聚合酶链式反应微芯片系统用于微量生物样品的快速扩增检测

基于MEMS微加工技术设计和制作了一种集成微加热器和温度传感器的聚合酶链式反应（PCR）微芯片。PCR微芯片结构通过有限元模拟验证分析。该芯片在PCR扩增过程中具有良好的制热效率和热传递均匀性。在微型温度控制电路装置下进行热循环反应,芯片的温度起伏小于1℃／s,升降温速度分别达到5℃／s和3℃／s,30个热循环耗时30min。此系统已经用于GUS基因的扩增检测,获得了良好的结果,极大的缩短了热循环的时间,可用于微量生物样品的快速扩增检测。     

环境友好型比率荧光微流控纸芯片快速检测苯醚甲环唑

本文构建了一种新型比率荧光印迹微流控纸芯片,将绿色荧光(NBD-APTES)作为对照荧光源,以半胱氨酸修饰后的碳量子点(CDs-Cys)的荧光变化来实现苯醚甲环唑的快速可视化检测.采用扫描电子显微镜和激光共聚焦显微镜等详细研究纸基芯片的检测性能.在优化条件下,该荧光传感器的线性范围为0.3~60μmol/L,检测限为75 nmol/L,样品回收率为102.1%~111.2%,准确度在3.1%~4.2%.与传统液相荧光传感材料相比,固相基质的比率荧光传感器具备更好的携带性和储存性,表现出令人满意的荧光检测特性.此外,该传感器用于分离和检测苯醚甲环唑时具有高度的特异性,已成功地应用于实际样品的检测,为新型比率荧光技术与微流控纸基芯片结合开辟了新途径,并为未来提供了潜在的即时医疗应用前景.     

纳米氧化锡修饰的微催化燃烧式氢气传感器的研制

研制了一种基于多孔纳米氧化锡(SnO₂)催化剂的微催化燃烧式气体传感芯片(Pellistor). 基于微机电系统(Micro- Electro-Mechanical Systems, MEMS)工艺制备硅基封闭膜式微催化燃烧式传感器, 通过气相沉积技术在Pt微加热电极和高温绝缘层表面制备三维纳米氧化锡催化膜, 利用催化膜对氢气良好的催化特性, 采用惠斯通电桥电路进行测量, 实现对空气环境中氢气在0～4%浓度范围内的快速检测, 响应时间和恢复时间分别达到0.65 s和2.32 s, 灵敏度达75.4 mV/1% H₂, 线性度为99.4%. 考察200 天内该传感芯片对氢气的检测能力, 传感芯片表现出良好的稳定性, 精确度保持在95%以上. 在绝缘层高温性能稳定的条件下, 将三维纳米氧化锡应用于微催化燃烧式传感器的氢气检测, 对催化燃烧式传感器性能的改进具有重要的意义.     

快速气相色谱控制系统的设计与应用

设计了一种用于快速气相色谱(Fast gas chromatography,FGC)的新型控制系统。该控制器主要由色谱柱温度控制系统、自动进样及气路压力控制系统组成。其温度控制范围为30~160℃,升温速度约为3℃/s,温度控制精度为±0.5℃,载气压力控制范围为0~0.5 MPa。将本控制器应用于自制快速色谱,并用色谱对由直链正构烷烃(C1~C8)以及甲苯9种物质组成的标准样品进行测试。结果显示,色谱能在100 s内将此9种物质完全分开。     

甲基苯丙胺电化学检测条件的优化

采用电化学方法检测甲基苯丙胺,探讨了不同扫描速度、初始浓度、pH值、电极处理温度及干扰物对检测的影响,获得了甲基苯丙胺电化学检测的最佳条件.结果表明,工作电极为掺硼金刚石薄膜电极,扫描速度为50~100 m V/s,初始浓度小于100 mg/L,pH=7.0及电极处理温度为100℃时,甲基苯丙胺的检测速度最快,检测结果最好,同时干扰物对于电化学方法检测甲基苯丙胺无明显影响.     

