测定癌胚抗原免疫芯片技术的研究

癌胚抗原的检测可作为肿瘤诊断的重要辅助方法之一,依据化学发光免疫分析技术,制备免疫芯片,以测定癌胚抗原.实验表明:本法检测限、线性范围和精密度、固定20 nL CEA-1ab时的合适浓度分别为1.0 ng/mL、1.0~64 ng/mL、<5.1%、0.8~1.0μg/mL,且未发现与AFP,Ferritin,HCG,PSA发生交叉反应.芯片于37℃存储7 d或2~8℃6个月,信号是稳定的.     

Phage display antibody-based proteomic device using resonance-enhanced detection

The combination of phage display antibody arrays with a novel nanotransducer technique based on resonant nanoparticles in a nanosandwiched film enables the sensitive parallel screening of proteins. Using the resonance of nanoparticles with their induced mirror dipoles in a thin-film structure, limitations of fluorophores, such as unspecific background and nonvisibility to the eye, can be overcome, thereby leading to an optical signal significantly more sensitive than that of standard colloid techniques. The signal can be both directly observed as a color change of a microdot at the sensor surface and tuned throughout the visible range of the spectrum. Here we report the application of an optical chip using scFv-antibody-antigen interactions. Artificial scFv-antibodies against a variety of proteins, including yeast enzymes and bovine serum albumin (as a standard), were constructed via Phage Display. These scFv-antibodies were then coated onto metal nanoclusters and bound to their antigens that were arrayed as nanodroplets at the resonance layer of the chip. ScFv-Antibody-antigen interaction resulted in a visible array of microdots. Using resonance-enhanced absorption, the absorption signal of the spots was amplified by one to two orders of magnitude (compared to colloid-based techniques). For quantitative analysis, either an 8-micron scanner or a CCD camera (resolution 4 microns) was employed to gain direct-reflection spectra rather than unspecific scatter data (prone to dust and unspecific interaction). Our results demonstrate that this device enables high-throughput proteomics to overcome some limitations of fluorescence, enzyme labels, and colloid techniques.     

Optical near-field mapping of plasmonic nanoprisms

The optical local-field enhancement on nanometer length scales provides the basis for plasmonic metal nanostructures to serve as molecular sensors and as nanophotonic devices. However, particle morphology and the associated surface plasmon resonance alone do not uniquely reflect the important details of the local field distribution. Here, we use interferometric homodyne tip-scattering near-field microscopy for plasmonic near-field imaging of crystalline triangular silver nanoprisms. Strong spatial field variation on lengths scales as short as 20 nm are observed sensitively depending on structural details and environment. The poles of the dipole and quadrupole plasmon modes, as identified by phase-sensitive probing and calculations performed in the discrete dipole approximation (DDA), reflect the particle symmetry. Together with the observation that the largest enhancement is not necessarily found to be associated with the tips of the nanoprisms, our results provide critical information for the selection of particle geometries as building blocks for plasmonic device applications.     

Optical near-field Raman imaging with subdiffraction resolution

We report optical near-field Raman imaging with subdiffraction resolution (approximately 120 nm) without field enhancement effects. Chemical discrimination on tetracyanoquinodimethane organic thin films showing localized salt complexes is accomplished by detailed Raman maps. Acquisition times that are much shorter than previously reported are due to the high Raman efficiency of the materials and to careful collection and detection of the optical signals in our near-field Raman spectrometer.     

