基于纳米磁珠技术的新型微全分析DNA芯片的研究

在微全分析系统的研究中,样品提取及DNA分析技术是非常重要的一个环节.也是目前国内外研究的热点之一.文中介绍了一种新型的基于单芯片的样品制备和扩增方法.采用多层微加工技术制作SU-8模具,通过注模成型,制作出有立体微柱结构的PDMS(聚二甲基硅氧烷)芯片,在芯片微池内填充超顺磁性磁珠,利用固相提取(solid phase extraction,SPE)法,将细胞裂解、DNA提取、PCR反应等功能集成在一个PDMS芯片上.整个流程快速有效,操作简便且易于芯片系统集成,提取产物可以不必洗脱,直接作为下一步PCR反应的模板,在同一芯片上进行扩增反应,实现了样品预处理、DNA提取和PCR扩增的集成.     

基于ADuC824的PCR仪测温系统的研制

从软件和硬件角度讨论了以新型单片机ADuC824为核心的荧光基因扩增仪(PCR仪)温度测量系统的没计。模拟试验表明，该温度测量系统的性能、精度指标能满足PCR热循环高精度温度控制的要求。     

空域PCR芯片/微装置中温度控制技术

基于微电子机械系统(MEMS)技术而发展起来的空域聚合酶链式反应(空域PCR)芯片/微装置由于其具有反应速度快、所需DNA样品体积小以及自动化程度高等优点,已经日益引起人们的关注。空域PCR芯片/微装置系统中精确的温度控制是DNA成功扩增的关键因素之一。主要介绍国内外空域PCR芯片/微装置中温度控制技术进展,包括空域PCR系统温度循环特征;薄膜电阻加热器/传感器的设计原则;空域PCR反应系统温度均一性等。     

核酸扩增仪中温度的控制研究

核酸扩增仪一直是各种生命科学研究中重要的基础仪器设备之一。在核酸扩增仪中温度的快速性、精确性是影响其扩增效果的重要因素。选取在核酸扩增仪中的96孔反应槽作为研究平台,采用半导体制冷制热片对系统进行加热和降温。目前,已有大量应用实践证明自抗扰控制器的控制精度、抗干扰能力、参数鲁棒性等方面性能优于PID控制器,通过LabVIEW进行自抗扰控制和传统PID控制算法编程,对核酸扩增仪中实验平台进行快速升降温实验,并以实验数据来判断两种控制在核酸扩增仪中控制效果的优劣。     

PCR生物芯片/微装置在微生物检测中应用研究

聚合酶链式反应(PCR)技术已经在分子生物学、生物化学、临床医学、遗传以及法医等领域得到广泛的应用.基于微电子机械系统技术开发而成的PCR生物芯片由于具有所需样品和反应混合物体积小、反应时间短、轻便等优点而日益受到人们的重视.介绍PCR生物芯片/微装置(包括反应池内固定扩增式和连续流动式)在微生物埃希氏大肠杆菌(E.coli)和微生物战剂检测中的应用.     

基于MEMS技术的PCR生物芯片的研究

聚合酶链式反应 (PCR)技术已在分子生物学、基因测序、医学诊断等方面得到广泛的应用。基于MEMS技术研制了微结构的集成PCR -DNA分子扩增生物芯片 ,芯片集成了加热子、温度传感器、反应室、进样通道等 ;还介绍了芯片的设计、制作、温度循环特性。     

集成型PCR芯片的研究

PCR芯片的温度控制部分的搭构是决定整个PCR芯片性能、尺寸、集成性的重要组成部分。利用薄膜技术与MEMS技术,将热电材料依照珀尔帖模型排列组合制作于微结构PCR芯片的反应室底部,可实现对反应室升温及降温的操作,并可通过改变电流方向,方便实现两者的切换,制作出集成了DNA反应室、温度传感器以及热电材料温控组件的集成型微结构PCR芯片。     

一种微腔型PCR集成芯片的设计及其热分析

设计了一种新型的硅微聚合酶链式反应(PCR)芯片.该芯片采用掺杂半导体作为加热电阻来提高加热效率,改善反应腔内的温度均匀性.集成在芯片底部的Pt温度传感器与微加热器组成温度控制单元,为PCR反应过程提供所需的三种特定温度.此外,为了便于温度校准,设计了敞开式的反应腔,其容积约1.78 μL.采用集总参数法计算了芯片在加...     

