Real-time PCR microfluidic devices with concurrent electrochemical detection

Electrochemistry-based detection methods hold great potential towards development of hand-held nucleic-acid analyses instruments. In this work, we demonstrate the implementation of in situ electrochemical (EC) detection method in a microfluidic flow-through EC-qPCR (FTEC-qPCR) device, where both the amplification of the target nucleic-acid sequence and subsequent EC detection of the PCR amplicon are realized simultaneously at selected PCR cycles in the same device. The FTEC-qPCR device utilizes methylene blue (MB), an electroactive DNA intercalator, for electrochemical signal measurements in the presence of PCR reagent components. Our EC detection method is advantageous, when compared to other existing EC methods for PCR amplicon analysis, since FTEC-qPCR does not require probe-modified electrodes, or asymmetric PCR, or solid-phase PCR. Key technical issues related to surface passivation, electrochemical measurement, PCR inhibition by metal electrode, bubble-free PCR, were investigated. By controlling the concentration of MB and the exposure of PCR mixture to the bare metal electrode, we successfully demonstrated electrochemical measurement of MB in solution-phase, symmetric PCR by amplifying a fragment of lambda phage DNA.

Expression profiles of penaeidin from Fenneropenaeus chinensis in response to WSSV and vibrio infection by real-time PCR

Penaeidin from Chinese shrimp (Fenneropenaeus chinensis) has proved to be one of the most important antimicrobial peptides in the bodies of animals. The relative quantitative real-time PCR method is developed to study through time, the mRNA expression profile of penaeidin in the muscle and haemocyte tissue of Chinese shrimp infected with vibrio (Vibrio anguillarum) and WSSV (white spot syndrome virus). Research results showed that the same pathogens infection experiments produced similar gene expression profile in different tissues while different expression profiles appeared in the same tissues infected by different exterior pathogens. In vibrio infection experiments, a "U" Re expression profile resulted. Expression levels of penaeidin increased and surpassed the non-stimulated level, indicating that penaeidin from Chinese shrimp has noticeable antimicrobial activities. In WSSV infection experiments, the expression profile appeared as an inverse "U" with the expression of penaeidin gradually decreasing to below baseline level after 24 h. The expression of antimicrobial peptides gene in mRNA level in response to virus infection in shrimp showed that international mechanisms of virus to haemocytes and microbial to haemocytes are completely different. Decline of penaeidins expression levels may be due to haemocytes being destroyed by WSSV or that the virus can inhibit the expression of penaeidins by yet undiscovered modes. The expression profiles of penaeidin in response to exterior pathogen and the difference of expression profiles between vibrio and WSSV infection provided some clues to further understanding the complex innate immune mechanism in shrimp.

Difterentially expressed genes of Tetrahymena thermophila in response to tributyltin (TBT) identified by suppression subtractive hybridization and real time quantitative PCR

Tributyltin (TBT) is widely used as antifouling paints, agriculture biocides, and plastic stabilizers around the world, resulting in great pollution problem in aquatic environments. However, it has been short of the biomonitor to detect TBT in freshwater. We constructed the suppression subtractive hybridization library of Tetrahymena thermophila exposed to TBT, and screened out 101 Expressed Sequence Tags whose expressions were significantly up- or down-regulated with TBT treatment. From this, a series of genes related to the TBT toxicity were discovered, such as glutathione-S-transferase gene (down-regulated), plasma membrane Ca2+ ATPase isoforms 3 gene (up-regulated) and NgoA (up-regulated). Furthermore, their expressions under different concentrations of TBT treatment (0.5-40 ppb) were detected by real time fluorescent quantitative PCR. The differentially expressed genes of T thermophila in response to TBT were identified, which provide the basic to make Tetrahymena as a sensitive, rapid and convenient TBT biomonitor in freshwater based on rDNA inducible expression system. (c) 2006 Elsevier B.V. All rights reserved.

