A simple, valveless microfluidic sample preparation device for extraction and amplification of DNA from nanoliter-volume samples

A glass microdevice has been constructed for the on-line integration of solid-phase extraction (SPE) of DNA and polymerase chain reaction (PCR) on a single chip. The chromatography required for SPE in the microfluidic sample preparation device (muSPD) was carried out in a silica bead/sol-gel SPE bed, where the purified DNA was eluted directly into a downstream chamber where conventional thermocycling allowed for PCR amplification of specific DNA target sequences. Through rapid, simple passivation of the PCR chamber with a silanizing reagent, reproducible DNA extraction and amplification was demonstrated from complex biological matrixes in a manner amenable to any research laboratory, using only a syringe pump and a conventional thermocycler. The muSPD allowed for SPE concentration of DNA from 600 nL of blood coupled to subsequent on-chip amplification that yielded a detectable amplicon; this simple device can be applied to a variety of routine genetic analyses without the need for sophisticated instrumentation. In addition, the applicability of these developments to nonconventional thermocycling was demonstrated through the use of noncontact, IR-mediated heating. This was exemplified with the isolation of DNA from an anthrax spore-spiked nasal swab and the subsequent on-chip amplification of target DNA sequences in a total processing time of only 25 min.     

The effects of gas pressure in plasma surface alloying process on testing accuracy of the photoelectric pyrometer

In order to control the specimen temperature precisely in a plasma surface alloying process, the measurements obtained using a photoelectric pyrometer were compared with a thermocouple system. The effect of pressure on the accuracy of the photoelectric pyrometer is discussed. The results indicate that at a specific temperature, as the working pressure increases, a larger emission coefficient ε should be used to keep the pyrometer values consistent with those of the thermocouple, which is considered to give the real temperature. The value of ε increases by about 0.1 as the working pressure increases by 5-10 Pa. With this adjustment, the error of the pyrometer is within ±3°C.     

0.9μm光电高温计应用单固定点延伸和多固定点内插的分度方法比较

单固定点延伸和多固定点内插是光电高温计的两种常用分度方法。针对新研制的0.9μm光电高温计进行了这两种分度方法的比较研究。在500~1800℃测温范围内,利用高温黑体炉作比较源,在整百度点与标准光电高温计进行量值验证,结果表明这两种分度方法的温度示值一致性优于0.2℃,与标准值的误差最大不超过0.6℃。两种分度方法的不确定度分别评估为0.2~1.3℃和0.2~0.8℃,k=2。相对于必须测量高温计光谱响应度及其响应度非线性的单固定点延伸方法,多固定点内插法简单并易操作,且不确定度可保持较优水平。该方法无论作为光电高温计的出厂分度还是周期示值校准,均可为大多数温度校准实验室提供参考。     

航空发动机热端表面温度场测量

航空发动机热端温度场的测量和监视对延长发动机的使用寿命具有重要意义.文章简要介绍了目前采用的各种温度测量方式,如热电偶、光电高温计、红外热像仪和示温漆,并详细介绍了多波长温度测量和谱色温度测量两种新型的温度测量方法.两种方法大大降低了温度测量对被测物体表面发射率的依赖性,适用于1000～3000℃温度范围内高温物体表面温度的测量.     

Integrated glass microdevice for nucleic acid purification, loop-mediated isothermal amplification, and online detection

A microdevice made of glass for genetic analysis has been fabricated, for the first time, for integration of extraction of nucleic acids and loop-mediated isothermal amplification (LAMP), followed by online fluorescence detection of amplification products on a single chip. The nucleic acid (NA) extraction region consists of a microfabricated serpentine channel in which micropillars were etched to increase the channel surface area and the capture efficiency of NAs. Nucleic acid molecules were bound to these pillars and channel surface in the presence of the chaotropic salt guanidine hydrochloride and eluted into a downstream amplification chamber with low ionic strength buffer where loop-mediated isothermal amplification was efficiently performed. Amplification can be detected online by the increase of fluorescence intensity at 540 nm when a low concentration of SYBR Green I, a fluorescent dsDNA intercalating dye, is employed. Flow control was accomplished by using laminar flow and differential channel flow resistances. Through passivation of the LAMP chamber and the channel between the extraction region and amplification domain, effective nucleic acid extraction and amplification were performed by just using a double-channel syringe pump and a heating block. By using this integrated microdevice, the purification of nucleic acids from complex biological matrixes and their subsequent amplification and detection online could be finished within 2 h.     

