This application is a continuation-in-part of U.S. application Ser. No. 09/081,260 filed May 19, 1998, now abandoned, and U.S. application Ser. No. 09/194,374 filed Jul. 25, 2000. This application is also related to U.S. application Ser. No. 09/275,061 filed Mar. 23, 1999. All of these applications are incorporated by reference herein.
本申请是1998年5月19日提交、现已放弃的美国申请号09/081,260和2000年7月25日提交的美国申请号09/194,374的部分延续。本申请还与 1999 年 3 月 23 日提交的美国申请 Ser.所有这些申请均并入本文作为参考。

This invention was made with Government support under contract DAAM01-96-C-0061 awarded by the U.S. Army. The Government has certain rights in the invention.
本发明是根据美国陆军授予的 DAAM01-96-C-0061 合同在政府支持下完成的。政府对本发明拥有某些权利。

The present invention relates generally to optical detection systems, and in particular to a multi-channel detection system for the real-time detection of a plurality of different analytes in a fluid sample.
本发明一般涉及光学检测系统，特别是用于实时检测流体样品中多种不同分析物的多通道检测系统。

There are many applications in the field of chemical processing in which it is desirable to precisely control the temperature of reaction mixtures (e.g., biological samples mixed with chemicals or reagents), to induce rapid temperature transitions in the mixtures, and to detect target analytes in the mixtures. Applications for such heat-exchanging chemical reactions may encompass organic, inorganic, biochemical and molecular reactions, and the like. Examples of thermal chemical reactions include nucleic acid amplification, thermal cycling amplification, such as polymerase chain reaction (PCR), ligase chain reaction (LCR), self-sustained sequence replication, enzyme kinetic studies, homogeneous ligand binding assays, and more complex biochemical mechanistic studies that require complex temperature changes.
在化学处理领域有许多应用，需要精确控制反应混合物（例如与化学品或试剂混合的生物样品）的温度，诱导混合物中的快速温度转换，并检测混合物中的目标分析物。此类热交换化学反应的应用可包括有机、无机、生化和分子反应等。热化学反应的例子包括核酸扩增、热循环扩增，如聚合酶链式反应（PCR）、连接酶链式反应（LCR）、自持序列复制、酶动力学研究、均相配体结合测定，以及需要复杂温度变化的更复杂的生化机理研究。

A preferred detection technique for chemical or biochemical analysis is optical interrogation, typically using fluorescence or chemiluminescence measurements. For ligandbinding assays, time-resolved fluorescence, fluorescence polarization, or optical absorption are often used. For PCR assays, fluorescence chemistries are often employed.
化学或生化分析的首选检测技术是光学检测，通常使用荧光或化学发光测量。配体结合检测通常使用时间分辨荧光、荧光偏振或光学吸收。在 PCR 检测中，通常会使用荧光化学试剂。

Conventional instruments for conducting thermal reactions and for optically detecting the reaction products typically incorporate a block of metal having as many as ninety-six conical reaction tubes. The metal block is heated and cooled either by a Peltier heating/cooling apparatus or by a closed-loop liquid heating/cooling system in which liquid flows through channels machined into the block. Such instruments incorporating a metal block are described in U.S. Pat. No. 5,038,852 to Johnson, U.S. Pat. No. 5,333,675 to Mullis, and U.S. Pat. No. $5,475,610$$5,475,610$5,475,6105,475,610 to Atwood.
用于进行热反应和光学检测反应产物的传统仪器通常包括一个金属块，上面有多达 96 个锥形反应管。金属块通过珀尔帖加热/冷却装置或闭环液体加热/冷却系统进行加热和冷却，在闭环液体加热/冷却系统中，液体通过加工到金属块中的通道流动。美国专利 No.No. 5,038,852 to Johnson、U.S. Pat.Mullis的美国专利第5,333,675号和U.S. Pat. $5,475,610$$5,475,610$5,475,6105,475,610 号。

These conventional instruments have several disadvantages. First, due to the large thermal mass of a metal block, the heating and cooling rates in these instruments are limited to about $1^{\circ }\mathrm{C}$$1^{\circ }\mathrm{C}$1^(@)C1^{\circ} \mathrm{C}./sec resulting in longer processing times. For example, in a typical PCR application, fifty cycles may require two or more hours to complete. With these relatively slow heating and cooling rates, it has been observed that some processes requiring precise temperature control are inefficient. For example, reactions may occur at the intermediate temperatures, creating unwanted and interfering side products, such as PCR “primer-dimers” or anomalous amplicons, which are detrimental to the analytical process. Poor control of temperature also results in over-consumption of reagents necessary for the intended reaction.
这些传统仪器有几个缺点。首先，由于金属块的热质量大，这些仪器的加热和冷却速度被限制在 $1^{\circ }\mathrm{C}$$1^{\circ }\mathrm{C}$1^(@)C1^{\circ} \mathrm{C} ./秒左右，导致处理时间延长。例如，在典型的 PCR 应用中，50 个循环可能需要两个或更长时间才能完成。由于加热和冷却速度相对较慢，人们发现一些需要精确温度控制的过程效率很低。例如，反应可能在中间温度下进行，产生不需要的干扰副产物，如 PCR "引物二聚体 "或异常扩增子，这对分析过程不利。温度控制不当还会导致预期反应所需的试剂消耗过多。

Another disadvantage of these conventional instruments is that they typically do not permit real-time optical detection or continuous optical monitoring of the chemical reaction. For example, in the Perkin Elmer 7700 (ATC) instrument, optical fluorescence detection is accomplished by guiding an optical fiber to each of ninety-six reaction sites in a metal block. A central high power laser sequentially excites each reaction site and captures the fluorescence signal through the optical fiber. Since all of the reaction sites are sequentially excited by a single laser and since the fluorescence is detected by a single spectrometer and photomultiplier tube, simultaneous monitoring of each reaction site is not possible.
这些传统仪器的另一个缺点是通常无法对化学反应进行实时光学检测或连续光学监控。例如，在珀金埃尔默 7700（ATC）仪器中，光学荧光检测是通过将光纤引向金属块中 96 个反应点中的每个反应点来实现的。中央高功率激光器依次激发每个反应点，并通过光纤捕捉荧光信号。由于所有反应点都是由单个激光器依次激发的，而且荧光是由单个光谱仪和光电倍增管检测的，因此无法同时监测每个反应点。

Some of the instrumentation for newer processes requiring real-time optical monitoring of a chemical reaction has only recently become available. One such instrument is the MATCI device disclosed by Northrup et al in U.S. Pat. No. $5,589,136$$5,589,136$5,589,1365,589,136. This device uses a modular approach to PCR thermal cycling and optical analysis. Each chemical reaction is performed in its own silicon sleeve and each sleeve has its own associated optical excitation source and fluorescence detector. Using a light-emitting diode (LED) and a solidstate detector, real-time optical data is obtained from a compact, low-power module. The device includes only one light source and one detector for each module, however, so that the simultaneous detection of multiple analytes is not possible.
一些用于需要对化学反应进行实时光学监控的较新工艺的仪器最近才问世。Northrup 等人在美国专利 $5,589,136$$5,589,136$5,589,1365,589,136 No. $5,589,136$$5,589,136$5,589,1365,589,136 。该设备采用模块化方法进行 PCR 热循环和光学分析。每个化学反应都在自己的硅套管中进行，每个套管都有自己的相关光学激发光源和荧光检测器。利用发光二极管（LED）和固态检测器，可从一个小巧、低功耗的模块中获得实时光学数据。不过，该装置每个模块只有一个光源和一个检测器，因此无法同时检测多个分析物。

