基于C8051F020的多通道手持式PCR扩增仪的设计

设计旨在研制一种新型的手持式PCR扩增仪,利用微电子机械系统(MEMS)技术制作PCR微加热芯片,采用聚二甲基硅氧烷(PDMS)材料制作一次性使用的微反应腔阵列,以C8051F020微控制器为核心搭建一个可以同时进行多路扩增的温控系统。它从根本上克服了传统台式PCR扩增仪的缺点,具有体积小、重量轻、反应速率快、节省反应试剂等诸多优点。     

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。     

聚二甲基硅氧烷球形微腔阵列的数字PCR芯片

数字聚合酶链式反应(dPCR)技术是一种核酸绝对定量技术,但现有dPCR平台因其昂贵的设备或复杂的操作限制了其实际应用.利用气体膨胀浇注成型技术,结合集成微腔阵列的模具,设计、制作了一种成本低廉、简单可靠的dPCR微流控芯片.独特的球形微腔为液滴存储提供了更稳定的几何形式,保证了样品数字离散化的可靠性和稳定性.同时,芯片还利用预脱气的聚二甲基硅氧烷(PDMS)实现自动进样和样品离散化,大大降低了对复杂昂贵设备的依赖,且提供了更高集成度.利用芯片进行了EGFR基因第19号外显子数字PCR定量检测,验证了该芯片的实用性.     

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。     

硅对聚合酶链式反应抑制作用的实时定量实验研究

采用实时定量PCR的方法研究了硅表面对乙肝病毒(HBV)DNA扩增过程的抑制作用.实验中采用HF处理的H终止硅和与自然氧化的硅片作为样品,按照1.0 mm2/μL和5.2 mm2/μL的面体比(硅片总表面积与PCR反应液的体积之比)与PCR反应液预混合(未加DNA样品,同时,Taq DNA聚合酶的浓度分别为0.2 U/35μL和1.0 U/35μL),充分混合预设的时间后,取出硅片,汲取上清液与DNA样品混合后采用实时定量PCR仪SIanTM进行扩增.扩增结果的荧光曲线表明:自然氧化的硅样品对PCR抑制作用更强,其对Taq聚合酶的抑制效果大于4.4 mU/mmz;高面体比条件下,即使在初始扩增阶段,也达不到理想的指数形式;缩短芯片反应时间有利于降低材料对扩增的抑制效应.     

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。     

单泵控制聚焦流形态的微流控芯片的研制

研究一种单动力源、聚焦流形态可控的用于细胞排队的微流控芯片。建立了样品沟道与鞘流沟道不同长度比例、不同夹角的模型并进行了不同负压条件下聚焦流形态仿真,运用SPSS软件进行了回归分析并进行了模型优化。在芯片的微加工过程中,利用印刷电路板(PCB)制作了母板,以聚二甲基硅氧烷(PDMS)为芯片主要材料,制作了PDMS—PDMS,PDMS—玻璃及PCB—PDMS三种芯片。制作的芯片能够在单个动力源条件下控制聚焦流宽度,使不同大小的微粒及细胞呈单个排列流动。研究结果为分析不同尺寸的细胞而选择合适的样品流沟道与鞘流沟道长度、夹角等条件提供了依据,所制作的芯片也达到了廉价且实用的目的。     

具有单一热源的对流双温PCR微流控装置的研究

为了克服传统连续流动聚合酶链式反应(PCR)微系统经常需要多个加热源来获取PCR所需要的温度,且集成或者非集成的泵系统也需要用来驱动PCR溶液连续流动,从而实现PCR扩增的缺点,研究了一种具有单一热源的对流双温PCR微系统.通过复合肋片技术在沟槽铜片上形成对流双温PCR所需要的温度,由流体密度差形成的自然对流来驱动闭环...     

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...     

基于微流体芯片结构的PDMS二次倒模工艺研究

研究了用于制作微流体芯片结构的聚二甲基硅氧烷(PDMS)与PDMS之间的倒模方法。首先,通过使用同一个微流体芯片模具倒出多个相同的PDMS负模结构;接着分别在各负模结构上溅射不同种类、不同厚度的金属,然后再对溅射过金属的负模上浇铸PDMS并固化以进行二次倒模,最后对二次倒模出的PDMS微流体结构表面粘连、结构完整性、尺寸等进行观测,从而通过比较得到倒模溅射所需的最佳金属和溅射金属薄膜的最优厚度。此方法倒出的PDMS微流体结构完整性好,不仅提出了一种全新的用于PDMS倒模的方法,而且解决了PDMS与PDMS之间直接倒模时所遇到的相互粘连和结构撕裂等难题。     

An integrated genetic analysis microfluidic platform with valves and a PCR chip reusability method to avoid contamination

Clinical diagnostics and genomic research often require performing numerous genetic tests. While microfluidic devices provide a low-cost alternative to such demands, integrated microfluidic devices are fabricated using expensive technology not always affordable for single use. However, carryover cross-contamination (CXC) concerns (i.e. either false positive or false negative PCR data) in PCR chips prevent reuse, defying much of the advantages of miniaturized systems developed using expensive MEMS processing. In this work, we present an integrated and reusable PCR–CE glass microfluidic chip capable of multi-chamber PCR and sequential CE, with emphasis on a unique chip reusability approach to avoid CXC. For reliable PCR, the surface of the chamber is re-configured from its virgin hydrophilic (CA < 20°) to hydrophobic (CA > 110°) by silanization. To then extend this silanization method as a chip reusability technique, the silanization coating is ‘stripped and re-silanized’ (SRS) to create a fresh coating prior to each successive PCR run. Experimental confirmation of the effectiveness of SRS method in avoiding the CXC is demonstrated using plasmid DNA and HIV-1 infected DNA samples. We also present passive plug microvalves incorporated in the chip to enable fluid/vapor retention during the PCR and controlled fluid flow from the PCR chamber to the CE section for further analysis.     