基于碳点的荧光纳米开关灵敏检测Cu(Ⅱ)离子和焦磷酸盐

利用羟基与氨基在高温条件下反应,不断聚合生成碳点（CDs）,该碳点可发射不依赖激发波长的明亮的红色荧光.将CDs作为目标敏感荧光团时,发现Cu²⁺可特异性猝灭碳点的荧光,而焦磷酸盐（PPi）可在一定程度上恢复上述体系的荧光.其中,Cu²⁺对CDs的荧光猝灭是由于Cu²⁺与CDs发生络合反应,从而发生静态猝灭过程;而加入PPi之后,由于它与CDs的结合能力更强,因此Cu²⁺离开CDs的表面,体系的荧光得以恢复.在此基础上构建了一种高选择性、高灵敏度的off-on荧光纳米开关传感体系,分别用于Cu²⁺和PPi的定量检测,检出限分别达2.14 μmol/L和1.85 μmol/L.该传感体系可应用于实际样品检测,拓宽了荧光CDs的传感应用,为其在生物体内目标分子的检测提供了可能性.     

基于荧光寿命机理的光纤温度传感器研究

为了实现恶劣环境下温度的测量,设计了一种基于荧光寿命机理的光纤温度传感系统.温度传感系统选用415 nm LED作为光源,以稀土荧光材料Y2O2S:Eu作为温度敏感材料,通过探测放大器和信号采集模块测量了敏感材料的荧光寿命,并由荧光寿命与温度的单调关系最终实现了温度的测量.采用油浴加热的方法进行温度实验,实验结果表明,温度传感系统在25~80℃实现了温度的测量,分辨率为0.5℃.     

光纤分导微梁阵列生化传感装置研制及检测应用

研制了一套基于光杠杆原理的微悬臂梁阵列传感器平台,并通过使用设计制作的微悬臂梁阵列芯片展示其在生物化学方面的检测应用.传感器平台使用光导纤维束分别与激光器耦合作为悬臂梁阵列的扫描光源,具有良好的检测稳定性,检测信号噪声水平约为2 nm;设计制作的微悬臂梁阵列芯片具有良好的平直度,温度响应均匀一致,各梁温度改变响应灵敏度偏差不超过5.0％.将整套传感系统被用于检测水溶液中的Hg2+,检测浓度范围为1 ～ 200 ng/mL;同一浓度下微悬臂梁阵列检测结果曲线一致性良好,平均偏差小于15％.在研制仪器平台上,分别实现了自制和国外商品化芯片对1.0和0.2 ng/mL样品的检测,结果表明,制作的微悬臂梁阵列芯片的检测灵敏度相对较低,需进一步改进悬臂梁阵列制作工艺.     

基于高荧光碳纳米粒子的Fe3+超灵敏检测

以廉价的豆腐残渣为碳源,用简单的水热合成法,在不同的温度下成功制备了水溶性的发射蓝光的碳纳米粒子(Carbon nanoparticles,CNPs)。通过AFM、Uv-vis、FTIR、NMR、XPS及荧光光谱对碳纳米粒子的结构和光学性能进行了研究。实验结果表明,随着制备温度升高,CNPs的荧光量子产率(Quantum yield,QY)增加,当温度达到240℃时,QY可达15.24%。该CNPs荧光探针对Fe3+的检测表现出了良好的灵敏度和选择性,检测限低至50 nmol/L。同时,CNPs荧光探针能够实现真实水样及细胞内Fe3+的检测,在传感及生物成像领域具有潜在的实际应用价值。     

基于低温等离子体辅助催化发光的乙烯传感器

将等离子体的高活化性能与催化发光的传感特性相结合,以成本低、合成简单的碱土金属纳米MgO为传感材料,构建了基于低温等离子体辅助的催化发光传感器,用于乙烯的快速检测.由于等离子体具有高活化性能,本方法的检测温度远低于传统的催化发光检测法的常用温度(300~500℃),无需加热装置,在室温下实现了对乙烯快速、灵敏的检测.室温(25℃)下,对乙烯的检出限为37 ng/mL (30 ppm),线性范围为112~4997 ng/mL (90~3998 ppm, R=0.97669),传感器具有良好的选择性和重现性.此传感器制备简单、稳定性高、低能耗、成本低,与传统的气体检测方法相比具有良好的实用性和普适性,为开发性能优异的新型催化发光传感器提供了策略.     