Another analysis instrument is available from Idaho Technologies and described by Wittwer et al. in “The LightCycler ${}^{\mathrm{TM}}$${}^{\mathrm{TM}}$^(TM)^{\mathrm{TM}} : A Microvolume Multisample Fluorimeter with Rapid Temperature Control”, BioTechniques, Vol. 22, pgs. 176-181, January 1997. The instrument includes a circular carousel with a stepper motor for holding up to twenty-four samples and for sequentially positioning each of the samples over an optics assembly. The temperature of the samples is controlled by a central heating cartridge and a fan positioned in a central chamber of the carousel.
Wittwer 等人在 "The LightCycler ${}^{\mathrm{TM}}$${}^{\mathrm{TM}}$^(TM)^{\mathrm{TM}} : A Microvolume Multisample Fluorimeter with Rapid Temperature Control"（《LightCycler${}^{\mathrm{TM}}$${}^{\mathrm{TM}}$^(TM)^{\mathrm{TM}}：一种具有快速温度控制功能的微量多样品荧光计》）一文中介绍了另一种分析仪器，该仪器由 Idaho Technologies 公司提供，《BioTechniques》第 22 卷第 176-181 页，1997 年 1 月。176-181 页，1997 年 1 月。该仪器包括一个带步进电机的圆形转盘，可容纳多达二十四个样品，并将每个样品按顺序放置在光学组件上。样品的温度由一个中央加热盒和一个位于转盘中央腔室的风扇控制。

In operation, the samples are placed in capillaries which are held by the carousel, and each sample is interrogated through a capillary tip by epi-illumination. The light source is a blue LED that is reflected off a first dichroic filter towards the sample. Light is focused to and collected from the capillary tip by an epi-illumination lens. Light emitted from the capillary tip passes through the first dichroic filter, is filtered by one or more additional dichroic filters, and is focused to photodiodes for detection.
在操作过程中，样品被放入由转盘固定的毛细管中，每个样品都通过毛细管尖端的外延照明进行检测。光源是一个蓝色发光二极管，从第一个二向色滤光片反射到样品上。光通过外延照明透镜聚焦到毛细管尖端并从毛细管尖端收集。从毛细管尖端发出的光穿过第一个二向色滤光片，再经过一个或多个二向色滤光片过滤，然后聚焦到光电二极管上进行检测。

Although this instrument permits detection of multiple analytes in a sample undergoing chemical reaction, it has several disadvantages. First, the illumination beams and the emitted light beams have relatively short optical path lengths through the sample volume and share the same path below the capillary tip. This may cause fluorescent emissions from the sample to be weak, leading to poor optical detection sensitivity. Second, the instrument only provides illumination light in one excitation wavelength range. Different fluorescent dyes have different optimal excitation wavelength ranges, however, so that the instrument cannot provide excitation beams in the optimal excitation wavelength range for each of multiple fluorescent dyes in the reaction fluid. Third, the use of dichroic filters may significantly decrease the optical sensitivity of the instrument. Each dichroic filter decreases the intensity of the emitted light by about half, so that the emitted light beams may be weak by the time they reach the detectors. For these reasons, the instrument may exhibit poor sensitivity in detecting fluorescently labeled analytes in the samples.
虽然这种仪器可以检测正在发生化学反应的样品中的多种分析物，但它也有一些缺点。首先，照明光束和发射光束穿过样品体积的光路长度相对较短，并且在毛细管尖端下方共享相同的路径。这可能会导致样品的荧光发射较弱，从而导致光学检测灵敏度较低。其次，仪器只提供一种激发波长范围的照明光。然而，不同的荧光染料有不同的最佳激发波长范围，因此仪器无法为反应液中的每一种多种荧光染料提供最佳激发波长范围内的激发光束。第三，使用二向色滤光片可能会大大降低仪器的光学灵敏度。每个二向色滤光片都会将发射光的强度降低一半左右，因此发射光束到达检测器时可能已经很弱了。因此，仪器在检测样品中的荧光标记分析物时可能会表现出较低的灵敏度。
U.S. Pat. No. 5,675,155 issued to Pentoney et al. discloses another detection system for sequentially and repetitively
美国专利由 Pentoney 等人获得的第 5,675,155 号专利披露了另一种检测系统，该系统可按顺序重复地进行检测。
scanning a plurality of sample volumes and for detecting radiation emitting from each of the samples. The system includes a plurality of coplanar side-by-side capillaries each containing a sample volume. The system also includes an electromagnetic radiation source, a mirror aligned to receive and reflect electromagnetic radiation, a scanner for moving the mirror, a filter wheel for filtering electromagnetic radiation collected from the samples, and a detector aligned to receive the filtered radiation. The sample volume in each capillary column contains fluorescently-labeled samples separated on an electrophoretic medium.
扫描多个样品体积，并检测每个样品发出的辐射。该系统包括多个共面并排毛细管，每个毛细管包含一个样品体积。该系统还包括一个电磁辐射源、一个用于接收和反射电磁辐射的反射镜、一个用于移动反射镜的扫描仪、一个用于过滤从样品中收集到的电磁辐射的过滤轮，以及一个用于接收过滤后辐射的检测器。每个毛细管柱中的样品体积包含在电泳介质上分离的荧光标记样品。

In operation, the radiation source, preferably a laser, directs an excitation beam onto the mirror. The reflected excitation beam passes through a focusing lens and onto a sample volume of a first capillary within the capillary array. Fluorescence emission radiation from the sample is collected and passed through a first filter of the filter wheel which is selected to block light at the wavelength of the laser source and to transmit fluorescence emitted by a first fluorescent dye in the sample volume. Fluorescence transmitted through the first filter is then detected by the detector. A motor then rotates the filter wheel to bring a second filter into the fluorescence emission beam. The second filter transmits fluorescence emitted by a second fluorescence dye, and the fluorescence is measured by the detector. The same process is repeated with third and fourth filters of the filter wheel to measure the fluorescent emission of third and fourth dyes in the sample volume. The entire four-step operation is then performed sequentially and repeatedly with each capillary column in the array.
在操作过程中，辐射源（最好是激光器）将激发光束照射到反射镜上。反射的激发光束穿过聚焦透镜，照射到毛细管阵列中第一个毛细管的样品体积上。样品发出的荧光辐射被收集起来，并通过滤光轮的第一个滤光片，该滤光片被选择用来阻挡激光源波长的光，并透射样品体积中第一种荧光染料发出的荧光。然后，检测器检测通过第一个滤光器的荧光。然后，电机转动滤光片轮，将第二个滤光片带入荧光发射光束。第二个滤光片透过第二种荧光染料发出的荧光，检测器对荧光进行测量。滤光片轮上的第三和第四个滤光片重复同样的过程，以测量样品体积中第三和第四种染料的荧光发射。整个四步操作在阵列中的每个毛细管柱上依次重复进行。

Although this system permits the detection of multiple fluorescent dyes in a sample volume, it has several disadvantages in its use of a moving mirror and a rotating filter wheel. These moving parts typically result in a high cost of the optical system, high maintenance requirements, low reliability, high power consumption, and potential vibratory interference with the optical measurements.
虽然这种系统可以检测一个样品体积中的多种荧光染料，但它有几个缺点，即使用移动镜和旋转滤光轮。这些移动部件通常会导致光学系统成本高、维护要求高、可靠性低、功耗大，以及对光学测量的潜在振动干扰。