Rapid detection of bacterial cell from whole blood: Integration of DNA sample preparation into single micro-PCR chip

A novel bacterial cell detection method from blood samples has been developed for molecular diagnostics. Functional integration of DNA sample preparation into polymerase chain reaction (PCR) chip enabled detection of pathogenic bacterial cells in a single microchip. Surface-modified micropillars possessing affinity for bacterial cells were fabricated inside a PCR chip, and reaction conditions were optimized to render the microchip with high surface-to-volume ratio PCR-compatible. After bacterial cells were captured on the micropillars from whole blood and PCR inhibitors were washed out, PCR mixture was injected to allow real-time amplification of DNA extracted from the isolated cells. Cell enrichment effect produced by volume reduction from large initial sample to small micro-PCR chip chamber led to increased detection sensitivity. Moreover, the developed method from sample preparation to detection of bacterial cells from whole blood took less than 1 h. These results demonstrated that the surface-modified pillar-packed microchip would be a practical approach for integration into Lab-On-a-Chip (LOC) to enable point-of-care genetic analysis.     

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。     

线阵电极电泳芯片与单片机控制系统

介绍了用于生化分析的一种微全分析控制系统,包括电泳芯片的设计制作、微机控制系统以及初步实验结果。新型电泳芯片基于线性阵列电极,可灵活设定分离时间、长度、电压等电泳的各项条件,满足多种分离需求。以C8051F020单片机为控制核心,扩展出大量并行I/O口,并与高压系统实现良好的控制与衔接。突出了单片机系统的高度集成、低功耗、高扩展性等特点,给出了扩展大量I/O口并灵活控制多路高压器件的实例。     

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.     

Fabrication of DNA purification microchip integrated with mesoporous matrix based on MEMS technology

This paper presents a novel microfabricated DNA purification microfluidic chip with enhanced performance based on the principle of micro solid phase extraction. The microchip comprises a layer of mesoporous material as solid phase matrix which is fabricated on the internal wall of the channel of microfluidic chip by electrochemical etching Si in an electrolyte. The conditions of electrochemical etching and porosity of the mesoporous matrix have been investigated. The properties of mesoporous matrix have been characterized by scanning electron microscopy and by BET (Brunauer, Emmet, and Teller) nitrogen adsorption analysis. The pore size of the mesoporous matrix is in the range of 10–30 nm, and the surface area is about 300 m²/g. Compared with the microfluidic chips with micropillar array matrix or non-porous matrix, the microchip with mesoporous matrix is able to extract enough polymerase chain reaction-amplifiable DNA from cultures of rat mesenchymal stem cells in 20 min. This highly efficient, effortless, and flexible technology can be used as a lab-on-a-chip component for initial biologic sample preparation.     

Numerical and experimental study of a droplet-based PCR chip

A two-temperature continuous-flow polymerase chain reaction (PCR) polymer chip has been constructed that takes advantage of droplet technology to avoid sample contamination and adsorption at the surface. Samples contained in aqueous droplets are continuously moved by an oil carrier-fluid through various temperature zones, introducing the possibility of real-time quantitative PCR. In the present paper, we investigate many of the factors affecting droplet-based PCR chip design, including thermal mass, flow rate, and thermal resistance. The study focuses particularly on the fluid and substrate temperature distribution within the PCR chip and the droplet residence times in critical temperature zones. The simulations demonstrate that the flow rate strongly affects the temperature field within the carrier-fluid. Above a critical flow rate, the carrier-fluid fails to achieve the required temperatures for DNA amplification. In addition, the thermal resistances of the different layers in the chip are shown to have a major impact on the temperature profile in the channel.     

On-chip PCR amplification of genomic and viral templates in unprocessed whole blood

Performing medical diagnosis in microfluidic devices could scale down laboratory functions and reduce the cost for accessible healthcare. The ultimate goal of such devices is to receive a sample of blood, perform genetic amplification (polymerase chain reaction—PCR) and subsequently analyse the amplified products. DNA amplification is generally performed with DNA purified from blood, thus requiring on-chip implementation of DNA extraction steps with consequent increases in the complexity and cost of chip fabrication. Here, we demonstrate the use of unprocessed whole blood as a source of template for genomic or viral targets (human platelet antigen 1 (HPA1), fibroblast growth factor receptor 2 (FGFR2) and BK virus (BKV)) amplified by PCR on a three-layer microfluidic chip that uses a flexible membrane for pumping and valving. The method depends upon the use of a modified DNA polymerase (Phusion™). The volume of the whole blood used in microchip PCR chamber is 30 nl containing less than 1 ng of genomic DNA. For BKV on-chip whole blood PCR, about 3000 copies of BKV DNA were present in the chamber. The DNA detection method, laser-induced fluorescence, used in this article so far is not quantitative but rather qualitative providing a yes/no answer. The ability to perform clinical testing using whole blood, thereby eliminating the need for DNA extraction or sample preparation prior to PCR, will facilitate the development of microfluidic devices for inexpensive and faster clinical diagnostics.