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1.
Suspension array technology for simultaneous detection of three kinds of veterinary drugs, chloramphenicol (CAP), clenbuterol and 17-beta-estradiol has been developed. Conjugates of chloramphenicol and clenbuterol coupled with bovine serum albumin were synthesized and purified. Probes of suspension array were constituted by coupling the three conjugates on the fluorescent microspheres/beads and the microstructures of the beads’ surface were observed by scanning electron microscopy which was a direct confirmation for the successful conjugates’ coupling. The optimal addition of conjugates and the amounts of antibodies were optimized and selected, respectively. Standard curves were plotted and the coefficient of determination-R2 was greater than 0.989 which suggested good logistic correlation. The detection ranges for the three veterinary drugs are 40-6.25 × 105 ng L−1, 50-7.81 × 105 ng L−1 and 1 × 103-7.29 × 105 ng L−1, respectively and the lowest detection limits (LDLs) of them are 40, 50 and 1000 ng L−1, respectively. The suspension array is specific and has no significant cross-reactivity with other chemicals. Meanwhile, unknown samples were detected by suspension array and ELISA in comparison with each other. The errors between found and real for the detection of the unknown samples were relatively small to both of the two methods, whereas, the detection ranges of suspension array are broader and sensitive than that of the traditional ELISA. The high-throughput suspension array is proved to be a novel method for multi-analysis of veterinary drugs with simple operation, high sensitivity and low cost.  相似文献   

2.
A method for preparing a glass surface containing an ordered array of nanowells is described. These nanowell arrays are prepared via a plasma-etch method using a nanopore alumina film as the etch mask. A replica of the pore structure of the alumina mask is etched into the glass. We demonstrate that chemical information in the form of negatively charged latex nanoparticles can be selectively stored within these nanowells and not indiscriminately deposited on the surface surrounding the nanowells. To accomplish this, the chemistry of the glass surfaces within these nanowells (walls and bottoms) must be different from the chemistry of the surface surrounding the nanowells. Two different procedures were developed to make the inside vs. surrounding surface chemistries different. Atomic force microscopy (AFM) was used to image the nanowells and, via friction-force measurements, to prove that the inner nanowell surfaces can be made chemically different from the surface surrounding the nanowells.  相似文献   

3.
A new arraying method is presented based on the properties of poly(dimethylsiloxane) (PDMS) polymer to entrap beads bearing biologically active compounds. It is shown that such beads could be spotted and dried at the surface of a poly(vinyl chloride) master and subsequently transferred at the PDMS interface by direct moulding of the polymer on the mask. Moreover, the use of the PDMS-assisted-immobilization enables the development of either a low density array (100 spots) or a micro-channel biochip with a direct incorporation of the sensing element in a fluidic system for the quantitative detection of enzyme substrates, antigens and oligonucleotides, depending on the immobilized sensing element. All biochip formats were revealed by a chemiluminescent reaction detected with a charge coupled device camera.As a result, arrays of beads bearing active enzymes, antibodies and oligonucleotides were successfully obtained and enabled the achievement of biochips for the chemiluminescent detection of enzyme substrates, protein antigens and oligonucleotides sequence with detection limit of 1 μM, 1.5×107 molecules and 108 molecules, respectively.  相似文献   

