Analysis of specific gene by integration of isothermal amplification and electrophoresis on poly(methyl methacrylate) microchips

We have successfully achieved the integration of isothermal amplification and the subsequent analysis of specific gene fragments on poly(methyl methacrylate) microchips. In our experiments, loop-mediated isothermal amplification, which can offer higher specificity and efficiency than PCR, has been performed at a constant temperature (65 degreesC). After amplification, products could be either examined by the integrated microchip-based electrophoresis or directly observed by naked eye with SYBR Green I added into the reaction solution. By such an integrated microsystem, the amplification and the subsequent analysis of prostate-specific antigen gene with template concentration at 23 fg/muL could be finished within 15 min, which demonstrates its advantages of high specificity, good reproducibility, and fast speed in gene detection.

Study on Broadband Emitting Self-Assembled Quantum-Dot Material and Devices

City Univ Hong Kong

Fabrication and spectroscopic broadening properties for 1.5 mu m amplification of Er3+-doped BaF2 nanoparticles

We show the potential application of Er3+-doped BaF2 nanoparticles prepared from microemulsion technology for 1.5 mu m amplification in telecommunication. Nanoparticles with different sizes of about 8, 10, and 20.5 nm were prepared. The XRD patterns showed the excursion of diffraction peaks. When the particle size is smaller or the diffraction angle is larger, this kind of excursion will be more serious.

[Ru(bpy)(2)(dcbpy)NHS] Labeling/Aptamer-Based Biosensor for the Detection of Lysozyme by Increasing Sensitivity with Gold Nanoparticle Amplification

A novel [Ru(bpy)(2) (dcbpy)NHS] labeling/aptamer-based biosensor combined with gold nanoparticle amplification for the determination of lysozyme with an electrochemiluminescence (ECL) method is presented. In this work, an aptamer, an ECL probe, gold nanoparticle amplification, and competition assay are the main protocols employed in ECL detection. With all the protocols used, an original biosensor coupled with an aptamer and [Ru(bpy)(2)(dcbpy)NHS] has been prepared. Its high selectivity and sensitivity are the main advantages over other traditional [Ru(bpy)(3)](2+) biosensors. The electrochemical impedance spectroscopy (EIS) and atomic force microscopy (AFM) characterization illustrate that this biosensor is fabricated successfully. Finally, the biosensor was applied to a displacement assay in different concentrations of lysozyme solution, and an ultrasensitive ECL signal was obtained. The ECL intensity decreased proportionally to the lysozyme concentration over the range 1.0 x 10-(13)-1.0 x 10(-8) mol L-1 with a detection limit of 1.0 x 10(-13) mol L-1.

Amplification of antigen-antibody interactions based on biotin labeled protein-streptavidin network complex using impedance spectroscopy

Antibody was covalently immobilized by amine coupling method to gold surfaces modified with a self-assembled monolayer of thioctic acid. The electrochemical measurements of cyclic voltammetry and impedance spectroscopy showed that the hexacyanoferrate redox reactions on the gold surface were blocked due to the procedures of self-assembly of thioctic acid and antibody immobilization. The binding of a specific antigen to antibody recognition layer could be detected by measurements of the impedance change. A new amplification strategy was introduced for improving the sensitivity of impedance measurements using biotin labeled protein- streptavidin network complex. This amplification strategy is based on the construction of a molecular complex between streptavidin and biotin labeled protein. This complex can be formed in a cross-linking network of molecules so that the amplification of response signal will be realized due to the big molecular size of complex. The results show that this amplification strategy causes dramatic improvement of the detection sensitivity of hIgG and has good correlation for detection of hIgG in the range of 2-10 mug/ml. (C) 2001 Elsevier Science B.V. All rights reserved.

Sensitive and Rapid Detection of Genetic Modified Soybean (Roundup Ready) by Loop-Mediated Isothermal Amplification

Using the LAMP method, a highly specific and sensitive detection system for genetically modified soybean (Roundup Ready) was designed. In this detection system, a set of four primers was designed by targeting the exogenous 35S epsps gene. Target DNA was amplified and visualized on agarose gel within 45 min under isothermal conditions at 65 degrees C. Without gel electrophoresis, the LAMP amplicon was visualized directly in the reaction tube by the addition of SYBR Green I for naked-eye inspection. The detection sensitivity of LAMP was 10-fold higher than the nested PCR established in our laboratory. Moreover, the LAMP method was much quicker, taking only 70 min, as compared with 300 min for nested PCR to complete the analysis of the GM soybean. Compared with traditional PCR approaches, the LAMP procedure is faster and more sensitive, and there is no need for a special PCR machine or electrophoresis equipment. Hence, this method can be a very useful tool for GMO detection and is particularly convenient for fast screening.