New polyaniline-based potentiometric biosensor for pesticides detection

A cholinesterase potentiometric sensor based on a glassy carbon electrode modified with processible polyaniline has been developed and explored for the detection of organophosphorus and carbamic pesticides. Acetyl- and butyryl-cholinesterase from various sources were immobilized on the surface of a polyaniline modified electrode by cross-linking with glutaraldehyde. Polyaniline modification made it possible to obtain a reversible pH response of about 86-mV/pH unit and to improve the analytical and operational characteristics of substrate and inhibitor detection. The sensitivity of pesticide detection depends on both the source of the enzyme and its activity and on pesticide hydrophobicity. The detection limits of the pesticides investigated (Trichlorfon, 1.5/spl middot/10/sup -7/ mol/l, Coumaphos, 5/spl middot/10/sup -9/ mol/l, Methiocarb, 8/spl middot/10/sup -7/ mol/l, Aldicarb, 2/spl middot/10/sup -7/ mol/l) were found lower than those obtained with other similar cholinesterase sensors. The possibility for the detection of the trace amounts of pesticides in river waters and grape juice with cholinesterase sensors developed has been established on model samples.     

Impedance endothelial cell biosensor for lipopolysaccharide detection

It is important to analyse endothelial cell adherence for the development of biomedical devices of antithrombogenic vascular grafts. Endothelial cells must be firmly attached to the biomaterials when cells are seeded in order to create a natural lining.

Polystyrene (PS) is presented as a reproducible implant model substrate for studying cell–material interactions. Polystyrene was deposited as a thin layer on a thiol functionalised gold electrode. Fibronectin, a protein promoting the endothelial cell adhesion was then adsorbed on PS surface. The different steps of this multilayer assembly were characterized by Electrochemical Impedance Spectroscopy (EIS). The charge transfer resistance and the capacitance of the total layer were modified at each step in agreement with the electrical properties of each layer. The electrical properties of the confluent layer of endothelial cells were determined: (i) a charge transfer resistance of 2 kΩ cm^− 2 shows no large defects in the cell layer, (ii) as the cells attach and spread on the gold electrode, the impedance increases.

EIS was used for testing behaviour of endothelial cells on substrate coated by fibronectin layer and in presence of cytotoxicants such as lipopolysaccharide (LPS). The impedance measurement may be a valuable method for the assessment of mechanisms of decreased endothelial barrier function occurring with inflammatory mediators. The results indicate that LPS causes a dose-dependent decrease in impedance of the endothelial cell monolayer, indicating widening of the paracellular pathways and increasing vascular endothelial permeability. This study is an increasing trend towards the development of impedimetric biosensors and designing cell sensor arrays for toxic and drug detection.     

Zinc oxide nanostructured biosensor for glucose detection

Zinc oxide （ZnO） nanocombs were fabricated by vapor phase transport, and nanorods and hierarchical nanodisk structures by aqueous thermal decomposition. Glucose biosensors were constructed using these ZnO nanostructures as supporting materials for glucose oxidase （GOX） loading. These ZnO glucose biosensors showed a high sensitivity for glucose detection and high affinity of GOX to glucose as well as the low detection limit. The results demonstrate that ZnO nanostructures have potential applications in biosensors.     

DNA biosensor for the detection of hydrazines

A double-stranded (ds) DNA-coated carbon paste electrode is employed as a remarkably sensitive biosensor for the detection of hydrazine compounds. The sensor relies on monitoring changes in the intrinsic anodic response of the surface-confined DNA resulting from its interaction with hydrazine compounds and requires no label or indicator. Short reaction times (1-10 min) are sufficient for monitoring part-per-billion levels of different hydrazines. Applicability to untreated natural water samples is illustrated. The response mechanism is discussed, along with prospects of using DNA biosensors for quantitaing other important molecules and elucidating DNA interactions and damage.     

Optical biosensor based on hollow integrated waveguides

The first absorbance biosensor based on pure silicon hollow integrated waveguides is presented in this work. With the use of horseradish peroxidase (HRP) as a model recognition element, an enzymatic sensor for the measurement of hydrogen peroxide was fabricated, numerically simulated, and experimentally characterized. Waveguides with widths ranging from 50 to 80 microm, having a depth of 50 microm and lengths up to 5 mm were easily fabricated by just one photolithographic step. These were further modified by covalent immobilization of HRP using silanization chemistry. Simulation studies of the proposed approach showed a sensor linear behavior up to 300 microM H2O2 and a sensitivity of 2.7 x 10(-3) AU/microM. Experimental results were in good agreement with the simulated ones. A linear behavior between 10 and 300 microM H2O2, a sensitivity of 3 x 10(-3) AU/microM, and a signal-to-noise ratio around 20 dB were attained. Also, kinetic studies of the activity of the immobilized enzyme on the silicon waveguide surface gave an apparent Michaelis-Menten constant of 0.44 mM. The simple technology proposed in this work enables the fabrication of cost-effective, easy-to-use, miniaturized biosensor generic platforms, these being envisioned as excellent candidates for the development of lab-on-a-chip systems.     