Design, Construction, and Evaluation of Ni�CC Eutectic Fixed Points

In this article the design, construction, and evaluation of eutectic fixed points for contact and non-contact measurements that have been recently developed at MIRS/UL/FE-LMK are presented. The design of the cells is made in such way that it can be used for only thermocouple calibration in vertical or horizontal furnaces, for calibration of pyrometers in a horizontal furnace, and for simultaneous calibration of contact and non-contact thermometers in a horizontal furnace. Special care has been taken to achieve repeatability and reproducibility of the transition temperatures and mechanical robustness of the cell. This includes also usage of a carbon sleeve in combination with carbon sheets, in a so-called hybrid design of the eutectic cell. With such a design, significant improvement in mechanical robustness of the cell and temperature stability of the cell has been achieved. All the measurements are performed within specially designed seven-zone furnaces within which different gradients, and different heating and cooling rates can be realized. The melting temperature of Ni?CC ((1328.44 ± 0.70) °C) measured with a thermocouple type R, calibrated at the zinc, aluminum, silver, copper, and palladium fixed points, and a non-contact thermometer, calibrated by comparison against different blackbodies, agree within their expanded uncertainties of 0.7 °C and 1.5 °C, respectively.     

基于波长函数的辐射温度计一点标定法及其精度的理论估计与实验验证

叙述了通过测量辐射温度计波长函数和一个温度点的输出信号,从而得到辐射温度计全量程标定方法估计了各项误差源对温度标定误差的影响,给出了重要结论。进一步以某多波长高温计为例,在黑体炉标定实验基础,进行了一点标定法的验证,结果证明该方法可以得到较高精度的标定。     

A New High-Temperature Oscillating Cup Viscometer

A new oscillating cup viscometer for temperatures up to 2,300°C has been constructed. A vacuum furnace with a graphite heater is used for heating the sample. The temperatures of the furnace and sample are measured by both a thermocouple and a pyrometer. The temperature is controlled with a stability better than 1 K. The oscillation of the cup is measured with a reflected laser beam using a position sensitive detector. The measured values of angle and time are then fitted to an analytical oscillation function. From the parameters of this function, the viscosity values are calculated using the Roscoe formalism. Measurements were carried out on pure metals at temperatures up to 1,700°C because of limitations of the thermocouple. The obtained viscosity values showed good agreement with literature data.     

利用材料发射率来确定玻璃高真空系统的烘烤温度

在高真空系统中，真空室的烘烤温度对获得要求的真空氛围有着重要的影响，对于玻璃制作的真空系统，目前对于该温度的确定缺少一种较为简单易行的方法，本文基于辐射换热分析提出了具有通用性的利用材料表面辐射率来计算玻璃表面烘烤温度的近似方法，将这难以直接测量的温度通过附设热电偶的示值和本文推荐的关系式来确定，于实际情况相比较为吻合．     

多区加热热电偶检定炉的温场研究

针对在热电偶检定过程中使用传统的检定炉用均温块所导致的控温滞后、恒温时间长、漏电及炉管弯曲等问题,研制出不用均温块温场也能满足相关规范要求的多区加热检定炉,以提高检测效率.根据JJF 1184-2007《热电偶检定炉温度场测试技术规范》对用均温块的传统单区加热检定炉与不用均温块的多区加热检定炉的温场分别进行测试;再用热...     