自热式连续流PCR芯片的模拟与设计

介绍了一种连续流式的聚合酶链式反应(PCR)芯片,采取主动加热、被动冷却的方式,可实现DNA片断的倍增。用ANSYS有限元分析软件对器件进行温场分布仿真及分析,在此基础上, 设计了一种基于玻璃-玻璃的PCR芯片,芯片上分布着宽90μm,深40μm的迂回微沟道。通过单侧局部区域加热的方法,即可形成3个较宽的恒温区(95, 72, 60℃)与PCR反应相对应,且恒温区内温差在5℃以内,可以满足PCR反应的需要。     

PCR仪模糊自整定PID温度控制算法的研究

论文针对PCR基因扩增仪对温度的要求提出了与其适应的算法—模糊自整定PID算法。首先给出了PCR反应的各个阶段对温度的响应速度及精度的要求。然后通过对系统建模仿真,分别得到变换隶属函数前后模糊PID与模糊自整定PID的两条曲线。最后给出了模糊PID与模糊自整定PID的对比关系。发现同模糊PID相比模糊自整定PID的响应速度快,超调量小,使系统更稳定。     

A new thermal cycling mechanism for effective polymerase chain reaction in microliter volumes

This study presents a new thermal cycler using infrared (IR) heating and water impingement cooling for polymerase chain reaction (PCR) amplification of 10 l samples in thin glass capillary tubes. The thermal cycling system can achieve a temperature ramping-up rate of 65 °C/s and a ramping-down rate of 80 °C/s. Two other cooling mechanisms, natural convection and forced air convection, can also be used in the present system to obtain a ramping-down rate of 2 °C/s and 6 °C/s, respectively. The amplification of the 439 fragment of hepatitis B virus (HBV) DNA was successful. The PCR amplified products were analyzed by agarose gel electrophoresis with ethidium bromide staining for visualization. A comparison of results of the amplification products at three different ramping-down rates was made and the rapid thermal cycling of the present system can run DNA required amplification in 29 min for 20 thermal cycles that is only 1/3 the time spent in the conventional PCR machine used in comparison.This work was supported by the National Taiwan University College of Medicine in preparing the HBV DNA samples. We thank our research assistance W. L. Chen and Dr. Chen for their technical advice. We gratefully acknowledge the financial support by the Nation Science Council, No. NSC-88–2212-E-002–025.     

Experimental implementation and analysis of a DNA computing readout method based on real-time PCR with TaqMan probes

A new readout approach for the Hamiltonian Path Problem (HPP) in DNA computing based on the real-time polymerase chain reaction (PCR) is experimentally implemented and analyzed. Several types of fluorescent probes and detection mechanisms are currently employed in real-time PCR, including SYBR Green, molecular beacons, and hybridization probes. In this study, real-time amplification performed using the TaqMan probes is adopted, as the TaqMan detection mechanism can be exploited for the design and development of the proposed readout approach. Double-stranded DNA molecules of length 140 base-pairs are selected as the input molecules, which represent the solving path for an HPP instance. These input molecules are prepared via the self-assembly of 20-mer and 30-mer single-stranded DNAs, by parallel overlap assembly. The proposed readout approach consists of two steps: real-time amplification in vitro using TaqMan-based real-time PCR, followed by information processing in silico to assess the results of real-time amplification, which in turn, enables extraction of the Hamiltonian path. The performance of the proposed approach is compared with that of conventional graduated PCR. Experimental results establish the superior performance of the proposed approach, relative to graduated PCR, in terms of implementation time.     

Enhancement of thermal uniformity for a microthermal cycler and its application for polymerase chain reaction

Thermal uniformity is essentially important for micro reactors which require precise control of critical reaction temperatures. Accordingly, we report a new approach to increase the temperature uniformity inside a microthermal cycler, especially for polymerase chain reaction (PCR). It enhances the thermal uniformity in the reaction region of a PCR chip by using new array-type microheaters with active compensation (AC) units. With this approach, the edges of the microthermal cyclers which commonly have significant temperature gradients can be compensated. Significantly, the array-type microheaters provide higher uniformity than conventional block-type microheaters. Besides, experimental data from infrared (IR) images show that the percentages of the uniformity area with a thermal variation of less than 1 °C are 63.6%, 96.6% and 79.6% for three PCR operating temperatures (94, 57 and 72 °C, respectively) for the new microheaters. These values are significantly better than the conventional block-type microheaters. Finally, the performance of this proposed microthermal cycler is successfully demonstrated by amplifying a detection gene associated with Streptococcus Pneumoniae (S. Pneumoniae). The PCR efficiency of the new microthermal cycler is statistically higher than the block-type microheaters. Therefore, the proposed microthermal cycler is suitable for DNA amplification which requires a high temperature uniformity and is crucial for micro reactors with critical thermal constraints.     