Optical near-field scanning by microparticles suspended in immersion fluid

A new method of obtaining images with a resolution exceeding the diffraction limit of an optical microscope is proposed and experimentally implemented. The method is based on monitoring the pattern of free motion of microparticles over the sample surface and an analysis of the intensity of light scattered by these particles. In experiment, a threefold increase in the ultimate resolution of a microscope has been achieved.     

Antimicrobial peptides for detection of bacteria in biosensor assays

Bacteria, plants, and higher and lower animals have evolved an innate immune system as a first line of defense against microbial invasion. Some of these organisms produce antimicrobial peptides (AMPs) as a part of this chemical immune system. AMPs exert their antimicrobial activity by binding to components of the microbe's surface and disrupting the membrane. The overall goal of this study was to apply the AMP magainin I as a recognition element for Escherichia coli O157:H7 and Salmonella typhimurium detection on an array-based biosensor. We immobilized magainin I on silanized glass slides using biotin-avidin chemistry, as well as through direct covalent attachment. Cy5-labeled, heat-killed cells were used to demonstrate that the immobilized magainin I can bind Salmonella with detection limits similar to analogous antibody-based assays. Detection limits for E. coli were higher than in analogous antibody-based assays, but it is expected that other AMPs may possess higher affinities for this target. The results showed that both specific and nonspecific binding strongly depend on the method used for peptide immobilization. Direct attachment of magainin to the substrate surface not only decreased nonspecific cell binding but also resulted in improved detection limits for both Salmonella and E. coli.     

An Au/Si hetero-nanorod-based biosensor for Salmonella detection

We present a novel and effective food-borne bacteria detection method. A hetero-structured silicon/gold nanorod array fabricated by the glancing angle deposition method is functionalized with anti-Salmonella antibodies and organic dye molecules. Due to the high aspect ratio nature of the Si nanorods, dye molecules attached to the Si nanorods produce an enhanced fluorescence upon capture and detection of Salmonella. This bio-functional hetero-nanorod detection method has great potential in the food safety industry as well as in biomedical diagnostics.     

Embedded DNA-polypyrrole biosensor for rapid detection of Escherichia Coli

The principal objective of this paper was to present the design and fabrication of a single-strand (ss) DNA biosensor for the detection of Escherichia coli (E. coli) DNA synthetic oligonucleotides as a model of rapid detection of bacterial select bioterrorism agents. Molecular biology and chemical electrodeposition techniques, such as cyclic voltammetry (CV), were combined to develop and test a model DNA-based biosensor on a platinum (Pt) electrode electropolimerized with polypyrrole (PPY). The hybridization on embedded DNA into PPY with complementary DNA samples was determined. The recognition element was a 25 base pair (bp) oligonucleotide specific for E. coli derived from the uidA gene that codes for the enzyme /spl beta/-D glucuronidase. CV scans between 0.0 and +0.70 V at a 50-mV/s scanning rate generated current versus potential graphs. A standard DNA concentration of 1 /spl mu/g//spl mu/L was used to determine the hybridization signal of the biosensor. The model biosensor generated distinctive CV signals between complementary and noncomplementary DNA oligonucleotides. The biosensor proved to be effective in the detection of complementary uidA 25-bp oligonucleotide for E. coli K-12.     