The present invention overcomes the disadvantages of the prior art by providing an improved system for thermally controlling and optically interrogating reaction mixtures (e.g., biological samples mixed with chemicals or reagents). In contrast to the prior art devices described above, the system of the present invention provides excitation light to each mixture in multiple, distinct excitation wavelength ranges. This ensures that the optimal excitation wavelength range is provided for each of a plurality of analytes in the mixture having different fluorescent, phosphorescent, chemiluminescent, or electrochemiluminescent labels. In addition, the system permits the simultaneous, real-time detection of multiple analytes in the mixture without requiring any moving parts, e.g., carousels or optical filter wheels. Because it has no moving parts, the system of the present invention typically has a lower cost, lower maintenance requirements, higher reliability, and lower power consumption than the prior art devices described above.
本发明克服了现有技术的缺点，提供了一种改进的系统，用于对反应混合物（例如，与化学品或试剂混合的生物样本）进行热控制和光学检测。与上述现有技术装置不同的是，本发明系统为每种混合物提供多个不同激发波长范围的激发光。这可确保为混合物中具有不同荧光、磷光、化学发光或电化学发光标签的多种分析物中的每一种提供最佳激发波长范围。此外，该系统还可同时实时检测混合物中的多种分析物，而无需任何移动部件，如转盘或滤光轮。由于没有移动部件，本发明的系统通常比上述现有技术设备成本更低、维护要求更低、可靠性更高、功耗更低。
The system of the present invention also overcomes the disadvantages of the prior art by providing for extremely rapid and accurate temperature changes of the reaction mixtures. Such tight control of temperature inhibits side reactions, such as the formation of unwanted bubbles or the degradation of components at certain temperatures, that would otherwise interfere with optical detection and analysis. The system is therefore useful in thermally sensitive chemical processes, such as polymerase chain reaction with the first set filters for transmitting the excitation beams in at least four excitation wavelength ranges, and the second optics assembly of each module includes at least four 5 detectors arranged with the second set of filters for detecting emitted light in at least four emission wavelength ranges. The system thus includes at least four separate optical channels for detecting up to four different analytes in each reaction mixture. Also in the preferred embodiment, the 0 system includes a base instrument for receiving the heatexchanging modules. The base instrument includes processing electronics for independently controlling the operation of each module. The system also preferably includes a computer programmed to control the processing electronics in the base instrument.
本发明的系统还克服了现有技术的缺点，可以极其快速准确地改变反应混合物的温度。这种严格的温度控制可抑制副反应，如在特定温度下形成不需要的气泡或成分降解，否则会干扰光学检测和分析。因此，该系统适用于对温度敏感的化学过程，如聚合酶链反应，第一组滤光片用于在至少四个激发波长范围内传输激发光束，每个模块的第二光学组件包括至少四个与第二组滤光片一起布置的探测器，用于检测至少四个发射波长范围内的发射光。因此，该系统包括至少四个独立的光学通道，用于检测每个反应混合物中多达四种不同的分析物。在优选的实施方案中，0 系统还包括一个用于接收热交换模块的基础仪器。基础仪器包括处理电子装置，用于独立控制每个模块的运行。系统最好还包括一台计算机，通过编程控制基础仪器中的处理电子装置。

Although it is presently preferred to position all of the light sources. in the first optics assembly and all of the
虽然目前倾向于将所有光源置于第一光学组件中，而将所有光源置于第二光学组件中。
detectors in the second optics assembly, it is also possible to include both one or more light sources and one or more detectors in each of the optics assemblies. According to a second embodiment of the invention, the first optics assembly comprises a first housing having a first optical window. The first optics assembly also includes a first light source for transmitting a first excitation beam to the reaction mixture through the first window and a first detector for receiving light emitted from the chamber through the first window. The first optics assembly further includes a first set of filters arranged in the first housing for filtering portions of the first excitation beam outside of a first excitation wavelength range, for filtering portions of the emitted light outside of a first emission wavelength range, and for directing the emitted light in the first emission wavelength range to the first detector. The first light source, the first set of filters, and the first detector are rigidly fixed in the first housing.
根据本发明的第二种实施方式，第一光学组件包括具有第一光学窗口的第一外壳，第二光学组件包括具有第一光学窗口的第二探测器，也可以在每个光学组件中同时包括一个或多个光源和一个或多个探测器。根据本发明的第二个实施方案，第一光学组件包括一个具有第一光学窗口的第一外壳。第一光学组件还包括用于通过第一窗口向反应混合物发射第一激发光束的第一光源，以及用于接收通过第一窗口从腔室发射的光的第一检测器。第一光学组件还包括布置在第一外壳中的第一组滤波器，用于过滤第一激发光束中第一激发波长范围之外的部分，过滤第一发射波长范围之外的部分发射光，并将第一发射波长范围内的发射光导向第一检测器。第一光源、第一组滤光器和第一探测器被刚性固定在第一外壳中。
Also according to the second embodiment, the second optics assembly comprises a second housing having a second optical window. The second optics assembly also includes a second light source for transmitting a second excitation beam to the reaction mixture through the second window and a second detector for receiving light emitted from the chamber through the second window. The second optics assembly further includes a second set of filters arranged in the second housing for filtering portions of the second excitation beam outside of a second excitation wavelength range different than the first excitation wavelength range, for filtering portions of the emitted light outside of a second emission wavelength range different than the first emission wavelength range, and for directing the emitted light in the second emission wavelength range to the second detector. The second light source, the second set of filters, and the second detector are rigidly fixed in the second housing, so that the optical system has no moving parts. In the second embodiment, each optics assembly may optionally include an additional detector and filter to provide four optical detection channels for detecting up to four different analytes in each reaction mixture.
同样根据第二实施例，第二光学组件包括具有第二光学窗口的第二外壳。第二光学组件还包括用于通过第二窗口向反应混合物发射第二激发光束的第二光源，以及用于接收通过第二窗口从腔室发射的光的第二检测器。第二光学组件还包括布置在第二壳体中的第二组滤波器，用于过滤第二激发光束中不同于第一激发波长范围的第二激发波长范围之外的部分，过滤不同于第一发射波长范围的第二发射波长范围之外的发射光的部分，并将第二发射波长范围内的发射光导向第二探测器。第二光源、第二组滤光片和第二探测器刚性固定在第二外壳中，因此光学系统没有移动部件。在第二个实施例中，每个光学组件可选择包括一个额外的检测器和滤波器，以提供四个光学检测通道，用于检测每个反应混合物中最多四个不同的分析物。

A more complete understanding of the system of the present invention may be gained upon consideration of the following description and accompanying drawings.
通过阅读以下说明和附图，可以更全面地了解本发明的系统。

FIG. 1 shows a partially exploded, perspective view of a reaction vessel according to the present invention in which the reaction chamber sidewalls are removed to show the interior of the chamber.
图 1 显示了根据本发明设计的反应容器的部分剖视透视图，其中反应室的侧壁被移除，以显示反应室的内部。

FIG. 2 is a front view of the vessel of FIG. 1.
图 2 是图 1 中容器的正视图。
FIG. $\mathbf{3}$$\mathbf{3}$3\mathbf{3} is a side view of the vessel of FIG. 1 inserted in a thermal sleeve formed by opposing thermal plates.
图 $\mathbf{3}$$\mathbf{3}$3\mathbf{3} 是图 1 中容器的侧视图，容器插入由相对的隔热板形成的隔热套中。

FIG. 4 is a side view of a heat-exchanging module according to the present invention having a thermal sleeve, a pair of optics assemblies, and a cooling system. The reaction vessel of FIG. 1 is inserted into the thermal sleeve.
图 4 是本发明热交换模块的侧视图，该模块具有一个热套筒、一对光学组件和一个冷却系统。图 1 中的反应容器被插入热套管中。
FIGS. 5A-D are a series of intensity vs. wavelength graphs in which:
图 5A-D 是一系列强度与波长的对比图，其中：
FIGS. 5A and 5B show the excitation and emission spectra, respectively, of four dyes typically used in thermal reactions.
图 5A 和 5B 分别显示了四种通常用于热反应的染料的激发光谱和发射光谱。
FIG. 5C shows the effects of filtering the outputs of green and blue LEDs to provide distinct excitation wavelength ranges; and
图 5C 显示了过滤绿色和蓝色 LED 输出以提供不同激发波长范围的效果；以及
FIG. 5D shows the effects of filtering light emitted from each of the four dyes to form distinct emission wavelength ranges. used herein the term “fluid sample” includes both gases and used herein, the term “fluid sample” includes both gases and liquids, preferably the latter. The sample may be an aqueous
图 5D 显示了过滤四种染料中每种染料发出的光以形成不同发射波长范围的效果。在此使用的术语 "流体样品 "包括气体和液体，最好是后者。样品可以是水
solution containing particles, cells, microorganisms, ions, or liquids, preferably the latter. The sample may be an aqueous
含有颗粒、细胞、微生物、离子或液体的溶液，最好是后者。样品可以是水溶液
solution containing particles, cells, microorganisms, ions, or small and large molecules, such as proteins and nucleic 5 acids, etc. In a particular use, the sample may be a bodily fluid, e.g., blood or urine, or a suspension, such as pulverized food.
含有颗粒、细胞、微生物、离子或大小分子（如蛋白质和核酸等）的溶液。在特定用途中，样本可以是体液，如血液或尿液，也可以是悬浮液，如粉碎的食物。

FIG. 8 is a schematic, plan view of an optical detection assembly of the module of FIG. 4.
图 8 是图 4 模块的光学检测组件的平面示意图。

