Measurement of enzyme kinetics using a continuous-flow microfluidic system

This paper describes a microanalytical method for determining enzyme kinetics using a continuous-flow microfluidic system. The analysis is carried out by immobilizing the enzyme on microbeads, packing the microbeads into a chip-based microreactor (volume approximately 1.0 nL), and flowing the substrate over the packed bed. Data were analyzed using the Lilly-Hornby equation and compared to values obtained from conventional measurements based on the Michaelis-Menten equation. The two different enzyme-catalyzed reactions studied were chosen so that the substrate would be nonfluorescent and the product fluorescent. The first reaction involved the horseradish peroxidase-catalyzed reaction between hydrogen peroxide and N-acetyl-3,7-dihydroxyphenoxazine (amplex red) to yield fluorescent resorufin, and the second the beta-galactosidase-catalyzed reaction of nonfluorescent resorufin-beta-D-galactopyranoside to yield D-galactose and fluorescent resorufin. In both cases, the microfluidics-based method yielded the same result obtained from the standard Michaelis-Menten treatment. The continuous-flow method required approximately 10 microL of substrate solution and 10(9) enzyme molecules. This approach provides a new means for rapid determination of enzyme kinetics in microfluidic systems, which may be useful for clinical diagnostics, and drug discovery and screening.     

Self-contained microelectrochemical immunoassay for small volumes using mouse IgG as a model system

A self-contained, microelectrochemical immunoassay on the smallest volumes reported to date (1 microL for the antigen, 1 microL for the secondary antibody-enzyme conjugate, and 200 nL for the electrochemically detected species) has been developed using mouse IgG as a model system in a sandwich-type enzyme-linked immunosorbant assay, which takes less than 30 min to both complete the assembly of immunoassay components onto the antibody-modified surface and detect enzymatically generated species (excluding time for electrochemical cleaning of electrodes). These studies demonstrate the advantage of the close proximity of electrodes to modified surfaces and their application in the analysis of small volumes. Using a 50 microm diameter x 8 microm deep cavity with individually addressable electrodes on a microfabricated chip, the primary antibody was selectively and covalently attached at a gold, recessed microdisk (RMD) at the bottom of the microcavity to the free end of SAMs of either 11-mercaptoundecanoic acid or 11-mercapto-1-undecanol using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride. Nonspecific adsorption to the surrounding material, polyimide, of the microcavity device was eliminated. Electrochemical desorption was used to confine the immunoassay activity at the RMD. Enzymatic conversion of the substrate p-aminophenyl phosphate top-aminophenol is detectable in less than 30 s using cyclic voltammetry at a gold, tubular nanoband electrode, which is on the wall of the microcavity and immediately adjacent to the modified RMD. A third electrode, also within the region of the microcavity, served as the pseudoreference/auxiliary electrode. Calibration curves obtained for 1-microL solutions of 5-100 ng/mL of IgG and for 200 nL-solutions of 5 microM to 4 mM of PAPR gave detection limits of 4.4 nM (6.4 ng/mL) or 880 fmol (129 pg) for PAPR and 56 fM (9 pg/mL) or 56 zmol (9 fg) for IgG. It is expected that the device may be suitable for analysis with volumes down to tens of picoliters.     

Measurement of small high-precision angles

Translated from Izmeritel'naya Tekhnika, No. 9, pp. 65–66, September, 1992.     

跟踪测量视频的高精度同步采集系统

设计了一种无损实时跟踪测量视频和测控数据同步采集系统。根据测控信息同步信号及其对视频采集时刻的规定,以测控信息采集卡上高精度定时器为时钟,由视频采集卡通过软件修正摄像机曝光信号,实现对曝光时刻的精密调节。采用高速总线实现数据无失真存储和转储、AVI技术组织文件,充分利用了测量视频图像空间。     

Active focal zone sharpening for high-precision treatment using histotripsy

The goal of this study is to develop a focal zone sharpening strategy that produces more precise lesions for pulsed cavitational ultrasound therapy, or histotripsy. Precise and well-confined lesions were produced by locally suppressing cavitation in the periphery of the treatment focus without affecting cavitation in the center. The local suppression of cavitation was achieved using cavitation nuclei preconditioning pulses to actively control cavitation in the periphery of the focus. A 1-MHz 513-element therapeutic array was used to generate both the therapy and the nuclei preconditioning pulses. For therapy, 10-cycle bursts at 100-Hz pulse repetition frequency with P-/P+ pressure of 21/76 MPa were delivered to the geometric focus of the therapeutic array. For nuclei preconditioning, a different pulse was delivered to an annular region immediately surrounding the focus before each therapy pulse. A parametric study on the effective pressure, pulse duration, and delivery time of the preconditioning pulse was conducted in red blood cell-gel phantoms, where cavitational damage was indicated by the color change resulting from local cell lysis. Results showed that a short-duration (20 μs) preconditioning pulse at a medium pressure (P-/P+ pressure of 7.2/13.6 MPa) delivered shortly before (30 μs) the therapy pulse substantially suppressed the peripheral damage by 77 ± 13% while complete fractionation in the focal center was maintained. High-speed imaging of the bubble cloud showed a substantial decrease in the maximum width of the bubble cloud by 48 ± 24% using focal zone sharpening. Experiments in ex vivo livers confirmed that highly confined lesions were produced in real tissues as well as in the phantoms. This study demonstrated the feasibility of active focal zone sharpening using cavitation nuclei preconditioning, allowing for increased treatment precision compared with the natural focal width of the therapy transducer.     

