CN107847931A - 数字pcr装置 - Google Patents

数字pcr装置 Download PDF

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CN107847931A
CN107847931A CN201680028255.6A CN201680028255A CN107847931A CN 107847931 A CN107847931 A CN 107847931A CN 201680028255 A CN201680028255 A CN 201680028255A CN 107847931 A CN107847931 A CN 107847931A
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digital pcr
heating region
independent heating
pcr device
well
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沈梾福
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Source Identification Private Ltd Co
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Abstract

一种数字PCR装置，其包括：由导热材料制成的独立加热区域，该独立加热区域设置在不导电的表面上，该独立加热区域包括配置为在其中容纳和隔开DNA样品的多个一体井，和至少一根导电迹线，其被配置为连接到直流电压源并且在连接到直流电压源时将多个一体井加热到均匀的温度，所述至少一根导电迹线以至少部分地围绕所述独立加热区域的起伏构型布置在表面上。

Description

数字PCR装置
技术领域
本发明涉及一种数字PCR装置。
发明背景
PCR是一种常用的方法，用于制备用于各种应用的DNA序列的多个拷贝，例如用于测序、诊断疾病、从DNA样品鉴定个体以及进行基因功能分析的DNA克隆。在PCR中，DNA序列的复制在多个热循环中进行，每个循环通常具有三个主要步骤：变性、退火和延长。在变性步骤中，将双链DNA模板加热至约94-98℃，保持20-30秒，以得到单链DNA。在退火步骤中，通过将温度降低至约50-65℃，保持20-40秒，将引物退火至单链DNA。在延长步骤中，使用DNA聚合酶(例如Taq)，通过在DNA聚合酶的最适活性温度下(对于Taq为75-80℃)，将已经退火的引物延伸至单链DNA，合成新的双链DNA。除了上述三个主要步骤之外，如果所用的DNA聚合酶是热激活的，则可能需要初始化步骤，且最后一个循环的最后延长步骤可以保持更长的时间(例如5-15分钟)，以确保没有剩余的单链DNA片段。
用于进行PCR的任何装置需要能够进行重复的热循环，以便进行变性、退火和延长的步骤。这涉及将反应物加热并冷却至所需温度，并将所需温度保持在必要的时间长度。鉴于温度上升到几乎和/或超过100℃，现有的微流体或芯片上实验室PCR装置通常需要外部热循环仪来提供必要的热量，从而限制了它们在使用过程中的真正便携性和尺寸。
通过PCR的DNA复制呈指数性，因为在循环中形成的新的双链DNA在下一个循环中经历变性、退火和延长，使得每个循环有效地使获得的DNA序列的数量增加一倍。因此PCR扩增循环的数目取决于反应开始时DNA的拷贝数。
相比之下，数字PCR是这样一种PCR形式：其中可以在不依赖于PCR循环的数量的情况下确定初始样品中的DNA的量，从而可以量化DNA的起始量而不必依赖不确定的指数数据。在数字PCR中，将一个DNA样品分配至多个孔中，并在所有孔中同时进行PCR。假定多孔内的DNA浓度遵循泊松分布。在进行PCR之后，每个孔将指示其中不存在(阴性)或存在(阳性)扩增的DNA。以这种方式，可以通过计数显示正的最终结果的孔的数量来实现绝对定量。
目前，像常规PCR系统一样，数字PCR系统也依赖于外部热循环仪向DNA分配至其中的每个孔提供必需的热量，从而类似地限制其尺寸和便携性。这限制了世界许多地方的医护人员能够有效地执行重要的传染病诊断来控制流行病，特别是在人口分布在大的面积并且使用检测设备受到差的基础设施和运输网络的阻碍的情况下。