FIG. 9 is an exploded view of the detection assembly of FIG. 8.
图 9 是图 8 检测组件的剖视图。

FIG. 10 is a perspective view of a multi-site reactor system having dynamic, independent, computerimplemented control of each reaction site.
图 10 是多反应位反应器系统的透视图，该系统对每个反应位进行动态、独立的计算机控制。

FIG. 11 is a schematic, block diagram of another multisite reaction system having multiple thermal cycling instruments daisy-chained to a computer and a power source.
图 11 是另一个多位点反应系统的原理框图，该系统中的多台热循环仪器通过菊花链连接到计算机和电源上。

FIG. 12 is a schematic, block diagram of a base instrument of the system of FIG. 10.
图 12 是图 10 系统的基础仪器原理框图。

FIG. 13 is a schematic, block diagram of the electronic components of the heat-exchanging module of FIG. 4.
图 13 是图 4 热交换模块电子元件的原理框图。

FIG. 14 is a schematic, block diagram illustrating the computer controller architecture for the control, diagnostics, programming, and operational functions of the system of FIG. 10.
图 14 是说明图 10 系统控制、诊断、编程和操作功能的计算机控制器结构的方框示意图。

FIG. 15 is a block diagram showing the architecture of FIG. 14 that is preferably reproduced on a graphical user interface for selection of a function by a user.
图 15 是显示图 14 结构的框图，该结构最好再现于用户选择功能的图形用户界面上。

FIGS. 16-18 are a series of sample graphic displays viewable on the user’s computer monitor according to the present invention.
图 16-18 是根据本发明可在用户计算机显示器上查看的一系列图形显示示例。

FIG. 16 illustrates a Program Menu Screen through which site profiles are created and executed.
图 16 展示了一个程序菜单屏幕，通过该屏幕可以创建和执行站点配置文件。

FIG. 17 illustrates an Instrument Menu Screen that displays current thermal cycling status.
图 17 展示了一个仪器菜单屏幕，显示当前的热循环状态。

FIG. 18 illustrates a Library Menu Screen through which desired temperature profiles may be retrieved from memory and executed and through which results may be displayed, transmitted to another computer, and/or printed.
图 18 展示了一个库菜单屏幕，通过该屏幕可以从内存中检索和执行所需的温度曲线，并显示、传输到另一台计算机和/或打印结果。

FIG. 19 is a flow diagram showing the overall control and operation of the system of FIG. $\mathbf{1}\mathbf{0}$$\mathbf{1}\mathbf{0}$10\mathbf{1 0}.
图 19 是显示图 $\mathbf{1}\mathbf{0}$$\mathbf{1}\mathbf{0}$10\mathbf{1 0} 系统整体控制和操作的流程图。

FIG. 20 is a flow diagram showing the steps for running a selected temperature profile on the system of FIG. $\mathbf{1}\mathbf{0}$$\mathbf{1}\mathbf{0}$10\mathbf{1 0}.
图 20 是一个流程图，显示了在图 $\mathbf{1}\mathbf{0}$$\mathbf{1}\mathbf{0}$10\mathbf{1 0} 的系统上运行选定温度曲线的步骤。

FIG. 21 is a flow diagram showing the steps for raising the temperature of a reaction mixture according to a preferred embodiment of the invention.
图 21 是一个流程图，显示了根据本发明的一个优选实施方案提高反应混合物温度的步骤。

FIG. 22 is a flow diagram showing the steps for lowering the temperature of a reaction mixture according to the preferred embodiment of the invention.
图 22 是显示根据本发明优选实施例降低反应混合物温度的步骤的流程图。

FIGS. 23A-23B are schematic, plan views of a pair of optics assemblies for use in the module of FIG. 4 according to a second embodiment of the invention.
图 23A-23B 是根据本发明的第二个实施例，用于图 4 模块中的一对光学组件的平面示意图。

FIGS. 24A-24B are schematic, plan views of another pair of optics assemblies for use in the module of FIG. 4 according to a third embodiment of the invention.
图 24A-24B 是根据本发明第三种实施方式，用于图 4 模块的另一对光学组件的平面示意图。

The present invention provides a system for thermally controlling and optically interrogating a reaction mixture, g., a fluid sample mixed with chemicals or reagents. As
本发明提供了一种对反应混合物（例如，与化学品或试剂混合的流体样品）进行热控制和光学检测的系统。如

FIG. 6 is a schematic, plan view of an optical excitation assembly of the module of FIG. 4.
图 6 是图 4 模块的光学激发组件的平面示意图。

FIG. 7 is an exploded view of the excitation assembly of FIG. 6.
图 7 是图 6 励磁组件的剖视图。

In a preferred embodiment, the system includes reaction vessels for holding the mixtures and heat-exchanging modules for receiving the vessels. Each heat-exchanging module includes a pair of opposing thermal plates between which one of the vessels is inserted for thermal processing, a fan positioned adjacent the plates for cooling the mixture, one or more temperature sensors for measuring the temperature of the plates, and a pair of optics assemblies for optically interrogating the mixture. The system also includes a base unit with processing electronics for receiving the heatexchanging modules and for independently controlling each module. The system further includes a controller, such as a personal computer or network computer, that provides a user interface and controls the operation of the processing electronics.
在一个优选的实施方案中，系统包括用于容纳混合物的反应容器和用于接收容器的热交换模块。每个热交换模块包括一对相对的热板，其中一个容器插入热板之间进行热处理；一个风扇位于热板附近，用于冷却混合物；一个或多个温度传感器，用于测量热板的温度；一对光学组件，用于对混合物进行光学检测。该系统还包括一个带有处理电子装置的基本单元，用于接收热交换模块和独立控制每个模块。系统还包括一个控制器，如个人计算机或网络计算机，提供用户界面并控制处理电子装置的运行。

FIGS. 1-22 illustrate a preferred embodiment of the invention. FIG. 1 shows a partially exploded view of a reaction vessel 2, and FIG. 2 shows a front view of the vessel 2. The vessel $\mathbf{2}$$\mathbf{2}$2\mathbf{2} includes a reaction chamber $\mathbf{1}\mathbf{0}$$\mathbf{1}\mathbf{0}$10\mathbf{1 0} for holding a reaction mixture. The vessel $\mathbf{2}$$\mathbf{2}$2\mathbf{2} is designed for optimal heat transfer to and from the reaction mixture and for efficient optical viewing of the mixture. The thin shape of the vessel contributes to optimal thermal kinetics by providing large surfaces for thermal conduction and for contacting thermal plates. In addition, the walls of the vessel 2 provide optical windows into the chamber $\mathbf{1}\mathbf{0}$$\mathbf{1}\mathbf{0}$10\mathbf{1 0} so that the entire reaction mixture can be optically interrogated.
图 1-22 说明了本发明的一个优选实施例。图 1 显示反应容器 2 的部分剖视图，图 2 显示容器 2 的正视图。容器 $\mathbf{2}$$\mathbf{2}$2\mathbf{2} 包括一个用于容纳反应混合物的反应室 $\mathbf{1}\mathbf{0}$$\mathbf{1}\mathbf{0}$10\mathbf{1 0} 。容器 $\mathbf{2}$$\mathbf{2}$2\mathbf{2} 的设计可实现反应混合物之间的最佳热传导，并可对混合物进行有效的光学观察。容器的薄型设计为热传导和热板接触提供了较大的表面，从而有助于优化热动力学。此外，容器 2 的壁提供了进入腔室 $\mathbf{1}\mathbf{0}$$\mathbf{1}\mathbf{0}$10\mathbf{1 0} 的光学窗口，从而可以对整个反应混合物进行光学检测。