4.
An alternative approach for fabricating a protein array at nanoscale is suggested with a capability of characterization and/or localization of multiple components on a nanoarray. Fluorescent micro- and nanobeads each conjugated with different antibodies are assembled by size-dependent self-assembly (SDSA) onto nanometer wells that were created on a polymethyl methacrylate (PMMA) substrate by electron beam lithography (EBL). Antibody-conjugated beads of different diameters are added serially and electrostatically attached to corresponding wells through electrostatic attraction between the charged beads (confirmed by zeta potential analysis) and exposed p-doped silicon substrate underneath the PMMA layer. This SDSA method is enhanced by vibrated-wire-guide manipulation of droplets on the PMMA surface containing nanometer wells. Saturation rates of antibody-conjugated beads to the nanometer patterns are up to 97% under one component and 58–70% under two components nanoarrays. High-density arrays (up to 40,000 wells) could be fabricated, which can also be multi-component. Target detection utilizes fluorescence resonance energy transfer (FRET) from fluorescent beads to fluorescent-tagged secondary antibodies to Octamer-4 (Oct4), which eliminates the need for multiple steps of rinsing. The 100 nm green beads are covalently conjugated with anti-Oct4 to capture Oct4 peptides (39 kDa); where the secondary anti-Oct4 and F(ab)2 fragment of anti-gIgG tagged with phycoerythrin are then added to function as an indicator of Oct4 detection. FRET signals are detected through confocal microscopes, and further confirmed by Fluorolog3 spectrofluorometer. The success rates of detecting Oct4 are 32% and 14% of the beads in right place under one and two component nanoarrays, respectively. Ratiometric FRET is used to quantify the amount of Oct4 peptides per each bead, which is estimated about 2 molecules per bead.  相似文献   

5.
A new suspension array built upon laboratory-prepared functional fluorescence-encoded polystyrene beads (FFPBs) was developed for multiplex immunodetection of tumor markers. The FFPBs were synthesized by copolymerizing rhodamine 6G (R6G) and carboxyl function groups on the surface of the seed beads forming a core-shell structure. The fabrication process was facile and the encoding fluorescence intensity of the beads can be precisely controlled by adjusting the quantity of R6G. In present work, we demonstrated that the quantity variation of impregnated R6G had negligible effect on the coupling efficiency of biomolecules onto the surface of the FFPBs. The R6G encoding fluorescence remained good monodispersity upon capture probe coupling and immunocomplex formation. No fluorescence resonance energy transfer was observed between the R6G doped in the bead shell and fluorophore used for antibody labeling. Under the optimal conditions, the proposed suspension array allowed simultaneous detection of α-fetoprotein, carcinoembryonic antigen, and prostate specific antigen in the ranges of 0.07-500 ng mL−1, 1-2000 ng mL−1, and 0.5-500 ng mL−1, respectively, with detection limits of 0.0626 ng mL−1, 0.554 ng mL−1, and 0.250 ng mL−1. Test on clinical serum samples demonstrated that the results obtained with suspension array were in good agreement with those of the reference electrochemiluminescence immunoassay method. We conclude that the laboratory-made FFPBs are sufficient as the microcarrier for the construction of suspension array in clinical diagnosis.  相似文献   

6.
A microchip capillary electrophoresis system with highly sensitive fluorescence detection is reported. The system was successfully constructed using an inverted fluorescence microscope, a highly sensitive photon counter, a photomultiplier tube (PMT) and a capillary electrophoresis microchip. This system can be applied to the fluorescence detection with various wavelengths (300-600 nm). Different fluorescence reagents require different excitation wavelengths. The wavelengths of UV light (300-385 nm), blue light (450-480 nm) and green light (530-550 nm) are employed to excite Titan yellow, fluorescence-5-isothiocyanate (FITC) and Rhodamine 6G, respectively. The detection limit (S/N = 3) of FITC is 7 × 10−10 M, which is 2-3 orders of magnitude lower than that obtained with the lamp-based fluorescence and PMT detection system and approaches the data gained by the laser-induced fluorescence detection. The linear relationship is excellent within the range of concentration 1.3 × 10−9 to 6.5 × 10−8 M FITC. It offers a new method to widen the application of the lamp-based fluorescence detection.  相似文献   

