that the antibodies bound irreversibly with respect to rinsing with protein-free solutions. In additional experiments, the consecutive incubation of human IgG and anti-human IgG was investigated. These results showed that, on average, approximately half of the surface immobilized anti-human IgG molecules are capable of binding to human IgG during its incubation. From the consecutive binding experiments it could also be concluded that antibodies present in the polyclonal anti-human IgG preparation were capable of binding to around four different epitopes on the human IgG. A final set of experiments addressed the stability of adsorbed human IgG layers with respect to drying and incubation with surfactant. The results revealed that the adsorbed antibody layer is relatively resistant to these treatments.     

A method of printing uniform protein lines by using flat PDMS stamps

In this paper, we report a method of printing uniform protein lines on glass slides by using UV-treated flat PDMS stamps. Unlike traditional microcontact printing (μCP) which requires microstructured PDMS stamps, this μCP method only requires a flat PDMS stamp, an UV lamp and a number of straight needles. Our results show that lines of bovine serum albumin (BSA), immunoglobin (IgG), anti-biotin, anti-human IgG and anti-mouse IgG can be printed evenly on glass slides by using this μCP method. We also demonstrate that the printed protein lines are suitable for applications such as microfluidic immunoassays.     

Swellable polymer films containing Au nanoparticles for point-of-care therapeutic drug monitoring using surface-enhanced Raman spectroscopy

Large (10 × 10 cm) sheets of surface-enhanced Raman spectroscopy (SERS) active polymer have been prepared by stabilising metal nanoparticle aggregates within dry hydroxyethylcellulose (HEC) films. In these films the aggregates are protected by the polymer matrix during storage but in use they are released when aqueous analyte droplets cause the films to swell to their gel form. The fact that these “Poly-SERS” films can be prepared in bulk but then cut to size and stored in air before use means that they provide a cost effective and convenient method for routine SERS analysis. Here we have tested both Ag and Au Poly-SERS films for use in point-of-care monitoring of therapeutic drugs, using phenytoin as the test compound. Phenytoin in water could readily be detected using Ag Poly-SERS films but dissolving the compound in phosphate buffered saline (PBS) to mimic body fluid samples caused loss of the drug signal due to competition for metal surface sites from Cl⁻ ions in the buffer solution. However, with Au Poly-SERS films there was no detectable interference from Cl⁻ and these materials allowed phenytoin to be detected at 1.8 mg L⁻¹, even in PBS. The target range of detection of phenytoin in therapeutic drug monitoring is 10–20 mg L⁻¹. With the Au Poly-SERS films, the absolute signal generated by a given concentration of phenytoin was lower for the films than for the parent colloid but the SERS signals were still high enough to be used for therapeutic monitoring, so the cost in sensitivity for moving from simple aqueous colloids to films is not so large that it outweighs the advantages which the films bring for practical applications, in particular their ease of use and long shelf life.     

Multiple detection of proteins by SERS-based immunoassay with core shell magnetic gold nanoparticles

A highly selective and sensitive surface-enhanced Raman scattering (SERS)-based immunoassay for the multiple detection of proteins has been developed. The proposed core shell magnetic gold (Au) nanoparticles allow for successful protein separation and high SERS enhancement for protein detection. To selectively detect a specific protein in a mixed protein solution, we employed the sandwich type SERS immunoassay with core shell magnetic Au nanoparticles utilizing specific antigen–antibody interactions. Based on this proposed SERS immunoassay, we can successfully detect proteins in very low concentrations (∼800 ag/mL of mouse IgG and ∼5 fg/mL of human IgG) with high reproducibility. Magnetically assisted protein separation and detection by this proposed SERS immunoassay would provide great potential for effective and sensitive multiple protein detection. This technique allows for the straightforward SERS-based bioassays for quantitative protein detections.     

Flow Injection Fluoroimmunoassay for Human Transferrin Using a Protein a Immunoreactor

Abstract

A protein A immunoreactor (immobilized protein A) incorporating flow injection technique was used for on-line fluoroimmunoassay of human transferrin. Antibody immobilized on protein A and antibody-antigen complex formation took place in phosphate buffered saline (PBS, PH 7.4). After washing off excess lucifer yellow VS labelled human transferrin, the antibody-antigen complex was eluted with acid buffer and detected. Experimental variables have been studied and the method has been used to determine the transferrin contents in human serum.     