基于DNA技术的加密方案

DNA密码是伴随着DNA计算的研究而出现的密码学新领域。利用DNA合成技术、PCR扩增技术以及DNA数字编码技术,结合传统密码学提出了一种基于DNA技术的加密方案。方案利用引物对于PCR扩增技术的特殊作用,提出要以引物和编码方式为密钥,采用传统的加密方法对明文进行加密预处理,可有效防止可能词作为PCR引物进行攻击。生物学困难问题和密码学计算困难问题为该方案提供了双重的安全保障,安全性分析表明该加密方案具有很强的保密强度。     

Simulation and experimental validation of a SU-8 based PCR thermocycler chip with integrated heaters and temperature sensor

We present a SU-8 based polymerase chain reaction (PCR) chip with integrated platinum thin film heaters and temperature sensor. The device is fabricated in SU-8 on a glass substrate. The use of SU-8 provides a simple microfabrication process for the PCR chamber, controllable surface properties and can allow on chip integration to other SU-8 based functional elements. Finite element modeling (FEM) and experiments show that the temperature distribution in the PCR chamber is homogeneous and that the chip is capable of fast thermal cycling. With heating and cooling rates of up to 50 and 30 °C/s, respectively, the performance of the chip is comparable with the best silicon micromachined PCR chips presented in the literature. The SU-8 chamber surface was found to be PCR compatible by amplification of yeast gene ribosomal protein S3 and Campylobacter gene cadF. The PCR compatibility of the chamber surfaces was enhanced by silanization.     

Planar microreactor for biochemical application made from silicon and polymer films

Recently the progress of life science has been increasing rapidly, and the importance of the microfluidics for DNA analysis systems has been widely recognized, especially in medical fields. The polymerase chain reaction (PCR) is an essential technique for DNA assay of various diseases and it has been a strong requirement to shorten the total of PCR cycles more and more. We developed the microreactor with a single cell for PCR using fabrication technologies of MEMS. The reactor body and cover were sealed using high thickness PDMS prototyping film without using adhesive in order to achieve repeat grabbing motion for direct sample injection, resumption and cleansing the reaction cell. Good reproducibility of the heat cycling was obtained. The heating rate and cooling rate during PCR was 6.8 and 2.7°C, respectively, which well corresponds to the design parameters. The homogenous temperature distribution of variance less than 2.0°C was obtained. It is demonstrated that amplification of the DNA was successfully achieved by using the microreactor.     

Coupled flow and reaction during natural convection PCR

Polymerase chain reaction (PCR) in a microfluidic Rayleigh–Benard convection cell represents a promising route towards portable PCR for point-of-care uses. In the present contribution, the coupled fluid mechanics and heat transport processes are solved numerically for a 2-D flow cell. The resultant velocity and temperature fields serve as the inputs to a convection-diffusion-reaction model for the DNA amplification, wherein the reaction kinetics are modeled by Gaussian distributions around the conventional bulk PCR reaction temperatures. These evolution equations are integrated to determine the exponential growth rate of the double-stranded DNA concentration. The predicted doubling time is approximately 10–25 s, increasing with the Péclet number. This effect is attributed to low velocity, slow kinetics “dead zones” located at the center of the reactor. The latter observation provides an alternative rationalization for the use of loop-based natural convection PCR systems.     

Highly sensitive identification of foodborne pathogenic Listeria monocytogenes using single-phase continuous-flow nested PCR microfluidics with on-line fluorescence detection

Highly sensitive detection of foodborne pathogens such as Listeria monocytogenes (L. monocytogenes) is crucial to the prevention and recognition of problems related to public health and legal repercussions, due to “zero tolerance” standard adopted for food safety in many countries. Here we first propose a single-phase continuous-flow nested polymerase chain reaction (SP-CF-NPCR) strategy for identification of the low level of L. monocytogenes on an integrated microfluidic platform. The PCR reactor is constructed by a disposable capillary embedded in the grooved heating column, coupled with a fluorescence microscopy for on-line semi-quantitative end-point fluorescence detection. As a proof-of-concept microfluidic system, the nested PCR is performed in a continuous-flow format without the need of any non-aqueous oil or solvent. On this device, the performance of nested PCR amplification has been evaluated by investigating the effect of reaction parameters, including polymerase concentration, flow rates, and template DNA concentration. In addition, the types of samples the presented system can accept, such as the unpurified DNA samples and artificially contaminated clinical stool samples were also evaluated. With the optimized reaction parameters, 0.2 copies/μL of genomic DNA from L. monocytogenes can be detected on the presented device. To our knowledge, this is the highest detection sensitivity in single-phase continuous-flow PCR microsystems reported so far. The high sensitivity of the analysis method, combined with the flexibility of reaction volumes and convenience of continuous operation, renders it to be further developed for potential analytical and diagnostic applications.