Real-Time Measurement on Deformation Fields of Hole-Excavated Samples under Impact Tension

In this paper, the real-time deformation fields are observed in two different kinds of hole-excavated dog-bone samples loaded by an SHTB, including single hole sample and dual holes sample with the aperture size of 0.8mm. The testing system consists of a high-speed camera, a He-Ne laser, a frame grabber and a synchronization device with the controlling accuracy of I microsecond. Both the single hole expanding process and the interaction of the two holes are recorded with the time interval of 10 mu s. The observed images on the sample surface are analyzed by newly developed software based on digital correlation theory and a modified image processing method. The 2-D displacement fields in plane are obtained with a resolution of 50 mu m and an accuracy of 0.5 mu m. Experimental results obtained in this paper are proofed, by compared with FEM numerical simulations.

Real-Time Measurement on Deformation Fields of Notched Samples Under Impact Tension

In this paper, a real-time and in situ optical measuring system is reported to observe high-velocity deformations of samples subjected to impact loading. The system consists of a high-speed camera, a He-Ne laser, a frame grabber, a synchronization device and analysis software based on digital correlation theory. The optical system has been adapted to investigate the dynamic deformation field and its evolution in notched samples loaded by an split Hopkinson tension bar, with a resolution of 50 pin and an accuracy of 0.5 mum. Results obtained in experiments are discussed and compared with numerical simulations. It is shown that the measuring system is effective and valid.

The Real-Time Mach-Zehnder Interferometer Used In Space Experiment

The optical interference method is a promising technique for measuring temperature, density, and concentration in fluids. The non-intrusive and non-invasive nature of its optical techniques to the measured section are its most outstanding features. However, the adverse experiment environment, especially regarding shaking and vibrating, greatly restricts the application of the interferometer. In the present work, an optical diagnostic system consisting of a Mach-Zehnder interferometer (named after physicists Ludwig Mach) and an image processor has been developed that increases the measuring sensitivity compared to conventional experimental methods in fluid mechanics. An image processor has also been developed for obtaining quantitative results by using Fourier transformation. The present facility has been used in observing and measuring the mass transfer process of a water droplet in EAFP protein solution under microgravity condition provided by the satellite Shi Jian No. 8.

Real-Time Measurement on Deformation Fields of Hole-Excavated Samples under Impact Tension

In this paper, the real-time deformation fields are observed in two different kinds of hole-excavated dog-bone samples loaded by an SHTB, including single hole sample and dual holes sample with the aperture size of 0.8mm. The testing system consists of a high-speed camera, a He-Ne laser, a frame grabber and a synchronization device with the controlling accuracy of I microsecond. Both the single hole expanding process and the interaction of the two holes are recorded with the time interval of 10 mu s. The observed images on the sample surface are analyzed by newly developed software based on digital correlation theory and a modified image processing method. The 2-D displacement fields in plane are obtained with a resolution of 50 mu m and an accuracy of 0.5 mu m. Experimental results obtained in this paper are proofed, by compared with FEM numerical simulations.

Readout of a real-time hologram in bacteriorhodopsin film with high diffraction efficiency and intensity

A scheme for the readout of a hologram recorded in bacteriorhodopsin film with high diffraction efficiency and intensity is suggested and demonstrated. Two weak coherent continuous beams function as the recording beams, and a strong light pulse is used to read the real-time hologram. The width of the readout light pulse is modulated to be short compared with the erase time of the reading beam; the time space between two adjacent pulses is ensured to be longer than the time the beams take to recover the hologram, and high diffraction efficiency and intensity (similar to 11 mW/cm(2)) can be obtained. (C) 1996 Optical Society of America.

Real-time micro-vibration measurement in sinusoidal phase-modulating interferometry

As there exist some problems with the previous laser diode (LD) real-time microvibration measurement interferometers, such as low accuracy, correction before every use, etc., in this paper, we propose a new technique to realize the real-time microvibration measurement by using the LD sinusoidal phase-modulating interferometer, analyze the measurement theory and error, and simulate the measurement accuracy. This interferometer utilizes a circuit to process the interference signal in order to obtain the vibration frequency and amplitude of the detective signal, and a computer is not necessary in it. The influence of the varying light intensity and light path difference on the measurement result can be eliminated. This technique is real-time, convenient, fast, and can enhance the measurement accuracy too. Experiments show that the repeatable measurement accuracy is less than 3.37 nm, and this interferometer can be applied to real-time microvibration measurement of the MEMS. (C) 2007 Elsevier GmbH. All rights reserved.