近来，已经开发了可以使用9伏或更小的电压加热到高达或高于100℃的直流加热器，并且其中直流加热器是1平方英寸或更小。直流加热器包括布置在非导电表面上的由导热材料制成的独立加热区域，和至少一根导电迹线，其被配置为连接到直流电压源，并且当连接到直流电压源时将独立加热区域加热到均匀的温度。所述至少一根导电迹线至少部分地围绕所述独立加热区域以起伏(undulating)构型布置在所述表面上。这种由低电压源供电的小型加热器在空间和电力节约方面为许多应用开辟了可能性，特别是对于便携式和甚至一次性使用。然而，对于数字PCR，使用这样的直流加热器仍然是一个挑战，因为在小区域内提供足够数量的隔离物或孔以容纳其中的DNA样品以便在通过这种小型直流加热器加热时进行有意义的反应方面受到尺寸限制。
发明内容
根据第一方面，提供了一种数字PCR装置，包括：由导热材料制成的独立加热区域，该独立加热区域布置在不导电的表面上，所述独立加热区域包括配置为在其中容纳和分配DNA样品的多个一体井；和至少一根导电迹线，所述至少一根导电迹线配置为连接到直流电压电源，并且在连接到直流电压源时将多个一体井加热到均匀的温度，所述至少一根导电迹线以至少部分地围绕所述独立加热区域的起伏构型布置在所述表面上。
所述多个一体井中的每一个可以由包括独立加热区域厚度的井壁限定。
所述多个一体井可以在独立加热区域中作为规则阵列提供，且其中阵列的同一排中的井通过井壁中的开口彼此流体连通。
开口可以沿着每排井共线。
数字PCR装置还可以包括顶层，该顶层设置在表面上、在包括多个一体井的独立加热区域上、以及在至少一根导电迹线上。
表面和顶层可以由至少一个柔性片材形成。
附图说明
为了可以完全理解本发明并且容易实施，现在将以非限制性示例的方式描述本发明的示例性实施例，该描述参照附图进行说明。
图1是数字PCR装置的第一示例性实施例的图示说明。
图2是数字PCR装置的导电迹线的起伏构型的各种实施例的图示说明。
图3是数字PCR装置的多个一体井的平面图的照片。
图4是数字PCR装置的多个井模型的尺寸和热分析的屏幕抓图。
具体实施方式
下面将参照图1至4描述数字PCR装置10的示例性实施例及其各种应用，其中相同的附图标记用于表示相同或相似的部件。
在如图1所示的数字PCR装置10的第一实施例中，数字PCR装置10包括由生物惰性的导热材料制成的独立加热区域20，所述独立加热区域20布置在不导电的表面30上，例如聚合物膜。独立加热区域20作为具有一定厚度的材料层布置在表面30上。表面30例如可以由聚合物材料制成，并且可以为相对刚性的(例如层压材料，诸如用于印刷电路板的那些)或柔性的(例如，柔性聚丙烯片、聚合物膜、卡片纸等)。
数字PCR装置10还包括导电迹线40，该导电迹线40被配置为连接到直流电压源50(在图1中用(+)和(-)表示))，并且当连接到直流电压源50时将独立加热区域20加热到均匀的温度。独立加热区域20不与导电迹线40导电接触。导电迹线40和独立加热区域20优选地由相同材料制成，例如金，其既导电又是生物惰性的。
导电迹线40至少部分地围绕独立加热区域20以起伏构型60布置在表面30上。在图1中，起伏构型60具有如图2所示的扇形41形状，包括一系列在相同方向上弯曲、呈角度汇合、向外形成点的环状拱形物，并且拱形部朝向独立加热区域20凸出。在该实施例中，独立加热区域是矩形的，并且起伏构型60仅设置在独立加热区域20的两个相对侧上。
在替换实施例中，起伏构型60可以是锯齿形42、波形43、方波形44、鸽尾形45、邮票边缘形46、或每个上述起伏构型60的变化形式，如图2所示。起伏构型60可以认为包括导电迹线40的重复的曲线或弯曲部单元，所述单元可以或可以不穿插有直的部分。已经发现，较高的温度点出现在导电迹线40曲线或弯曲物的任何位置的外部或凸出边缘或角落处。因此，在表面30上提供导电迹线40的适当起伏构型60导致多个较高温度点定位成靠近独立加热区域20。以这种方式，可以使用例如可以由笔记本电脑的USB端口提供的仅5伏的直流电压，通过导电迹线40的起伏构型60产生足够的热量，以将具有例如仅3mm×10mm尺寸的独立加热区域20加热到所需的高温，例如100℃或者更高。
直流电压源50可以是具有9伏或更小电压的任何合适的电源，取决于配置数字PCR装置10的应用。例如，直流电压源50可以是电池的形式，或者如上所述，笔记本电脑或计算机的USB端口或本身具有电源和5伏或更小的电压的其它主机设备。以这种方式，数字PCR装置10易于携带，因为它可以由可以提供数字PCR装置10使用的低直流电压的电池或其他便携式装置供电。
通过将独立加热区域20配置为与导电迹线40的任何部分没有电接触，发现当数字PCR装置10达到稳态加热时，所有独立加热区域20具有均匀的温度。均匀加热的这个特征是特别重要的，因为独立加热区域20被配置为加热DNA样品分配至其中以进行数字PCR的数字PCR装置10的多个一体井29，并且重要的是，所有的井均匀加热，并且没有井暴露于可能导致DNA样品损坏的过高温度或温度峰值。