7.
Shen H  Fang Q 《Talanta》2008,77(1):269-272
An improved microfluidic chip-based sequential-injection trapped-droplet array liquid-liquid extraction system with chemiluminescence (CL) detection was developed in this work. Two recess arrays were fabricated on both sides of the extraction channel to produce droplet arrays of organic extractant. A chip integrated monolithic probe was fabricated at the inlet of the extraction channel on the glass chip instead of the capillary probe connected to the microchannel, in order to improve the system stability and reliability. A slotted-vial array system coupled with the monolithic probe was used to sequentially introduce sample and different solvents and reagents into the extraction channel for extraction and CL detection. The performance of the system was demonstrated in the determination of Al3+ using Al3+-dihydroxyazobenzene (DHAB) and tributyl phosphate (TBP) extraction system. The operation conditions, including extraction time, concentration and flow rate of the CL reagents, were optimized. Within one analysis cycle of 12 min, an enrichment factor of 85 was obtained in the extraction stage with a sample consumption of 1.8 μL. The consumption of CL reagent, bis(2-carbopentyloxy-3,5,6-trichlorophenyl)oxalate (CPPO), was 120 nL/cycle. The detection limit of the system for Al3+ was 1.6 × 10−6 mol/L with a precision of 4.5% (R.S.D., n = 6).  相似文献   

8.
A.K.M. Kafi 《Talanta》2009,79(1):97-37
We report on a novel amperometric biosensor for detecting phenolic compounds based on the co-immobilization of horseradish-peroxidase (HRP) and methylene blue (MB) with chitosan on Au-modified TiO2 nanotube arrays. The titania nanotube arrays were directly grown on a Ti substrate using anodic oxidation first; a gold thin film was then coated onto the TiO2 nanotubes by an argon plasma technique. The morphology and composition of the fabricated Au-modified TiO2 nanotube arrays were characterized by scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS). Cyclic voltammetry and amperometry were used to study the proposed electrochemical biosensor. The effect of pH, applied electrode potential and the concentration of H2O2 on the sensitivity of the biosensor have been systemically investigated. The performance of the proposed biosensor was tested using seven different phenolic compounds, showing very high sensitivity; in particular, the linearity of the biosensor for the detection of 3-nitrophenol was observed from 3 × 10−7 to 1.2 × 10−4 M with a detection limit of 9 × 10−8 M (based on the S/N = 3).  相似文献   

9.
Based on a boron dipyrromethene (BODIPY) derivative containing an N, O and S tridentate ligand, a Cu2+ fluorescent probe BTCu was developed. The detection mechanism was verified as Cu2+-promoted oxidative dehydrogenation of an amine moiety, leading to a formation of a fluorescent Cu+-Schiff base complex. Free BTCu exhibited a maximum absorption wavelength at 496 nm, and a very weak maximum emission at 511 nm. Upon addition of various metals ions, it showed large fluorescence enhancement toward Cu2+ (417-fold in MeCN and 103-fold in MeCN/HEPES solution, respectively) with high selectivity. The detection limits are as low as 1.74 × 10−8 M and 4.96 × 10−8 M in the two different solutions, respectively. And BTCu could work in a wide pH range with an extraordinary low pKa of 1.21 ± 0.06. Using fluorescence microscopy, the probe was shown to be capable of penetrating into living cells and imaging intracellular Cu2+ changes.  相似文献   

10.
Chih-Hsin Tsai 《Talanta》2007,72(2):368-372
A capillary electrophoresis Raman spectroscopy (CE-RS) method based on the stacking and sweeping modes are described. A non-fluorescent compound (malachite green, MG; crystal violet, CV) and a doubled Nd:YAG laser (532 nm, 300 mW) were selected as the model compound and light source, respectively. In order to carry out a quantitative and analysis of MG, a monochromator was used to collect the specific Raman line at 1616 cm−1 (the N-φ and C-C stretching, corresponding to 582 nm when the wavelength of the exciting source is 532 nm). The limit of detection (LOD) for MG was 1.6 × 10−5 and 1.1 × 10−5 M, respectively, based on the CZE and MEKC modes. This could be improved to 3.4 × 10−7 and 5.3 × 10−9 M, respectively, when the stacking and sweeping modes were applied. The method was also extended to the determination of MG in an actual sample.  相似文献   