T 90 Measurement of Co-C, Pt-C, and Re-C High-Temperature Fixed Points at the NIM

The blackbodies of high-temperature fixed points (HTFPs), namely, Co-C, Pt-C, and Re-C eutectic points, were gradually established at the National Institute of Metrology (NIM) of China after 2007, and their characteristics were studied. Recently, the primary standard pyrometer was improved with the lower size-of-source effect, distance effect, and partial temperature controls. The pyrometer was characterized at the new facility for the calibration of the spectral responsivity. The measurement of its nonlinearity extended to a primary standard pyrometer (PSP) reading of approximately 2680 °C for the HTFP measurements. The International Temperature Scale of 1990 above the silver point was realized at the NIM by an improved scheme, the fixed-point blackbody pyrometer assembly. Two cells each for Co-C, Pt-C, and Re-C points were assigned associated uncertainties (k = 2) of 0.22 °C, 0.37 °C, and 0.75 °C, respectively, in accordance with the NIM scale. These HTFP blackbodies are being adopted for the study and calibration of radiation thermometers at the NIM.     

Thin-filament pyrometry with a digital still camera

A novel thin-filament pyrometer is presented. It involves a consumer-grade color digital still camera with 6 megapixels and 12 bits per color plane. SiC fibers were used and scanning-electron microscopy found them to be uniform with diameters of 13.9 micro m. Measurements were performed in a methane-air coflowing laminar jet diffusion flame with a luminosity length of 72 mm. Calibration of the pyrometer was accomplished with B-type thermocouples. The pyrometry measurements yielded gas temperatures in the range of 1400-2200 K with an estimated uncertainty of +/-60 K, a relative temperature resolution of +/-0.215 K, a spatial resolution of 42 mum, and a temporal resolution of 0.66 ms. Fiber aging for 10 min had no effect on the results. Soot deposition was less problematic for the pyrometer than for the thermocouple.     

以“固定点—高温计”方式传递 961.78 ℃以上国际温标

中国计量科学研究院在持续对高温基准装置研究工作的基础上,提升了光电高温计信噪比、非线性和辐射源尺寸效应等关键性能,并完善了光电高温计性能参数测量平台。与之前高温温标通过钨带灯传递不同,以“参考固定点—光电高温计”方式进行了银点以上高温温标的复现和传递,该方式可以避免光电高温计长期稳定性的影响,并将高温温度上限从2 200 ℃延伸至2 474 ℃,实验分析得到961.78～2 474 ℃的温标扩展不确定度(k=2)为0.08～0.62 ℃。2009年中英西三国温标复现比对结果验证了三国国际温标的一致性,并支持了上述不确定度评定。新传递方法可为精密光电高温计提供最高水平校准,并可扩展应用于900 nm等红外高温计校准。     

A microfluidic platform using molecular beacon-based temperature calibration for thermal dehybridization of surface-bound DNA

This work presents a simple microfluidic device with an integrated thin-film heater for studies of DNA hybridization kinetics and double-stranded DNA melting temperature measurements. The heating characteristics of the device were evaluated with a novel, noninvasive indirect technique using molecular beacons as temperature probes inside reaction chambers. This is the first microfluidic device in which thermal dehybridization of surface-bound oligonucleotides was performed for measurement of double-stranded DNA melting temperatures with +/- 1 degrees C precision. Surface modification and oligonucleotide immobilization were performed by continuously flowing reagents through the microchannels. The resulting reproducibility of oligonucleotide surface densities, at 9% RSD, was better than for the same modification chemistries on glass slides in unstirred reagent solutions (RSD=20%). Moreover, the surface density of immobilized DNA probe molecules could be varied controllably by changing the concentration of the reagent solution used for immobilization. Thus, excellent control of surface characteristics was made possible, something which is often difficult to achieve with larger devices. Solid-phase hybridization reactions, a fundamental aspect of microarray technologies often taking several hours in conventional systems, were reduced to minutes in this device. It was also possible to determine forward rate constants for hybridization, k. These varied from 820,000 to 72,000 M(-1) s(-1), decreasing as surface densities increased. Surface densities could therefore be optimized to obtain rapid hybridization using such an approach. Taken together, this combined microfluidic/small-volume heating approach represents a powerful tool for surface-based DNA analysis.     