值得注意的是，如图3所示，数字PCR装置10的多个井29与独立加热区域20成一体。这可以通过在独立加热区域20中蚀刻井29、或者打印独立加热区域20以包括多个一体井29、或者通过与独立加热区域20一体形成多个井29的任何其它合适的方法来获得。在如图3所示的示例性实施例中，多个一体井29中的每一个具有约420μm长度乘以220μm宽的内部尺寸。
如图4所示，每个井29由具有大约60μm壁高度(即独立加热区域20的厚度)的井壁27限定。如图3可见，多个一体井29优选地以规则阵列提供。更优选地，阵列的同一行中的井29配置为通过井壁27中的开口28彼此流体连通，以允许DNA样品流入所有井29。在图3所示的实施例中，开口宽约为180μm。开口28优选地沿着每排井共线，以促进DNA样品通过阵列的流动。以这种方式，每个井29被配置为用作隔离物，以保留流入井29的DNA样品的一部分。
在使用中，DNA样品流过多个一体井29的阵列，使得每个井29可以保留DNA样品的一部分。适当地向导电迹线40提供直流电压并使之循环导致独立加热区域20根据所提供的直流电压的循环被均匀地加热，使得在多个一体井29中的每一个中进行相同数量的PCR循环。对于所有多个一体井29，存在于每个井29中的任何DNA被扩增相同的次数。通过向所有的井29施加荧光或任何其它合适的标签，在PCR开始时存在大量DNA的井将显示阳性结果。将显示阳性结果的井计数，因此允许通过调整井29中扩增的PCR循环次数来量化样品中的DNA。
数字PCR装置10优选地具有设置在表面30上、在独立加热区域20上和导电迹线40上的顶层(未示出)，以便保护它们免受由于暴露于环境而造成的损坏。顶层优选还提供用于用户安全的电绝缘功能。因此，表面30和顶层可以将导电迹线40和包括多个一体井29的独立加热区域20包封在小于3mm厚的包装中。
顶层应具有至少一个用于将DNA样品置于多个一体井29中的样品收集开口，并且可以具有一个或多个用于将试剂添加到多个一体井29中的其它开口。顶层还可以具有被配置为用于观察多个一体井29的结果窗口的透明部分。
如果数字PCR装置10自己具有由USB主机设备提供的直流电压源，则数字PCR装置10可以另外设置有突片(tab)(未示出)，其中导电迹线40配置有USB接口，用于与USB主机设备连接。
在一个示例性实施例中，数字PCR装置10包括独立加热区域20，该独立加热区域20具有约3mm×10mm的面积，并且包括800个用于在4μl样品上进行数字PCR的一体井29。数字PCR装置10允许从这样的小样品中量化DNA的能力在只有小DNA样品可以得到的法医应用中特别有利。因此，数字PCR装置10提供了用于使用小DNA样品的各种DNA扩增应用的便携式甚至一次性的解决方案。设想数字PCR装置10所需的样品的体积可以低至皮升范围，使得等温扩增或恒温PCR循环成为可能。这使得DNA扩增更容易进行，这对于传染病诊断特别有利，具有低成本的巨大优点，使临床医生能够进行诊断，而不必大量投入昂贵的设备。
虽然在前面的描述中已经描述了本发明的示例性实施例，但是本领域的技术人员将理解，在不脱离本发明的情况下，可以对设计、构造和/或操作的细节进行许多变化。例如，根据可能需要的数字PCR装置的实际应用，可以设想提供独立加热区域20和起伏构型60的备选结构，而不限于这些特征的可能形状和尺寸。井的数量和尺寸、壁高度和独立加热区域的尺寸均可以从上述示例中变化，并根据期望的数量和尺寸进行配置，这取决于具体应用和待用装置进行的PCR类型。

Claims (6)

1.一种数字PCR装置，包括：
由导热材料制成的独立加热区域，该独立加热区域设置在不导电的表面上，该独立加热区域包括配置为在其中容纳和分配DNA样品的多个一体井；和
至少一根导电迹线，其配置为连接到直流电压电源并且在连接到直流电压源时将多个一体井加热到均匀的温度，所述至少一根导电迹线以至少部分地围绕所述独立加热区域的起伏构型布置在所述表面上。
2.根据权利要求1所述的数字PCR装置，其中所述多个一体井中的每一个由包括独立加热区域厚度的井壁限定。
3.根据权利要求2所述的数字PCR装置，其中所述多个一体井在所述分散加热区域中以规则阵列提供，并且其中所述阵列的同一排中的井通过所述井壁中的开口彼此流体连通。
4.根据权利要求3所述的数字PCR装置，其中所述开口沿着每排井共线。
5.根据前述权利要求中任一项所述的数字PCR装置，其还包括顶层，该顶层设置在所述表面上、在包括所述多个一体井的独立加热区域上、和在所述至少一根导电迹线上。
6.根据前述权利要求中任一项所述的数字PCR装置，其中所述表面和顶层由至少一个柔性片材形成。
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