11.
A new nano-sized silver(I) ion-imprinted polymer (IIP) was prepared via precipitation copolymerization using ethyleneglycol dimethacrylate, as a cross-linking agent in the presence of Ag+ and an aza-thioether crown containing a 1,10-phenanthroline subunit as a highly selective complexing agent. The imprint silver(I) ion was removed from the polymeric matrix using a 1.0 M HNO3 solution. The resulting powder material was characterized using IR spectroscopy and scanning electron microscopy. The SEM micrographs showed colloidal nanoparticles of about 52 nm and 75 nm in diameter and slightly irregular in shape for leached and unleached IIPs, respectively. The optimal pH for quantitative enrichment was 6.0 and maximum sorbent capacity of the prepared IIP for Ag+ was 18.08 μmol g−1. The relative standard deviation and limit of detection (LOD = 3Sb/m) for flame atomic absorption spectrometric determination of silver(I) ion, after its selective extraction by the prepared IIP nanobeads, were evaluated as 2.42% and 2.2 × 10−8 M, respectively. The new Ag+-IIP was also applied as a suitable sensing element to the preparation of highly selective and sensitive voltammetric and potentiometric sensors for ultra trace detection of silver(I) ion in water samples, with limits of detection of 9.0 × 10−10 and 1.2 × 10−9 M, respectively.  相似文献   

12.
Xiluan Yan  Masaaki Kai 《Talanta》2009,79(2):383-4519
We have developed a novel sensitive chemiluminescence (CL) aptasensor for the target assay as exemplified by using adenosine as a model target. In this work, we have demonstrated the signaling mechanism to make detection based on magnetic separation and 3,4,5-trimethoxyl-phenylglyoxal (TMPG), a special CL reagent as the signaling molecule, which reacts instantaneously with guanine nucleobases (G) of adenosine-binding aptamer strands. Briefly, amino-functioned capture DNA sequences are immobilized on the surface of carboxyl-modified magnetic beads, and then hybridized with label-free G-rich (including 15 guanine nucleobases) adenosine-binding aptamer strands to form our CL aptasensor. Upon the introduction of adenosine, the aptamer on the surface of magnetic beads is triggered to make structure switching to the formation of the adenosine/aptamer complex. Consequently, G-rich aptamer strands are forced to dissociate from magnetic beads sensing interface, resulting in a decrease of CL signal. The decrement of peak signal is proportional to the amount of adenosine. The effects of the amounts of capture DNA, aptamer, magnetic beads are investigated and optimized. It was found that the CL intensity had a linear dependency on the concentration of adenosine in the range of 4 × 10−7 to 1 × 10−5 M. With a low detection limit of 8 × 10−8 M and simplicity in CL detection, this novel technique will offer a great promise for future target/aptamer analysis.  相似文献   

13.
Terbium sensitized fluorescence was used as a post-column detection system to develop a simple, sensitive and rapid high-performance liquid chromatographic method for the simultaneous determination of catecholamines norepinephrine (NE), epinephrine (E) and dopamine (DA).Catecholamines were separated by an ion-pair reversed-phase chromatography on a BDS-Hypersil analytical column with a mobile phase of methanol and 50 mmol l−1 acetate buffer (pH 4.7) containing 1.1 mmol l−1 SOS and 0.11 mmol l−1 EDTA (15+85 v/v).Catecholamines and the internal standard (3,4-dihydroxybenzylamine, DHBA) were post-column derivatized by the addition to the eluent of an alkaline solution containing a stoichiometric mixture of terbium(III) chloride and EDTA. Fluorescence detection (λex=300 nm, λem=545 nm) is based on the sensitization of terbium ion fluorescence after complexation with catecholamines.The chemical compatibility between the eluent and the post-column reagent was studied and the analytical characteristics of the method were established. Detection limits found were 1.0×10−8, 4.0×10−8 and 7.0×10−8 mol l−1 for NE, E and DA, respectively. The method has been successfully applied to the determination of catecholamines in urine samples after solid-phase extraction (SPE) pre-treatment. Recoveries from urine spiked with NE (4.0×10−7, 2.0×10−6 and 4.0×10−6 mol l−1), E (8.2×10−8, 4.1×10−7 and 8.2×10−7 mol l−1) and DA (1.0×10−6, 5.0×10−6 and 1.0×10−5 mol l−1) varied from 98 to 100% (mean=99.3%), from 106 to 107% (mean=106.3%) and from 98 to 101% (mean=99.3%), respectively. The between-run precision (relative standard deviation, R.S.D.) for the method for three urine samples at different concentration levels of each catecholamine varied from 3.6 to 7.0%.  相似文献   