PCR微池芯片的设计、制作及实现样品扩增

设计并制作了一种石英玻璃材料的PCR(Polymerase Chain Reaction)微池芯片，使用商用热循环仪控温。通过实时测量芯片微池内温度，改进温度控制程序，使用BSA(牛血清白蛋白)处理微池内壁等优化工作，实现了DNA片段的有效PCR扩增。     

Thermoelectric Characteristic of High-Temperature Thermocouples W5%RE/W20%RE

In the temperature range (900 to 2800) K, there has been confirmed compliance with the existing national standards for thermocouple wires W5% Re/W20%Re (type A) produced in Russia. The homogeneity within a lot of wires was evaluated by measuring the emf deviations from the corresponding reference function of thermocouples constructed from the front and the rear sections of paired coils of wires. The diameter of the wires amounted to 0.35 mm and 0.5 mm. Stability indicators were thermal emf changes after annealing for 2 hours at 1773 K. It was found that the inhomogeneity of thermoelements did not exceed (4 to 5) K for paired wire coils with a thermoelectric stability within a temperature equivalent of (1.0 to 1.5) K. EMF deviations from the reference table values for the thermocouples investigated did not exceed 1 % in the temperature range of (900 to 2773) K. Such deviations meet the requirements of the new draft of IEC standards 60584-1 and 2. Thermocouples were calibrated in four laboratories by comparison with various standard temperature gauges (type B thermocouple, radiation pyrometer, standard specimens of thermoelements). Measurements were carried out under vacuum, argon, and hydrogen. Depending on the calibration method, the expanded uncertainty of the measurements at 1773 K varied from (2.8 to 8) K.     

Quantitative polymerase chain reaction using infrared heating on a microfluidic chip

The IR-mediated polymerase chain reaction (IR-PCR) in microdevices is an established technique for rapid amplification of nucleic acids. In this report, we have expanded the applicability of the IR-PCR to quantitative determination of starting copy number by integrating fluorescence detection during the amplification process. Placing the microfluidic device between an IR long-pass filter and a hot mirror reduced the background to a level that enabled fluorescence measurements to be made throughout the thermal cycling process. The average fluorescence intensity during the extension step showed the expected trend of an exponential increase followed by a plateau phase in successive cycles. PUC19 templates at different starting copy numbers were amplified, and the threshold cycle showed an increase for decreasing amounts of starting DNA. The amplification efficiency was 80%, and the gel separation indicated no detectable nonspecific product. A melting curve was generated using IR heating, and this indicated a melting temperature of 85 °C for the 304 bp amplicon, which compared well to the melting temperature obtained using a conventional PCR system. This methodology will be applicable in other types of IR-mediated amplification systems, such as isothermal amplification, and in highly integrated systems that combine pre- and post-PCR processes.     

Assessment of Temperature Measurements Using Thermocouples at NIS-Egypt

Thermocouples are increasingly used in industry and research. For many industrial heating processes, particularly those carried out at high temperatures, a thermocouple is the most convenient and simple instrument for temperature measurement. In some instances, it is the only feasible method. The aim of this study is to select and recommend the best thermocouples from both base and noble metals to users in industrial and scientific institutions. Different types of thermocouples and calibration methods are described. From this work, the Nicrosil–Nisil thermocouple has been proposed as the best base metal thermocouple and the Au/Pt thermocouple is the most recommended as a substandard up to 1,000 °C.     

气流温度传感器时间常数关键影响因素分析

通过对气流温度传感器在热校准风洞上进行动态响应校准,得到不同温度传感器在不同工况条件下的时间常数,并提取焊球直径、偶丝直径、屏蔽罩结构、气流马赫数、气流温度以及温度阶跃量等关键影响因素,对其影响机制进行分析,得到了温度传感器时间常数的影响规律。结果表明:减小焊球直径、偶丝直径、屏蔽罩进出气口面积比和温度阶跃量,增大气流马赫数和气流温度,以及采用进出气口对开结构,均可减小温度传感器的时间常数;其中当偶丝直径由0.8mm减小到0.5mm时,温度传感器的时间常数减小142.9%。研究结果可为气流温度传感器的设计、使用与动态补偿等提供必要的依据。