In vitro corrosion study of different TiO2 nanotube layers on titanium in solution with serum proteins

Titanium oxide nanotubes prepared by anodization have received considerable attention in the biomaterials domain. The objective of this study was to demonstrate the electrochemical behavior of different diameter TiO(2) nanotube layers on titanium in phosphate buffered saline (PBS) and Dulbecco's minimum essential medium+10% fetal calf serum (D-FCS) using open circuit potentials (OCP), electrical impedance spectroscopy (EIS), and a potentiodynamic polarization test. The results showed that the nanotubes had higher OCP, higher resistance of the inter barrier layer (R(b)), and lower I(pass) in the two test solutions compared to the smooth Ti, especially the 30 nm diameter nanotubes. The corrosion resistance of the nanotubes in D-FCS was higher than in PBS because of protein adsorption from the D-FCS solution as suggested by scanning electron microscope (SEM) images. In addition, protein aggregates of 30 nm diameter nanotubes caused the model of EIS spectra to transform from two-layer to three-layer. The corrosion behavior of the nanotubes for use as a dental implant material is discussed.     

Magnetron sputtering of silver nanowires using anodic aluminum oxide template: a new active substrate of surface enhanced Raman scattering and an investigation of its enhanced mechanism

A high quality anodic aluminum oxide (AAO) template with ordered apertures about 50-80 nm was fabricated by anodizing aluminum in electrolytes through a two-step method, and silver nanowires with diameters from 40nm to 70nm were prepared on this AAO template by magnetron sputtering. On the glass covered with silver nanowires, high quality surface enhanced Raman scattering (SERS) spectra of sudan II (C₁₈H₁₆N₂O) with enhancement factors of 10⁵ were obtained. And comparison of SERS spectra on silver nanowires with the SERS spectra of silver colloids indicates that main enhanced mode is lightning rod effect of nanorods on the Sudan II/silver nanowires system.     

Layered zinc hydroxide salts: delamination, preferred orientation of hydroxide lamellae, and formation of ZnO nanodiscs

Herein, we report our analysis of the surface modification of polystyrene (PS) when treated under ambient conditions with a common biological buffer such as phosphate buffered saline (PBS) or aqueous solutions of the ionic constituents of PBS. Attenuated total reflection Fourier transform infrared spectroscopy was used for the analysis because the resultant spectra are very sensitive to minor changes in the chemical and structural properties of PS films. In addition, ultraviolet-visible spectroscopy was applied to characterize the surface modifications of PS. Treatment with PBS resulted in the most significant chemical and structural surface modifications of the PS films, as compared with each of the solutions of the constituents of PBS, which were tested separately. A multistep mechanism for the wet modification of PS is discussed. We postulate that the observed surface modifications are the result of photo-oxidation/reduction, swelling, and conformational changes and re-arrangement of the polymer chain. The resultant surface modifications could be similar to those produced by commonly used dry processes such as plasma treatments and electron, ion or ultraviolet irradiation. We found that the modifications that occurred in PBS were more stable than those initiated by dry processes. The formation of active groups on the surface of PS can be controlled by adsorption of bovine serum albumin or thermal annealing of PS before PBS treatment. This approach provides a simple and efficient method for the surface modification of PS for biomedical applications.     

Spatially focused deposition of capillary electrophoresis effluent onto surface-enhanced Raman-active substrates for off-column spectroscopy