高精度气体微流量计温度控制系统的研制

采用虚拟仪器技术开发了一个温度的数据采集系统.并在高精度气体微流量计中得到的应用.详细介绍了系统的硬件组成和软件的设计.系统的硬件部分使用研华公司的PCI-1716多功能数据采集卡和ADAM3013热电阻变送模块,软件部分采用美国NI公司的LabVIEW图形化编程语言创建,通过调用设计的温度计实现了温度的采集及数据的自动保存.并且界面简单、方便使用、解决了多通道监测、实时性等问题.     

Quantifying particle coatings using high-precision mass measurements

We present a general method to quantify coatings on microparticle surfaces based on the additional mass. Particle buoyant mass is determined in a solution with a density that is nearly equivalent to that of the core particle, reducing the magnitude and uncertainty of the measurement. Under these conditions, added material with a different density than that of the core is a larger fraction of the total buoyant mass of the coated particle. This method can resolve a buoyant mass difference between uncoated and coated particles of ~1 fg. For the protein layer on the 3 μm polystyrene spheres measured herein, this is equivalent to 1/10th of a full layer.     

Genetic algorithms for high-precision reconstructions of three-dimensional topographies using stereo fractographs

A software is developed which enables reconstruction of the three-dimensional (3D) shape of fracture surfaces without human assistance. It is based upon computer image processing and pattern recognition techniques by using a stereo-pair of scanning electron micrographs. The processing consists of two subprocesses: searching the matching points between two images; and computation of heights using the relative shift of the matching points. By using the previously developed system, some mismatches were inevitable in the search process, in particular, for low-contrast SEM images, e.g. striations, intergranular facets, etc. In order to improve the accuracy of the search, a genetic algorithm (GA) was implemented into the developed system. By using the GA method, the 3D shapes of a wide variety of fracture surfaces including cleavage failures, intergranular cracking, dimples and fatigue striations, were successfully reconstructed with sufficient accuracy. The searching processes by the GA method and the previously developed two-step algorithm of coarse and close searching were compared. These proved that the GA method has both the advantage of accuracy in the searching process and a short run-time. A detailed 3D shape, of more than a 120 × 120 reconstructed point-sized shape, was thus obtained with sufficient accuracy and with a relatively short run-time.     

高精度水下倾角测量系统的设计

为满足深海资源的探测开发中倾斜角度精确测量的要求,设计一种高精度的倾角测量系统。系统包括以内置高精度温度传感器的加速度计MS9001.D和微处理器STM32为核心的硬件电路,以24bit A/D转换器ADS1251U采样、算法温度补偿和全双工异步通信为核心的下位机软件以及以NI为核心的Lab VIEW上位机软件。实现基于MODBUS协议的RS485接口连接上位机,实时显示并将采集到的角度值按照系统时间存储于上位机。编写IIR数字滤波算法,充分抑制振动噪声干扰。实测结果表明:该系统倾角测量精度可达0.01°,输出响应频率为2 Hz,具备环境温度补偿功能,满足实际深海作业需求。     

A digital microfluidic system for the investigation of pre-steady-state enzyme kinetics using rapid quenching with MALDI-TOF mass spectrometry

A digital microfluidic system based on electrowetting has been developed to facilitate the investigation of pre-steady-state reaction kinetics using rapid quenching and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The device consists of individually addressable electrodes arranged to allow the combination of liquid droplets at well-defined time intervals and an integrated, electrohydrodynamically driven mixer. The device combines two droplets to initiate a reaction, then, with precise timing, combines a third droplet to quench the reaction, and finally combines a fourth droplet to form a matrix. Improvements to throughput when compared to traditional laboratory-scale methods, and previous MALDI-TOF MS digital microfluidic systems, were made. The device was tested against a model protein tyrosine phosphatase system, and results agreed well with published data. The system therefore allows for the analysis of reaction kinetics that were previously too rapid to analyze using MALDI-TOF MS.     

Fluctuations in small volumes of liquid 3He

We calculate fluctuation contributions to the thermodynamic properties in a small volume of liquid ³He. Functional integration techniques are employed to derive the fluctuation free-energy functional for temperatures just above the superfluid transition temperature and for zero temperature. Due to finite-size effects, the critical regime is estimated to be large enough to warrant an experimental observation of fluctuations. At zero temperature we find two kinds of quasicollective gapless excitations, which correspond to fluctuations of phase and rotations of the order parameter in spin space, respectively. These excitations result in a periodic time dependence of the pair correlation function. Finally, the experimental observability of the effects considered are contemplated.     

FDM型3D打印机的高精度进料系统研究

为了提高打印成型件的精度和质量,对FDM型3D打印机进料系统及其具体实现方法进行了改进.系统采用功率管IRF530N、加热管和PWM组成加热模块,采用铂热电阻Pt100、差动放大电路和PID算法组成温度测控模块.既简化了标度变化的计算量,又降低了电阻值温漂对测量结果的影响.针对基于步进电机结构的送料控制模块,给出了减少步进电机失步的措施.系统实现了180~250℃,±1℃精度的恒温控制,为FDM型3D打印机系统的恒温和高精度进料提供了保障,并且在简单调整后,还可对湿度、压力、位移和角度等物理量进行控制.     

Supercritical hydrothermal synthesis and in situ organic modification of indium tin oxide nanoparticles using continuous-flow reaction system

ITO nanoparticles were synthesized hydrothermally and surface modified in supercritical water using a continuous flow reaction system. The organic modification of the nanoparticles converted the surface from hydrophilic to hydrophobic, making the modified nanoparticles easily dispersible in organic solvent. The addition of a surface modifier into the reaction system impacted the crystal growth and particle size as well as dispersion. The particle size was 18 nm. Highly crystalline cubic ITO with a narrow particle size distribution was obtained. The advantages of short reaction time and the use of a continuous reaction system make this method suitable for industrial scale synthesis.