A thermostat chip of indium tin oxide glass substrate for static polymerase chain reaction and in situ real time fluorescence monitoring

A thermostat chip of indium-tin oxide glass substrate for static chip polymerase chain reaction (PCR) is, for the first time, introduced in this paper. The transparent conductive layer was used as an electro-heating element. Pulse width modulation and fuzzy proportional integration-differentiation algorithm were adopted in the temperature programming of the chip. The temperature distribution was investigated, and a dynamic control precision within ±2 °C was achieved. The highest ramping rates were 37 °C s⁻¹ for heating and 8 °C s⁻¹ for cooling with an electric fan. The PCR reaction vials were constructed with polyethylene tubes or poly(dimethylsiloxane) directly on the thermostat chip; the chip had a typical size of 25 mm × 25 mm and a thickness of 1.1 mm. Static chip PCR was successfully demonstrated either in a single vial or in an up to 8-parallel array vials. In situ real time fluorescence monitoring during PCR of a λ DNA fragments (236 bp) with SYBR Green I was demonstrated using a blue light emission diode as a light source and a photomultiplier as a detector. The method proposed here is characterized by open access, easy fabrication and low cost. This work could be the basis for developing a portable real time PCR system with disposable chips for point of care tests.     

Optimization of direct whole blood PCR amplification with applications on a static thermostat chip

In this paper, direct whole blood PCR amplifications on a static chip thermostat without sample purifications are demonstrated; in these amplifications, problems such as cross-interferences and contaminations could be avoided. The amplification conditions, such as the compositions of reagents and thermal programs, were investigated systematically by a GeneAmp PCR system with a native p53 gene segment (about543 bp) of human genome and an exterior lambda DNA segment (about 500 bp) as targets. Direct amplifications of p53 and K-ras (about 157 bp) gene segments from 0.5 μL blood samples were successfully demonstrated by a static PCR chip with an indium tin oxide glass substrate. The chip thermostat has a typical size of 25 mm × 25 mm, and a polyethylene tube was used as the PCR vial on the glass surface of the chip. Fuzzy proportional integration–differentiation algorithms were adopted in temperature controls of the chip with an aid of a micro-Pt100 sensor. In the direct PCR with the thermostat chip, the whole process only involves automatic thermal programs. This work demonstrated that a chip PCR for field test without desktop facilities is possible either for a point of care test or for forensic analysis. Figure Photo of the glass static thermostat chip with 2 PCR reaction vials Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Fully integrated PCR-capillary electrophoresis microsystem for DNA analysis

A fully integrated genomic analysis microsystem including microfabricated heaters, temperature sensors, and PCR chambers directly connected to capillary electrophoretic separation channels has been constructed. Valves and hydrophobic vents provide controlled and sensorless sample positioning and immobilization into 200 nL PCR chambers. The use of microfabricated heating and temperature sensing elements improves the heating and cooling rates for the PCR reaction to 20 degree C s(-1). The amplified PCR product, labeled on-column with an intercalating fluorescent dye, is injected into the gel-filled capillary for electrophoretic analysis. Successful sex determination using a multiplex PCR reaction from human genomic DNA is demonstrated in less than 15 min. This device is an important step toward a microfabricated genomic microprocessor for use in forensics and point-of-care molecular medical diagnostics.     