Surface-enhanced Raman scattering (SERS) is employed to obtain distinctive spectra of compounds that are efficiently separated by capillary electrophoresis (CE) and deposited onto planar SERS-active substrates. A simple method is described that explains how to prepare SERS-active substrates by depositing a silver-colloid solution onto frosted-glass microscope slides, using a high-efficiency nebulizer. Scanning electron micrographs reveal a layered coating of fairly uniform-sized, 100-nm silver nanoparticles with interstitial spaces ranging from a few to tens of nanometers. The on-column separation is monitored by laser-induced fluorescence, while electrofilament depositing the CE effluent onto a moving SERS-substrate. Subsequently, the SERS spectra and off-column electropherograms are obtained with a simple confocal Raman spectrometer. The test compounds used to demonstrate this technique include compounds of biological significance: benzyloxyresorufin, riboflavin, and resorufin. CE and Raman conditions are evaluated to determine their affects on the SERS signals. An average off-column efficiency of 100,000 plates/m and a signal reproducibility of 11% relative standard deviation were achieved. Characteristic spectra with major Raman bands exhibiting signal-to-noise ratios of greater than 3 were obtained for a 3.2-nL injection of 10(-6) M (706 fg) resorufin. Forming a self-assembled monolayer (SAM) on the substrate increases the sensitivity of the SERS technique and decreases the on-substrate broadening. Calibration plots for both plain- and SAM-SERS substrates are demonstrated.     

Gold nanoparticle multilayer films based on surfactant films as a template: preparation, characterization, and application

Highly ordered gold nanoparticle multilayer films were achieved conveniently using didodecyldimethylammonium bromide (DDAB) films as a template. The template was produced by casting DDAB chloroform solution onto the surface of a (3-aminopropyl)trimethoxysilane-modified indium tin oxide substrate and then evaporating the organic solvent. Gold nanoparticle multilayer films were prepared by soaking the template in 2.6 nm colloidal gold solution for 120 min. The well-ordered superlattice structure of the DDAB template and the gold nanoparticle multilayer films was identified by x-ray diffraction. The characterizations of the gold nanoparticle multilayer films by UV-vis spectroscopy, atomic force microscopy, and cyclic voltammerty were described in detail. The application of the as-prepared gold nanoparticle multilayer films in surface-enhanced Raman spectroscopy (SERS) was investigated by using Rhodamine 6G as a probe molecule. It was found that the colloidal gold nanoparticle multilayer films exhibit remarkable enhancement ability and can be used as SERS substrates.     

Laser-MBE of nickel nanowires using AAO template: a new active substrate of surface enhanced Raman scattering

The highly ordered anodic aluminum oxide (AAO) template was fabricated using aluminum anodizing in electrolytes with two-step method, which apertures were about 50-80nm. The nickel nanowires with about 40-70nm in diameter was prepared on the AAO template by laser-MBE (molecular beam epitaxy). And high quality Raman spectra of SudanII were obtained on the glass covered with the nickel nanowires. On the nickel nanowires there are both surface enhanced Raman scattering (SERS) and tip enhanced Raman scattering (TERS). The new observations not only enlarge the range of SERS applications, but also imply a possible new enhancement mechanism. Otherwise the Raman and SERS frequencies of SudanII molecule were calculated using, respectively, DFT and B3PW91.     

Microspectrometric investigation of active substrates for surface enhanced Raman scattering

The results of investigations of several new active silver substrates and some previously reported active silver substrates for surface enhanced Raman spectrometry (SERS) using a Raman microprobe are given. Filter-papers of different composition and porosity, silver membranes and glass slides are evaluated as supports for SERS active substrates. Methods of silver preparation include evaporation and chemical reduction. The Raman microprobe facilitates the acquisition of SER spectra of the adsorbate over the metal microstructure being observed on a TV monitor. This capability allows the establishment of practical relationships between the surface morphology and SERS activity which can be used as guidelines for SERS experiments with the microprobe. For the most monodisperse substrates, it is possible to establish a linear relationship between SERS intensity and adsorbate concentration. In the lower extreme of the calibration graph, the amount of adsorbate being observed under the microscope objective is only 0.3 amol or 1.9 × 10⁵ molecules.     

A comparative analysis of polyurethane hydrogel for immobilization of IgG on chips

Hydrogels are considered an optimum material for protein chip surfaces, since they provide a quasi-liquid environment which allows protein activity to be maintained and shows good spot morphology as well as excellent immobilization capacity. In the following, we present a polyurethane (PU) chip that electrostatically binds IgG. The PU surface is optimized with regard to layer thickness (∼200 nm), hydrogel (2%) and immobilized antibody concentration (0.5 mg mL⁻¹; 0.3 ng spot⁻¹), pH and ionic strength of the print buffer as well as to blocking solution. Evaluation is done in a direct IgG immunoassay using the Nexterion slide H as a reference. It is shown that higher IgG loading is achieved on the PU chip than on slide H, no matter whether 1× PBS (pH 7.2), Sörensen (pH 5.8) or Nexterion buffer was used as a spotting solution. Moreover, the crossreactivity with goat IgG, human IgG and monoclonal anti-CRP spotted in Nexterion buffer was as low as ≤0.74% (slide H: ≤3.34%).     