Nine-analyte detection using an array-based biosensor

A fluorescence-based multianalyte immunosensor has been developed for simultaneous analysis of multiple samples. While the standard 6 x 6 format of the array sensor has been used to analyze six samples for six different analytes, this same format has the potential to allow a single sample to be tested for 36 different agents. The method described herein demonstrates proof of principle that the number of analytes detectable using a single array can be increased simply by using complementary mixtures of capture and tracer antibodies. Mixtures were optimized to allow detection of closely related analytes without significant cross-reactivity. Following this facile modification of patterning and assay procedures, the following nine targets could be detected in a single 3 x 3 array: Staphylococcal enterotoxin B, ricin, cholera toxin, Bacillus anthracis Sterne, Bacillus globigii, Francisella tularensis LVS, Yersiniapestis F1 antigen, MS2 coliphage, and Salmonella typhimurium. This work maximizes the efficiency and utility of the described array technology, increasing only reagent usage and cost; production and fabrication costs are not affected.     

Optical properties of microfabricated fully-metal-coated near-field probes in collection mode

A study of the optical properties of microfabricated, fully-metal-coated quartz probes collecting longitudinal and transverse optical fields is presented. The measurements are performed by raster scanning the focal plane of an objective, focusing azimuthally and radially polarized beams by use of two metal-coated quartz probes with different metal coatings. A quantitative estimation of the collection efficiencies and spatial resolutions in imaging both longitudinal and transverse fields is made. Longitudinally polarized fields are collected with a resolution approximately 1.5 times higher as compared with transversely polarized fields, and this behavior is almost independent of the roughness of the probe's metal coating. Moreover, the coating roughness is a critical parameter in the relative collection efficiency of the two field orientations.     

Transfer function and near-field detection of evanescent waves

We consider characterization of a near-field optical probe in terms of detection efficiency of different spatial frequencies associated with propagating and evanescent field components. The former are both detected with and radiated from an etched single-mode fiber tip, showing reciprocity of collection and illumination modes. Making use of a collection near-field microscope with a similar fiber tip illuminated by an evanescent field, we measure the collected power as a function of the field spatial frequency in different polarization configurations. Considering a two-dimensional probe configuration, numerical simulations of detection efficiency based on the eigenmode expansion technique are carried out for different tip apex angles. The detection roll-off for high spatial frequencies observed in the experiment and obtained during the simulations is fitted using a simple expression for the transfer function, which is derived by introducing an effective point of (dipolelike) detection inside the probe tip. It is found to be possible to fit reasonably well both the experimental and the simulation data for evanescent field components, implying that the developed approximation of the near-field transfer function can serve as a simple, rational, and sufficiently reliable means of fiber probe characterization.     

Polyaniline-based highly sensitive microbial biosensor for selective detection of lindane

A highly sensitive, selective, and rapid, whole-cell-based electrochemical biosensor was developed for detection of the persistent organochlorine pesticide γ-hexachlorocyclohexane (γ-HCH), commonly known as lindane. The gene linA2 encoding the enzyme γ-hexachlorocyclohexane (HCH) dehydrochlorinase (LinA2), involved in the initial steps of lindane (γ-HCH) biotransformation, was cloned and overexpressed in Escherichia coli . The lindane-biodegrading E. coli cells were immobilized on polyaniline film. The rapid and selective degradation of lindane and concomitant generation of hydrochloric acid by the recombinant E. coli cells in the microenvironment of polyaniline led to a change in its conductivity, which was monitored by pulsed amperometry. The biosensor could detect lindane in the part-per-trillion concentration range with a linear response from 2 to 45 ppt. The sensor was found to be selective to all the isomers of hexachlorocyclohexane (HCH) and to pentachlorocyclohexane (PCCH) but did not respond to other aliphatic and aromatic chlorides or to the end product of lindane degradation, i.e., trichlorobenzene (TCB). The sensor also did not respond to other commonly used organochlorine pesticides like DDT and DDE. On the basis of experimental results, a rationale has been proposed for the excellent sensitivity of polyaniline as a pH sensor for detection of H(+) ions released in its microenvironment.