PDMS-glass hybrid microreactor array with embedded temperature control device. Application to cell-free protein synthesis

A microreactor array was developed which enables high-throughput cell-free protein synthesis. The microreactor array is composed of a temperature control chip and a reaction chamber chip. The temperature control chip is a glass-made chip on which temperature control devices, heaters and temperature sensors, are fabricated with an ITO (indium tin oxide) resistive material. The reaction chamber chip is fabricated by micromolding of PDMS (polydimethylsiloxane), and is designed to have an array of reaction chambers and flow channels for liquid introduction. The microreactor array is assembled by placing the reaction chamber chip on the temperature control chip. The small thermal mass of the reaction chamber resulted in a short thermal time constant of 170 ms for heating and 3 s for cooling. The performance of the microreactor array was examined through the experiments of cell-free protein synthesis. By measuring the fluorescence emission from the products, it was confirmed that GFP (Green Fluorescent Protein) and BFP (Blue Fluorescent Protein) were successfully synthesized using Escherichia coli extract.     

Bulk-micromachined submicroliter-volume PCR chip with very rapid thermal response and low power consumption

The current paper describes the design, fabrication, and testing of a micromachined submicroliter-volume polymerase chain reaction (PCR) chip with a fast thermal response and very low power consumption. The chip consists of a bulk-micromachined Si component and hot-embossed poly(methyl methacrylate)(PMMA) component. The Si component contains an integral microheater and temperature sensor on a thermally well-isolated membrane, while the PMMA component contains a submicroliter-volume PCR chamber, valves, and channels. The micro hot membrane under the submicroliter-volume chamber is a silicon oxide/silicon nitride/silicon oxide (O/N/O) diaphragm with a thickness of 1.9 microm, resulting in a very low thermal mass. In experiments, the proposed chip only required 45 mW to heat the reaction chamber to 92 degrees C, the denaturation temperature of DNA, plus the heating and cooling rates are about 80 degrees C s(-1) and 60 degrees C s(-1), respectively. We validated, from the fluorescence results from DNA stained with SYBR Green I, that the proposed chip amplified the DNA from vector clone, containing tumor suppressor gene BRCA 1 (127 base pairs at 11th exon), after 30 thermal cycles of 3 s, 5 s, and 5 s at 92 degrees C, 55 degrees C, and 72 degrees C, respectively, in a 200 nL-volume chamber. As for specificity of DNA products, owing to difficulty in analyzing the very small volume PCR results from the micro chip, we vicariously employed the larger volume PCR products after cycling with the same sustaining temperatures as with the micro chip but with much slower ramping rates (3.3 degrees C s(-1) when rising, 2.5 degrees C s(-1) when cooling) within circa 20 minutes on a commercial PCR machine and confirmed the specificity to BRCA 1 (127 base pairs) with agarose gel electrophoresis. Accordingly, the fabricated micro chip demonstrated a very low power consumption and rapid thermal response, both of which are crucial to the development of a fully integrated and battery-powered instrument for a lab-on-a-chip DNA analysis.     

Instrumentation of a PLC-regulated temperature cycler with a PID control unit and its use for miniaturized PCR systems with reduced volumes of aqueous sample droplets isolated in oil phase in a microwell

We have developed a temperature cycler for polymerase chain reaction (PCR) in a microwell fabricated on a polymer/glass chip. The entire system consisted of three subsystems, which included (1) a thermal conditioner, (2) a proportional-integral-derivative (PID) control signal conditioner and (3) a data acquisition subsystem. The subsystems were regulated coordinately by a ladder logic program written for the programmable logic control (PLC) so that an actual sample temperature could be timed, changed and maintained according to the programmed temperature cycles. The present temperature control system showed high accuracy, stability and minimum overshoot with reduced heating and cooling transition rates. Applicability of the temperature controller to the miniaturized PCR system with reduced volumes of aqueous sample droplets isolated in an oil phase was confirmed by successful amplifications of a target DNA sequence in the microwell.     

P(NIPAM-co-NVP)共聚物分子链水溶液中的可逆聚集研究

用溶液聚合方法合成了线型聚(N-异丙基丙烯酰胺-co-N-乙烯基吡咯烷酮)共聚物,通过弹性光散射(elastic light scattering,ELS)、荧光光谱与动态光散射研究了共聚物水溶液分子链可逆聚集的温度和时间依赖性.研究表明,升温时,ELS强度增加,分子链聚集;降温时,ELS强度降低,聚集的分子链解离.荧...     