In more detail to FIGS. 1-2, the reaction vessel $\mathbf{2}$$\mathbf{2}$2\mathbf{2} includes a rigid frame 16 that defines the perimeter of the reaction chamber 10. The frame 16 also includes a port 4 and a channel 8 that connects the port 4 to the reaction chamber 10. Thin, flexible walls 18 (shown in FIG. 1 exploded from the frame 16) are coupled to opposite sides of the frame 16 to form the sidewalls of the chamber $\mathbf{1}\mathbf{0}$$\mathbf{1}\mathbf{0}$10\mathbf{1 0}.
更详细地参见图 1-2，反应容器 $\mathbf{2}$$\mathbf{2}$2\mathbf{2} 包括一个刚性框架 16，它限定了反应室 10 的周边。框架 16 还包括一个端口 4 和一个连接端口 4 与反应室 10 的通道 8。薄的柔性壁 18（如图 1 所示，与框架 16 分开）连接到框架 16 的相对侧，形成反应室 $\mathbf{1}\mathbf{0}$$\mathbf{1}\mathbf{0}$10\mathbf{1 0} 的侧壁。

The walls 18 facilitate optimal thermal conductance to the reaction mixture contained in the chamber $\mathbf{1}\mathbf{0}$$\mathbf{1}\mathbf{0}$10\mathbf{1 0}. The flexible nature of the walls 18 allows for maximum contact with thermal plates. The walls are conformable to the surface of the plates to prevent or minimize gaps between surfaces. Furthermore, the flexible walls continue to conform to the thermal surface if the surface shape changes during the course of the heat-exchanging operation.
壁 18 有利于腔室 $\mathbf{1}\mathbf{0}$$\mathbf{1}\mathbf{0}$10\mathbf{1 0} 中反应混合物的最佳热传导。壁 18 的柔性使其能够与隔热板最大程度地接触。壁可与隔热板表面贴合，以防止或尽量减少表面之间的间隙。此外，如果表面形状在热交换操作过程中发生变化，柔性壁可继续与热表面保持一致。

FIG. 3 shows contact between the reaction vessel and a pair of opposing thermal plates $\mathbf{3}\mathbf{4}\mathbf{A},34\mathrm{B}$$\mathbf{3}\mathbf{4}\mathbf{A},34\mathrm{B}$34A,34B\mathbf{3 4 A}, 34 \mathrm{~B}. At least one of the plates, and preferably both of the plates, includes a heating element, such as a resistor, for heating the reaction mixture in the vessel. The plates 34A, 34B also preferably include temperature sensors, such as thermistors $36\mathrm{A},36\mathrm{B}$$36\mathrm{A},36\mathrm{B}$36A,36B36 \mathrm{~A}, 36 \mathrm{~B}. When the vessel 2 is inserted between the plates, the inner surfaces of the plates contact walls 18. In this position, minimal or no gaps are found between the plate surfaces and the walls 18 of the reaction chamber. For good thermal conductance, the thickness of each wall 18 is preferably between about 0.003 to 0.5 mm , more preferably 0.01 to 0.15 mm , and most preferably 0.025 to 0.08 mm . Each wall 18 may be a film, sheet, or a molded, machined extruded or cast piece, or other convenient thin and flexible structure.
图 3 显示了反应容器与一对相对的热板 $\mathbf{3}\mathbf{4}\mathbf{A},34\mathrm{B}$$\mathbf{3}\mathbf{4}\mathbf{A},34\mathrm{B}$34A,34B\mathbf{3 4 A}, 34 \mathrm{~B} 之间的接触。其中至少一块板，最好是两块板都包括加热元件，如电阻器，用于加热容器中的反应混合物。板 34A、34B 最好还包括温度传感器，如热敏电阻 $36\mathrm{A},36\mathrm{B}$$36\mathrm{A},36\mathrm{B}$36A,36B36 \mathrm{~A}, 36 \mathrm{~B} 。当容器 2 插入板之间时，板的内表面与壁 18 接触。在此位置，板面与反应室壁 18 之间的间隙极小或没有间隙。为了达到良好的导热效果，每个壁 18 的厚度最好在 0.003 至 0.5 毫米之间，更理想的是 0.01 至 0.15 毫米，最理想的是 0.025 至 0.08 毫米。每个壁 18 可以是薄膜、薄片、模压件、机械挤压件或浇铸件，或其他方便的薄而柔性的结构。
The material composing the walls $\mathbf{1}\mathbf{8}$$\mathbf{1}\mathbf{8}$18\mathbf{1 8} may be a polyalcohol including polypropylene, polyethylene, polyester, and other polymers, laminates or homogenous polymers, metals or metal laminates, or other materials which may be thin, flexible, conformable and permit high heat transfer and is preferably in the form of a film or sheet. Where the frame 16 of the vessel is a particular material, such as polypropylene, the sidewalls are preferably the same material, such as polypropylene, so that the heat expansion and cooling rates of the walls are substantially the same as the frame. dyes prior to being added to the chamber 10. Alternatively, the sample may be introduced to reagents and dyes in the chamber 10. As shown in FIG. 3, the vessel 2 is placed between the thermal plates $34\mathrm{A},34\mathrm{B}$$34\mathrm{A},34\mathrm{B}$34A,34B34 \mathrm{~A}, 34 \mathrm{~B} so that the walls $\mathbf{1}\mathbf{8}$$\mathbf{1}\mathbf{8}$18\mathbf{1 8} of the vessel press against and conform to the inner surfaces of the plates. The reaction mixture is exposed to variations in temperature by activating the heating elements on the plates
构成壁 $\mathbf{1}\mathbf{8}$$\mathbf{1}\mathbf{8}$18\mathbf{1 8} 的材料可以是聚醇，包括聚丙烯、聚乙烯、聚酯和其他聚合物、层压材料或均质聚合物、金属或金属层压材料，或其他材料，它们可以很薄、柔韧、顺应性好并允许高热传导，最好是薄膜或薄片形式。如果容器的框架 16 是聚丙烯等特定材料，侧壁最好也是聚丙烯等相同材料，这样侧壁的热膨胀率和冷却率基本上与框架相同。或者，也可以将样品引入腔室 10 中的试剂和染料中。如图 3 所示，将容器 2 放置在热板 $34\mathrm{A},34\mathrm{B}$$34\mathrm{A},34\mathrm{B}$34A,34B34 \mathrm{~A}, 34 \mathrm{~B} 之间，使容器壁 $\mathbf{1}\mathbf{8}$$\mathbf{1}\mathbf{8}$18\mathbf{1 8} 紧贴热板内表面并与之保持一致。通过激活板上的加热元件，使反应混合物受到温度变化的影响

34A, 34B. The reaction mixture is then optically interrogated, preferably through the optically transmissive bottom walls 32A, 32B of the frame 16, as shown in FIG. 2. Arrows A in FIG. 2 represent illumination beams entering the chamber 10 through wall 32 A and arrows B represent emitted light exiting the chamber 10 through wall 32B.
34A、34B。然后对反应混合物进行光学检测，最好是通过框架 16 的光学透射底壁 32A、32B，如图 2 所示。图 2 中的箭头 A 代表通过壁 32 A 进入腔室 10 的照明光束，箭头 B 代表通过壁 32B 从腔室 10 出来的发射光。
The walls 32A, 32B are angularly offset from each other to allow optical excitation of labeled analytes in the reaction mixture through the first wall 32 A and optical detection of the labeled analytes through the second wall 32B. It is usually preferred that the walls 32A, 32B are offset at an angle of about ${90}^{\circ }$${90}^{\circ }$90^(@)90^{\circ}. A ${90}^{\circ }$${90}^{\circ }$90^(@)90^{\circ} angle between excitation and detection paths assures that a minimum amount of excitation radiation entering through wall 32 A will exit through wall 32B. Also the ${90}^{\circ }$${90}^{\circ }$90^(@)90^{\circ} angle permits a maximum amount of emitted radiation, e.g. fluorescence, to be collected through wall 32B. In alternative embodiments, the angle between the optical walls may be larger or smaller than ${90}^{\circ }$${90}^{\circ }$90^(@)90^{\circ}, depending upon the efficiency and sensitivity of the excitation and detection optics. For example, where a detection system effectively discriminates between excitation and emitted light, an angle of less than ${90}^{\circ }$${90}^{\circ }$90^(@)90^{\circ} between walls may be desired. Conversely, where a detection system fails to efficiently discriminate between excitation and emitted light, an angle greater than ${90}^{\circ }$${90}^{\circ }$90^(@)90^{\circ} may be of interest.
壁 32A 和 32B 在角度上相互偏移，以便通过第一壁 32A 对反应混合物中的标记分析物进行光激发，并通过第二壁 32B 对标记分析物进行光检测。通常优选的是，壁 32A、32B 以大约 ${90}^{\circ }$${90}^{\circ }$90^(@)90^{\circ} 的角度偏移。激发路径和检测路径之间的 ${90}^{\circ }$${90}^{\circ }$90^(@)90^{\circ} 夹角可确保通过壁 32A 进入的激发辐射从壁 32B 流出的量最小。另外， ${90}^{\circ }$${90}^{\circ }$90^(@)90^{\circ} 角允许通过壁 32B 收集到最大数量的发射辐射，例如荧光。在其他实施例中，光学壁之间的角度可以大于或小于 ${90}^{\circ }$${90}^{\circ }$90^(@)90^{\circ} ，这取决于激发和检测光学器件的效率和灵敏度。例如，如果检测系统能有效区分激发光和发射光，则光壁之间的夹角可能需要小于 ${90}^{\circ }$${90}^{\circ }$90^(@)90^{\circ} 。相反，如果检测系统无法有效区分激发光和发射光，则可能需要大于 ${90}^{\circ }$${90}^{\circ }$90^(@)90^{\circ} 的角度。