14.
Several covalent immobilization methods, which have been routinely used with proteins and antibodies, were studied for their ability to immobilize genetically engineered Escherichia coli cells to glass beads. The cells used in this study expressed a metal binding peptide that binds cadmium (Cd) and mercury (Hg). The initial work focused on a method employing 2.5% aminopropyltrimethoxy silane and 2.5% glutaraldehyde for covalent immobilization of cells onto porous glass beads. Scanning electron microscopy (SEM) demonstrated cell attachment (average of 3.0×108 cells per bead) to the irregular surface. Columns containing cells immobilized with the 2.5% aminosilane and 2.5% glutaraldehyde removed more than 90% of the Cd from solutions with 50 ppb and 1 ppm levels. Following removal of the bound Cd with HCl elution and regeneration to pH 6.0, the columns were shown to effectively bind additional cadmium. Various concentrations of aminosilane and glutaraldehyde were tested for improved cell density.Glutaraldehyde is a universal and convenient cross-linker, but there are some concerns with its effects on the cells and proteins, therefore, two additional covalent techniques were examined. One method employed the aminopropyltrimethoxy silane and carbodiimide, and the other used mercaptopropyltrimethoxy silane and the heterobifunctional cross-linker GMBS. Some comparisons of these two immobilization methods to the method employing glutaraldehyde are described.  相似文献   

15.
According to the current demands of Green Analytical Chemistry and regarding the need for lower reagent consumption with improved analytical performance, an automatic methodology with a flow-through optosensor incorporating solid-phase spectrophotometric detection was developed. The sensor used in this work was based on the redox state of thionine whose oxidized form is blue and reduced form is colorless with monitoring carried out at 621 nm. This redox indicator was immobilized on gel beads and subsequently packed into a flow-through cell. It was then assembled into a sequential injection system and was shown to be an excellent alternative to monitor enzymatic redox reactions automatically as the redox catalysis is performed by glucose dehydrogenase. This enzyme is a representative dehydrogenase enzyme and uses NAD+ as cofactor, promoting the oxidation of glucose to glucono-lactone and reduction of NAD+ to NADH. The produced NADH promotes color depletion on the surface of the sensor. The calibration graph for glucose was linear between 5.74 × 10−4 and 2.00 × 10−3 mol L−1 and detection limit was 1.72 × 10−4 mol L−1. Glucose concentration in different samples including sera, salines, perfusion solutions, powder for preparing oral solutions and solutions for hemodialysis was determined. The method proved to be reproducible with a RSD < 5% for glucose determinations.  相似文献   

16.
Pore structure variation as a function of pH was investigated for the pore-filled pH-sensitive poly(acrylic acid)-poly(vinylidene fluoride) membranes. The pore radius reduced drastically as the poly(acrylic acid) gel incorporated inside the nascent substrate, which is from 113 nm of nascent substrate to as low as 7.0 nm of pore-filled membranes at pH acidic. For the membranes, the pore radii at pH neutral estimated by the extend Nernst–Planck equation (2.76–4.20 nm) and by the Spiegler–Kedem model with the steric-hindrance pore model (3.4–4.1 nm) are close to each other and comparable with that calculated from the poly(acrylic acid) gel correlation length (1.79–2.93 nm). The calculated pore density at pH neutral (49–258 × 1014 m−2) is much higher than that at pH acidic (2.8–39.8 × 1014 m−2). The results are interpreted in terms of the gel structure in the pore-filled membranes.  相似文献   