Microchannel chips for the multiplexed analysis of human immunoglobulin G–antibody interactions by surface plasmon resonance imaging

We report the multiplexed, simultaneous analysis of antigen–antibody interactions that involve human immunoglobulin G (IgG) on a gold substrate by the surface plasmon resonance imaging method. A multichannel, microfluidic chip was fabricated from poly(dimethylsiloxane) (PDMS) to selectively functionalize the surface and deliver the analyte solutions. The sensing interface was constructed using avidin as a linker layer between the surface-bound biotinylated bovine serum albumin and biotinylated anti-human IgG antibodies. Four mouse anti-human IgG antibodies were selected for evaluation and the screening was achieved by simultaneously monitoring protein–protein interactions under identical conditions. Antibody–antigen binding affinities towards human immunoglobulin were quantitatively compared by employing Langmuir adsorption isotherms for the analysis of SPRi responses obtained under equilibrium conditions. We were able to identify two IgG samples with higher affinities towards the target, and the determined binding kinetics falls within the typical range of values reported in the literature. Direct measurement of proteins in serum samples by SPR imaging was achieved by developing methods to minimize nonspecific adsorption onto the avidin-functionalized surface, and a limit of detection (LOD) of 6.7 nM IgG was obtained for the treated serum samples. The combination of SPR imaging and multichannel PDMS chips offers convenience and flexibility for sensitive and label-free measurement of protein–protein interactions in complex conditions and enables high-throughput screening of pharmaceutically significant molecules. Figure Microchannel SPR imaging for protein–protein interactions     

Porous GaN as a template to produce surface-enhanced Raman scattering-active surfaces

Surface-enhanced Raman spectroscopy (SERS) substrates have been prepared by depositing Au or Ag on porous GaN (PGaN). The PGaN used as the template for the metal deposition in these studies was generated by a Pt-assisted electroless etching technique. PGaN was chosen as a potential SERS template due to its nanostructured surface and high surface area, two characteristics that are important for SERS substrates. Metal films were deposited either by solution-based electroless deposition or by thermal vacuum evaporation. SERS spectra were recorded at lambda = 752.5 nm for Au films and at lambda = 514.5 nm for Ag films deposited on PGaN. The SERS signal strength across the metal coated PGaN substrates was uniform and was not plagued by "hot" or "cold" spots on the surface, a common problem with other SERS surfaces. The Ag film deposited by electroless deposition had the highest overall SERS response, with an enhancement factor (EF) relative to normal Raman spectroscopy of 10(8). A portion of the increase in EF relative to typical SERS-active substrates can be assigned to the large surface area characteristic of the PGaN-Ag structures, but some of the enhancement is intrinsic and is likely related to the specific morphology of the metal-nanopore composite structure.     

Facile fabrication of silver nanoparticles on silicon for surface‐enhanced infrared and Raman analysis

A facile and economic method to fabricate and immobilize silver nanoparticles on a thin Si wafer (AgNP/Si) is reported for an analytical template in ambient environment by surface‐enhanced infrared/Raman spectroscopy. The protocol involves immersion of the Si wafer in a solution containing silver nitrate and hydrofluoric acid. To screen appropriate conditions for preparing AgNP/Si for SEIRAS application, different combinations of AgNO₃ and HF solutions were examined with paranitrobenzoic acid (PNBA) used as the probe molecule in transmission measurements. These SEIRA‐active substrates were also promising for SERS application, as demonstrated with high quality SERS spectra of iron (III) protoporphyrin adlayer on AgNP/Si with a red excitation line. The AgNP/Si substrates prepared under different conditions were examined by SEM for qualitative correlation of enhancements with morphologies. Copyright © 2008 John Wiley & Sons, Ltd.