The walls 32A, 32B may be joined to form a “V” shaped point at the bottom of the chamber $\mathbf{1}\mathbf{0}$$\mathbf{1}\mathbf{0}$10\mathbf{1 0}. Alternatively, the interface of the angled walls need not connect to form a point, but may be separated by an intermediary portion, such as another wall or various mechanical or fluidic features which do not interfere with the thermal and optical performance of the vessel. For example, the angled walls may meet at a port which leads to another processing area in communication with the chamber 10, such as an integrated capillary electrophoresis area. In the presently preferred embodiment, a locating tab 17 extends below the intersection of walls 32A, 32B. The locating tab 17 is used to properly position the vessel 2 in a heat-exchanging module described below with reference to FIG. 4.
壁 32A、32B 可以连接起来，在腔室 $\mathbf{1}\mathbf{0}$$\mathbf{1}\mathbf{0}$10\mathbf{1 0} 的底部形成一个 "V "形点。或者，斜壁的接口不必连接形成一个点，而可以由一个中间部分隔开，例如另一个壁或各种机械或流体特征，这些特征不会干扰容器的热性能和光学性能。例如，倾斜壁可以在一个端口处相接，该端口通向与腔室 10 相通的另一个处理区，如集成毛细管电泳区。在目前优选的实施方案中，定位片 17 延伸至壁 32A 和 32B 的交叉点下方。定位片 17 用于在下面参照图 4 描述的热交换模块中正确定位容器 2。

Optimum optical sensitivity may be attained by maximizing the optical sampling path length of both the light beams exciting the labeled analytes in the reaction mixture and the emitted light that is detected, as represented by the equation:
将激发反应混合物中标记分析物的光束和检测到的发射光的光采样路径长度最大化，可达到最佳光学灵敏度，如公式所示：

where ${\mathrm{I}}_o$${\mathrm{I}}_o$I_(o)\mathrm{I}_{o} is the illumination output of the emitted light in volts, photons or the like, C is the concentration of analyte to be detected, ${\mathrm{I}}_i$${\mathrm{I}}_i$I_(i)\mathrm{I}_{i} is the input illumination, L is the path length, and A is the intrinsic absorptivity of the dye used to label the analyte.
其中， ${\mathrm{I}}_o$${\mathrm{I}}_o$I_(o)\mathrm{I}_{o} 是以伏特、光子或类似单位表示的发射光的照明输出，C 是要检测的被分析物的浓度， ${\mathrm{I}}_i$${\mathrm{I}}_i$I_(i)\mathrm{I}_{i} 是输入照明，L 是路径长度，A 是用于标记被分析物的染料的本征吸收率。

The thin, flat reaction vessel 2 of the present invention optimizes detection sensitivity by providing maximum optical path length per unit analyte volume. In particular, the vessel $\mathbf{2}$$\mathbf{2}$2\mathbf{2} is preferably constructed such that each of the sides of the chamber $\mathbf{1}\mathbf{0}$$\mathbf{1}\mathbf{0}$10\mathbf{1 0} has a length in the range of 1 to 15 mm , the chamber has a thickness in the range of 0.5 to 5 mm , and the ratio of the length of each side of the chamber to the thickness of the chamber is at least $2:1$$2:1$2:12: 1. These parameters are presently preferred to provide a vessel having a relatively large optical path length through the chamber, i.e. 1 to 15 mm on average, while still keeping the chamber sufficiently thin to allow for extremely rapid heating and cooling of the reaction mixture contained in the chamber.
本发明的薄平反应容器 2 通过提供单位分析物体积的最大光路长度来优化检测灵敏度。特别是，容器 $\mathbf{2}$$\mathbf{2}$2\mathbf{2} 最好构造成：腔室 $\mathbf{1}\mathbf{0}$$\mathbf{1}\mathbf{0}$10\mathbf{1 0} 的每个侧面的长度在 1 至 15 毫米之间，腔室的厚度在 0.5 至 5 毫米之间，腔室每个侧面的长度与腔室厚度之比至少为 $2:1$$2:1$2:12: 1 。目前优选这些参数是为了提供一个具有通过腔室的相对较大光路长度（即平均 1 至 15 毫米）的容器，同时仍然保持腔室足够薄，以便对腔室中的反应混合物进行极快的加热和冷却。
More preferably, the vessel $\mathbf{2}$$\mathbf{2}$2\mathbf{2} is constructed such that each of the sides of the chamber 10 has a length in the range of 5 to 12 mm , the chamber has a thickness in the range of 0.5 retainers 40. Additionally, the plates may be spring-biased towards each other as described in U.S. application Ser. No. $09/194,374$$09/194,374$09//194,37409 / 194,374 filed Nov. 24, 1998, the disclosure of which is incorporated by reference herein. The housing 38 includes a 0 slot above the plates $\mathbf{3}\mathbf{4}\mathbf{A},\mathbf{3}\mathbf{4}\mathbf{B}$$\mathbf{3}\mathbf{4}\mathbf{A},\mathbf{3}\mathbf{4}\mathbf{B}$34A,34B\mathbf{3 4 A}, \mathbf{3 4 B} so that the vessel 2 may be inserted through the slot and between the plates.
更优选的是，容器 $\mathbf{2}$$\mathbf{2}$2\mathbf{2} 的结构使腔室10的每个侧面的长度在5至12毫米的范围内，腔室的厚度在0.5个保持器40的范围内。此外，如 1998 年 11 月 24 日提交的美国申请 Ser.外壳 38 包括一个位于板 $\mathbf{3}\mathbf{4}\mathbf{A},\mathbf{3}\mathbf{4}\mathbf{B}$$\mathbf{3}\mathbf{4}\mathbf{A},\mathbf{3}\mathbf{4}\mathbf{B}$34A,34B\mathbf{3 4 A}, \mathbf{3 4 B} 上方的 0 形槽，以便容器 2 可以通过槽插入板之间。