Platinum nanoparticle-facilitated reflective surfaces for non-contact temperature control in microfluidic devices for PCR amplification

The polymerase chain reaction (PCR) is critical for amplification of target sequences of DNA or RNA that have clinical, biological or forensic relevance. While extrinsic Fabry-Perot interferometry (EFPI) has been shown to be adequate for non-contact temperature sensing, the difficulty in defining a reflective surface that is semi-reflective, non-reactive for PCR compatibility and adherent for thermal bonding has limited its exploitation. Through the incorporation of a reflective surface fabricated using a thermally driven self-assembly of a platinum nanoparticle monolayer on the surface of the microfluidic chamber, an enhanced EFPI signal results, allowing for non-contact microfluidic temperature control instrumentation that uses infrared-mediated heating, convective forced-air cooling, and interferometic temperature sensing. The interferometer is originally calibrated with a miniature copper-constantan thermocouple in the PCR chamber resulting in temperature sensitivities of -22.0 to -32.8 nm·°C(-1), depending on the chamber depth. This universal calibration enables accurate temperature control in any device with arbitrary dimensions, thereby allowing versatility in various applications. Uniquely, this non-contact temperature control for PCR thermocycling is applied to the amplification of STR loci for human genetic profiling, where nine STR loci are successfully amplified for human identification using the EFPI-based non-contact thermocycling.     

Non-adiabatic thin-film (chip) nanocalorimetry

To study the kinetics of processes on a millisecond time scale a thin-film nanocalorimeter based on a commercially available microchip (thermal conductivity vacuum gauge, TCG 3880, from Xensor Integration, NL) was constructed. The gauge consists of a submicron silicon nitride membrane with a film-thermopile and a film-heater, which are located at the 100 μm × 100 μm central part of the membrane. Controlled fast cooling is possible in addition to fast heating at essentially non-adiabatic conditions. To allow fast cooling the measurements are performed in an ambient gas atmosphere. It is proved that the maximum rate of the controlled cooling can be achieved with a gas cooling agent, rather than in a system with a solid heat-sink. The advantage of the gauge TCG 3880 is that its central heated region is small enough to be considered as a point source of the heat-flow into the gas, which essentially simplifies the calorimeter calibration. The maximum cooling rate is inversely proportional to the radius of the heated region. The gauge is placed in a thermostat with controlled gas pressure and temperature to be utilized as a device for fast scanning calorimetry of sub microgram samples with sensitivity 1 nJ/K and time resolution ca. 5 ms.     

A large volume, portable, real-time PCR reactor

A point-of-care, diagnostic system incorporating a portable thermal cycler and a compact fluorescent detector for real-time, polymerase chain reaction (PCR) on disposable, plastic microfluidic reactors with relatively large reaction volume (ranging from 10 μL to 100 μL) is described. To maintain temperature uniformity and a relatively fast temperature ramping rate, the system utilizes double-sided heater that features a master, thermoelectric element and a thermal waveguide connected to a second thermoelectric element. The waveguide has an aperture for optical coupling between a miniature, fluorescent reader and the PCR reaction chamber. The temperature control is accomplished with a modified, feedforward, variable structural proportional-integral-derivative controller. The temperature of the liquid in the reaction chamber tracks the set-point temperature with an accuracy of ± 0.1 °C. The transition times from one temperature to another are minimized with controllable overshoots (< 2 °C) and undershoots (< 5 °C). The disposable, single-use PCR chip can be quickly inserted into a thermal cycler/reader unit for point-of-care diagnostics applications. The large reaction chamber allows convenient pre-storing of dried, paraffin-encapsulated PCR reagents (polymerase, primers, dNTPs, dyes, and buffers) in the PCR chamber. The reagents are reconstituted "just in time" by heating during the PCR process. The system was tested with viral and bacterial nucleic acid targets.