Real-time studies on microalgae under microgravity

Using remote sensing technique, we investigated real-time Nostoc sphaeroides Kiltz (Cyanobacterium) in Closed System under microgravity by SHENZHOU-2 spacecraft in January 2001. The experiments had 1g centrifuges in space for control and ground control group experiments were also carried out in the same equipments and under the same controlled condition. The data about the population growth of Nostoc sp. of experiments and temperature changes of system were got from spacecraft every minute. From the data, we can find that population growth of Nostoc sp. in microgravity group was higher than that of other groups in space or on ground, even though both the control I g group in space and I g group on ground indicated same increasing characteristics in experiments. The growth rate of 1.4g group (centrifuged group on ground) was also promoted during experiment. The temperature changes of systems are also affected by gravity and light. Some aspects about those differences were discussed. From the discussion of these results during experiment, it can be found that gravity is the major factor to lead to these changes. (C) 2004 Elsevier Ltd. All rights reserved.

A programmable SIMD vision chip for real-time vision applications

A programmable vision chip for real-time vision applications is presented. The chip architecture is a combination of a SIMD processing element array and row-parallel processors, which can perform pixel-parallel and row-parallel operations at high speed. It implements the mathematical morphology method to carry out low-level and mid-level image processing and sends out image features for high-level image processing without I/O bottleneck. The chip can perform many algorithms through software control. The simulated maximum frequency of the vision chip is 300 MHz with 16 x 16 pixels resolution. It achieves the rate of 1000 frames per second in real-time vision. A prototype chip with a 16 x 16 PE array is fabricated by the 0.18 mu m standard CMOS process. It has a pixel size of 30 mu m x 40 mu m and 8.72 mW power consumption with a 1.8 V power supply. Experiments including the mathematical morphology method and target tracking application demonstrated that the chip is fully functional and can be applied in real-time vision applications.

Investigation of the polyetherketone guest-host DR1/PEK-c polymer films by real-time monitoring of the second-harmonic generation

The real-time monitoring of the second-harmonic generation (SHG) was used to optimize the poling condition and to study the nonlinear optical (NLO) properties of the polyetherketone (PEK-c) guest-host polymer films. The high second-order NLO coefficient chi(33)((2)) = 11.02 pm/v measured at 1.064 mu m was achieved when the weight percent of DR1 guest in the polymer system is 20%. The NLO activity of the poled DR1/PEK-c polymer film can maintain more than 80% of its initial value when temperature is under 100 degrees C, and the normalized second-order NLO coefficient can maintain more than 85% after 2400 s at 80 degrees C. (C) 2000 Elsevier Science Ltd. All rights reserved.

Pipeline Architecture and Parallel Computation-Based Real-Time Stereovision Tracking System for Surgical Navigation

This paper studies the development of a real-time stereovision system to track multiple infrared markers attached to a surgical instrument. Multiple stages of pipeline in field-programmable gate array (FPGA) are developed to recognize the targets in both left and right image planes and to give each target a unique label. The pipeline architecture includes a smoothing filter, an adaptive threshold module, a connected component labeling operation, and a centroid extraction process. A parallel distortion correction method is proposed and implemented in a dual-core DSP. A suitable kinematic model is established for the moving targets, and a novel set of parallel and interactive computation mechanisms is proposed to position and track the targets, which are carried out by a cross-computation method in a dual-core DSP. The proposed tracking system can track the 3-D coordinate, velocity, and acceleration of four infrared markers with a delay of 9.18 ms. Furthermore, it is capable of tracking a maximum of 110 infrared markers without frame dropping at a frame rate of 60 f/s. The accuracy of the proposed system can reach the scale of 0.37 mm RMS along the x- and y-directions and 0.45 mm RMS along the depth direction (the depth is from 0.8 to 0.45 m). The performance of the proposed system can meet the requirements of applications such as surgical navigation, which needs high real time and accuracy capability.