The heat-exchanging module $\mathbf{3}\mathbf{7}$$\mathbf{3}\mathbf{7}$37\mathbf{3 7} also preferably includes a cooling system, such as a fan 42, for cooling the reaction mixture in the vessel 2 . When the vessel 2 is positioned 55 between the plates $\mathbf{3}\mathbf{4}\mathbf{A},\mathbf{3}\mathbf{4}\mathbf{B}$$\mathbf{3}\mathbf{4}\mathbf{A},\mathbf{3}\mathbf{4}\mathbf{B}$34A,34B\mathbf{3 4 A}, \mathbf{3 4 B}, the chamber of the vessel is cooled by the air circulating from the fan 42 . Alternatively, the cooling system may comprise a Peltier device or other
热交换模块 $\mathbf{3}\mathbf{7}$$\mathbf{3}\mathbf{7}$37\mathbf{3 7} 最好还包括冷却系统，如风扇 42，用于冷却容器 2 中的反应混合物。当容器 2 位于板 $\mathbf{3}\mathbf{4}\mathbf{A},\mathbf{3}\mathbf{4}\mathbf{B}$$\mathbf{3}\mathbf{4}\mathbf{A},\mathbf{3}\mathbf{4}\mathbf{B}$34A,34B\mathbf{3 4 A}, \mathbf{3 4 B} 之间 55 时，容器腔由风扇 42 循环的空气冷却。或者，冷却系统可以包括一个珀尔帖装置或其他设备。
refrigeration system for carrying a refrigerant or compressed fluid past the reaction vessel. These and other cooling systems are well known in the art.
制冷系统，用于携带制冷剂或压缩流体通过反应容器。这些冷却系统和其他冷却系统都是众所周知的。
The heat-exchanging module 37 further includes an optical excitation assembly 46 and an optical detection assembly 48 for optically interrogating the reaction mixture contained in the vessel 2. The excitation assembly 46 includes a first circuit board $\mathbf{5}\mathbf{0}$$\mathbf{5}\mathbf{0}$50\mathbf{5 0} for holding its electronic components, and the detection assembly 46 includes a second circuit board 52 for holding its electronic components. The excitation assembly 46 includes multiple light sources, such as LEDs, for exciting fluorescently-labeled analytes in the vessel $\mathbf{2}$$\mathbf{2}$2\mathbf{2}. The excitation assembly 46 also includes one or more lenses for collimating the light from the light sources, as well as filters for selecting the excitation wavelength ranges of interest. The detection assembly 48 includes multiple detectors, such as photodiodes, for monitoring the light emitted from the vessel 2. The detection assembly 48 also includes one or more lenses for focusing and collimating the emitted light, as well as filters for selecting the emission wavelength ranges of interest. The specific components of the optics assemblies 46, 48 are described in greater detail below with reference to FIGS. 6-9.
热交换模块 37 还包括光学激发组件 46 和光学检测组件 48，用于对容器 2 中的反应混合物进行光学检测。激发组件 46 包括用于容纳其电子元件的第一电路板 $\mathbf{5}\mathbf{0}$$\mathbf{5}\mathbf{0}$50\mathbf{5 0} ，检测组件 46 包括用于容纳其电子元件的第二电路板 52。激发组件 46 包括多个光源，如 LED，用于激发容器 $\mathbf{2}$$\mathbf{2}$2\mathbf{2} 中的荧光标记分析物。激发组件 46 还包括一个或多个用于准直来自光源的光的透镜，以及用于选择相关激发波长范围的滤波器。检测组件 48 包括多个检测器，如光电二极管，用于监测容器 2 发出的光。检测组件 48 还包括一个或多个用于聚焦和准直发射光的透镜，以及用于选择相关发射波长范围的滤波器。下文将参照图 6-9 更详细地描述光学组件 46 和 48 的具体组件。
The optics assemblies $\mathbf{4}\mathbf{6},48$$\mathbf{4}\mathbf{6},48$46,48\mathbf{4 6}, 48 are positioned in the housing 38 such that when the vessel $\mathbf{2}$$\mathbf{2}$2\mathbf{2} is placed between the plates $34\mathrm{A},34\mathrm{B}$$34\mathrm{A},34\mathrm{B}$34A,34B34 \mathrm{~A}, 34 \mathrm{~B}, the first optics assembly 46 is in optical communication with the first optically transmissive bottom wall 32A of the vessel and the second optics assembly 48 is in optical communication with the second optically transmissive bottom wall 32B of the vessel (see FIG. 2). In the preferred embodiment, the optics assemblies 46, 48 are placed in optical communication with the bottom walls of the vessel 2 by simply locating the optics assemblies next to the thermal plates $\mathbf{3}\mathbf{4}\mathbf{A},34\mathrm{B}$$\mathbf{3}\mathbf{4}\mathbf{A},34\mathrm{B}$34A,34B\mathbf{3 4 A}, 34 \mathrm{~B} so that when the vessel is placed between the plates, the optics assemblies 46,48 are physically adjacent the first and second bottom walls of the vessel, respectively.
光学组件 $\mathbf{4}\mathbf{6},48$$\mathbf{4}\mathbf{6},48$46,48\mathbf{4 6}, 48 位于外壳38中，当容器 $\mathbf{2}$$\mathbf{2}$2\mathbf{2} 放置在板 $34\mathrm{A},34\mathrm{B}$$34\mathrm{A},34\mathrm{B}$34A,34B34 \mathrm{~A}, 34 \mathrm{~B} 之间时，第一光学组件46与容器的第一光学透射底壁32A进行光学通信，第二光学组件48与容器的第二光学透射底壁32B进行光学通信（见图2）。在优选实施例中，光学组件 46、48 与容器 2 的底壁光学通信，只需将光学组件置于热板 $\mathbf{3}\mathbf{4}\mathbf{A},34\mathrm{B}$$\mathbf{3}\mathbf{4}\mathbf{A},34\mathrm{B}$34A,34B\mathbf{3 4 A}, 34 \mathrm{~B} 旁即可，这样当容器置于热板之间时，光学组件 46、48 分别与容器的第一和第二底壁物理相邻。
Additionally, the longitudinal axes of the optics assemblies $\mathbf{4}\mathbf{6},48$$\mathbf{4}\mathbf{6},48$46,48\mathbf{4 6}, 48 are preferably angularly offset from each other by an angle of about ${90}^{\circ }$${90}^{\circ }$90^(@)90^{\circ}, and the assemblies 46,48 are preferably positioned such that when the vessel 2 is placed between the plates, the longitudinal axis of the optics assembly 46 is orthogonal to the first bottom wall and the longitudinal axis of the optics assembly 48 is orthogonal to the second bottom wall. A ${90}^{\circ }$${90}^{\circ }$90^(@)90^{\circ} angle between excitation and detection paths assures that a minimum amount of excitation radiation entering through the first bottom wall of the vessel exits through the second bottom wall. Also the ${90}^{\circ }$${90}^{\circ }$90^(@)90^{\circ} angle permits a maximum amount of emitted radiation to be collected through the second wall.
此外，光学组件 $\mathbf{4}\mathbf{6},48$$\mathbf{4}\mathbf{6},48$46,48\mathbf{4 6}, 48 的纵轴最好在角度上相互偏移约 ${90}^{\circ }$${90}^{\circ }$90^(@)90^{\circ} 的角度，组件46、48最好定位成当容器2放置在板之间时，光学组件46的纵轴与第一底壁正交，光学组件48的纵轴与第二底壁正交。激发路径和检测路径之间的 ${90}^{\circ }$${90}^{\circ }$90^(@)90^{\circ} 夹角可确保从容器第一底壁进入的激发辐射从第二底壁排出的量最小。此外， ${90}^{\circ }$${90}^{\circ }$90^(@)90^{\circ} 角还允许通过第二底壁收集到最大量的发射辐射。
Optionally, a gel or fluid may be used to establish or improve optical communication between each optics assembly and the vessel 2. The gel or fluid should have a refractive index close to the refractive indexes of the elements that it is coupling. In alternative embodiments, optical communication may be established between the optics assemblies and the walls of the vessel via optical fibers; light pipes, wave guides, or similar devices. One advantage of these devices is that they eliminate the need to locate the optics assemblies 46,48 physically adjacent to the thermal plates $34\mathrm{A},34\mathrm{B}$$34\mathrm{A},34\mathrm{B}$34A,34B34 \mathrm{~A}, 34 \mathrm{~B}. This leaves more room around the plates in which to circulate cooling air or refrigerant, so that cooling may be improved.
可选择使用凝胶或流体来建立或改善每个光学组件与容器 2 之间的光通信。凝胶或流体的折射率应接近其耦合元件的折射率。在其他实施方案中，可通过光纤、光导管、波导管或类似装置在光学组件和容器壁之间建立光通信。这些装置的一个优点是无需将光学组件 46、48 安装在热板 $34\mathrm{A},34\mathrm{B}$$34\mathrm{A},34\mathrm{B}$34A,34B34 \mathrm{~A}, 34 \mathrm{~B} 附近。这就在隔热板周围留出了更多空间来循环冷却空气或制冷剂，从而改善冷却效果。
In the preferred embodiment, the vessel 2 includes a locating tab 17 (see FIG. 2) that fits into a slot formed between the optics assemblies $\mathbf{4}\mathbf{6}\mathbf{,}\mathbf{4}\mathbf{8}$$\mathbf{4}\mathbf{6}\mathbf{,}\mathbf{4}\mathbf{8}$46,48\mathbf{4 6 , 4 8} to ensure proper positioning of the vessel 2 for optical detection. For improved
在优选实施例中，容器 2 包括一个定位片 17（见图 2），该定位片与光学组件 $\mathbf{4}\mathbf{6}\mathbf{,}\mathbf{4}\mathbf{8}$$\mathbf{4}\mathbf{6}\mathbf{,}\mathbf{4}\mathbf{8}$46,48\mathbf{4 6 , 4 8} 之间形成的槽相匹配，以确保容器 2 的正确定位，从而进行光学检测。为了改进
detection, the module 37 also preferably includes a lighttight lid (not shown) that is placed over the top of the vessel 2 and sealed to the housing $\mathbf{3}\mathbf{8}$$\mathbf{3}\mathbf{8}$38\mathbf{3 8} after the vessel is inserted between the plates 34A, 34B.
通过检测，模块 37 最好还包括一个密封盖（未显示），盖子盖在容器 2 的顶部，并在容器插入板 34A、34B 之间后密封到外壳 $\mathbf{3}\mathbf{8}$$\mathbf{3}\mathbf{8}$38\mathbf{3 8} 上。