17.
A simple and accurate way of determining atom number densities in dense rubidium vapors is presented. The method relies on the experimental finding that the reduced absorption coefficients of the Rb triplet satellite bands between 740 nm and 750 nm and the triplet diffuse band between 600 nm and 610 nm are not temperature dependent in the range between 600 K and 800 K. Therefore, the absolute values of the reduced absorption coefficients of these molecular bands can provide accurate information about atomic number density of the vapor. The rubidium absorption spectrum was measured by spatially resolved white-light absorption in overheated rubidium vapor generated in a heat pipe oven. The absolute values for the reduced absorption coefficients of the triplet bands were determined at lower vapor densities, by using an accurate expression for the reduced absorption coefficient in the quasistatic wing of the Rb D1 line, and measured triplet satellite bands to the resonance wing optical depth ratio. These triplet satellite band data were used to calibrate in absolute scale the reduced absorption coefficients of the triplet diffuse band at higher temperatures. The obtained values for the reduced absorption coefficient of these Rb molecular features can be used for accurate determination of rubidium atomic number densities in the range from about 5 × 1016 cm− 3 to 1 × 1018 cm− 3.  相似文献   

18.
Monoliths formed by photopolymerization are excellent means for fabricating functional elements in miniaturized microdevices such as microextraction tips which are becoming important for sample preparation. Various silica-based and polymer-based materials have been used to fabricate monoliths with through pores of several nm to 4 μm. However, the back pressure created by such methods is still considered to be high for microtips that use suction forces to deliver the liquid. In this study, we demonstrated that emulsion techniques such as oil-in-water can be used to form monoliths with large through pores (>20 μm), and with rigid structures on small (10 μL) and large (200 μL) pipette tips by photopolymerization. We further showed that, with minor modifications, various functionalized particles (5–20 μm) can be added to form stable emulsions and successfully encapsulated into the monoliths for qualitative and quantitative solid-phase microextractions for a diverse application. Due to high permeability and large surface area, quick equilibration can be achieved by pipetting to yield high recovery rates. Using tryptic digests of ovalbumin as the standard, we obtained a recovery yield of 90–109% (RSD: 10–16%) with a loading capacity of 3 μg for desalting tips immobilized with C18 beads. Using tryptic digests of β-casein and α-casein as standards, we showed that phosphopeptides were substantially enriched by tips immobilized with immobilized metal affinity chromatography or TiO2 materials. Using estrogenic compounds as standards, we obtained a recovery yield of 95–108% (RSD: 10–12%) and linear calibration curves ranging from 5 to 100 ng (R2 > 0.99) for Waters Oasis HLB tips immobilized with hydrophilic beads.  相似文献   

19.
The electrochemical detection of cell lines of MCF-7 (human breast cancer) has been reported, using magnetic beads for the separation tool and high-affinity DNA aptamers for signal recognition. The high specificity was obtained by using the magnetic beads and aptamers, and the good sensitivity was realized with the signal amplification of DNA capped CdS or PbS nanocrystals. The ASV (anodic stripping voltammetry) technology was employed for the detection of cadmic cation and lead ions, for electrochemical assay of the amount of the target cells and biomarkers on the membrane of target cells, respectively. This electrochemical method could respond to as low as 100 cells mL−1 of cancer cells with a linear calibration range from 1.0 × 102 to 1.0 × 106 cells mL−1, showing very high sensitivity. Moreover, the amounts of HER-3 which were overexpressed on MCF-7 cells were calculated correspond to be 3.56 × 104 anti-HER-3 antibody molecules. In addition, the assay was able to differentiate between different types of target and control cells based on the aptamers and magnetic beads used in the assay, indicating the wide applicability of the assay for early and accurate diagnose of cancers.  相似文献   

20.
The UV and IR spectra of CxF2x+1CHO (x = 1-4) were investigated using computational and experimental techniques. CxF2x+1CHO (x = 1-4) have broad UV absorption features centered at 300-310 nm. The maximum absorption cross-section increases significantly and shifts slightly to the red with increased length of the CxF2x+1 group: CF3CHO, 3.10 × 10−20 (300 nm); C2F5CHO, 6.25 × 10−20 (308 nm); C3F7CHO, 8.96 × 10−20 (309 nm); and C4F9CHO, 10.9 × 10−20 (309 nm). IR spectra for CxF2x+1CHO were recorded, calculated, and assigned. Results are discussed with respect to the literature data and to the atmospheric fate of CxF2x+1CHO.  相似文献   

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