The housing $\mathbf{3}\mathbf{8}$$\mathbf{3}\mathbf{8}$38\mathbf{3 8} may be molded from a rigid, highperformance plastic, or other conventional material. The primary functions of the housing $\mathbf{3}\mathbf{8}$$\mathbf{3}\mathbf{8}$38\mathbf{3 8} are to provide a frame for holding the plates $34\mathrm{A},34\mathrm{B}$$34\mathrm{A},34\mathrm{B}$34A,34B34 \mathrm{~A}, 34 \mathrm{~B} and optics assemblies 46,48 and to provide flow channels and ports for directing cooling fluid, e.g. air or freon, and efficiently guiding the fluid flow across the surface of the plates $\mathbf{3}\mathbf{4}\mathbf{A},\mathbf{3}\mathbf{4}\mathbf{B}$$\mathbf{3}\mathbf{4}\mathbf{A},\mathbf{3}\mathbf{4}\mathbf{B}$34A,34B\mathbf{3 4 A}, \mathbf{3 4 B} and reaction vessel 2.
外壳 $\mathbf{3}\mathbf{8}$$\mathbf{3}\mathbf{8}$38\mathbf{3 8} 可由硬质高性能塑料或其他常规材料模制而成。外壳 $\mathbf{3}\mathbf{8}$$\mathbf{3}\mathbf{8}$38\mathbf{3 8} 的主要功能是提供一个用于固定板 $34\mathrm{A},34\mathrm{B}$$34\mathrm{A},34\mathrm{B}$34A,34B34 \mathrm{~A}, 34 \mathrm{~B} 和光学组件46、48的框架，以及提供用于引导冷却流体（例如空气或氟利昂）的流道和端口，并有效地引导流体流过板 $\mathbf{3}\mathbf{4}\mathbf{A},\mathbf{3}\mathbf{4}\mathbf{B}$$\mathbf{3}\mathbf{4}\mathbf{A},\mathbf{3}\mathbf{4}\mathbf{B}$34A,34B\mathbf{3 4 A}, \mathbf{3 4 B} 和反应容器2的表面。

The heat-exchanging module $\mathbf{3}\mathbf{7}$$\mathbf{3}\mathbf{7}$37\mathbf{3 7} also includes a PC board 54 for holding the electronic components of the module and an edge connector 58 for connecting the module 37 to a base instrument, as will be described below with reference to FIG. 10. The heating elements and thermistors 36A, 36B on the plates $\mathbf{3}\mathbf{4}\mathbf{A},\mathbf{3}\mathbf{4}\mathbf{B}$$\mathbf{3}\mathbf{4}\mathbf{A},\mathbf{3}\mathbf{4}\mathbf{B}$34A,34B\mathbf{3 4 A}, \mathbf{3 4 B}, as well as the optical boards $\mathbf{5}\mathbf{0}$$\mathbf{5}\mathbf{0}$50\mathbf{5 0} and $\mathbf{5}\mathbf{2}$$\mathbf{5}\mathbf{2}$52\mathbf{5 2}, are connected to the PC board $\mathbf{5}\mathbf{4}$$\mathbf{5}\mathbf{4}$54\mathbf{5 4} by flex cables (not shown in FIG. 4 for clarity of illustration). The module $\mathbf{3}\mathbf{7}$$\mathbf{3}\mathbf{7}$37\mathbf{3 7} may also include a grounding trace 56 for shielding the optical detection circuit. The module 37 also preferably includes an indicator, such as an LED 44, for indicating to a user the current status of the module such as “ready to load sample”, “ready to load reagent,” “heating,” “cooling,” “finished,” or “fault”.
热交换模块 $\mathbf{3}\mathbf{7}$$\mathbf{3}\mathbf{7}$37\mathbf{3 7} 还包括用于容纳模块电子元件的 PC 板 54 和用于将模块 37 连接到基础仪器的边缘连接器 58，下文将参考图 10 进行描述。板 $\mathbf{3}\mathbf{4}\mathbf{A},\mathbf{3}\mathbf{4}\mathbf{B}$$\mathbf{3}\mathbf{4}\mathbf{A},\mathbf{3}\mathbf{4}\mathbf{B}$34A,34B\mathbf{3 4 A}, \mathbf{3 4 B} 上的加热元件和热敏电阻 36A、36B 以及光学板 $\mathbf{5}\mathbf{0}$$\mathbf{5}\mathbf{0}$50\mathbf{5 0} 和 $\mathbf{5}\mathbf{2}$$\mathbf{5}\mathbf{2}$52\mathbf{5 2} 通过柔性电缆连接到 PC 板 $\mathbf{5}\mathbf{4}$$\mathbf{5}\mathbf{4}$54\mathbf{5 4} 上（为便于说明，图 4 中未显示）。模块 $\mathbf{3}\mathbf{7}$$\mathbf{3}\mathbf{7}$37\mathbf{3 7} 还可包括用于屏蔽光检测电路的接地线 56。模块 37 最好还包括一个指示灯，如 LED 44，用于向用户指示模块的当前状态，如 "准备装载样品"、"准备装载试剂"、"加热"、"冷却"、"完成 "或 "故障"。

FIGS. 5A and 5B show the fluorescent excitation and emission spectra, respectively, of four fluorescent dyes of interest. These dyes are standard fluorescent dyes used with the TaqMan® chemistry (available from the Perkin-Elmer Corporation, Foster City, Calif.) and are well known by their acronyms FAM, TET, TAMRA, and ROX. Although the preferred embodiment is described with reference to these four dyes, it is to be understood that the system of the present invention are not limited to these particular dyes or to the TaqMan® ${}^{\circledR }$${}^{\circledR }$^(®){ }^{\circledR} chemistry. The system may be used with any fluorophores including, but not limited to, fluorescent dyes used with the Beacons chemistry, dyes used with the Sunrise ${}^{\circledR }$${}^{\circledR }$^(®){ }^{\circledR} chemistry, and interculating dyes such as ethidium bromide. Fluorescent dyes and labeling chemistries for labeling analytes in a reaction mixture are well known in the art and need not be discussed further herein. Further, although fluorescence detection is presently preferred, the detection system of the present invention is not limited to detection based upon fluorescent labels. The system may be applicable to detection based upon phosphorescent labels, chemiluminescent labels, or electrochemiluminescent labels
图 5A 和 5B 分别显示了四种相关荧光染料的荧光激发光谱和发射光谱。这些染料是与 TaqMan® 化学反应（可从加州福斯特市的珀金-埃尔默公司购买）一起使用的标准荧光染料，其缩写为 FAM、TET、TAMRA 和 ROX。尽管本发明的优选实施方案是参照这四种染料描述的，但应该理解的是，本发明的系统并不局限于这些特定的染料或 TaqMan® ${}^{\circledR }$${}^{\circledR }$^(®){ }^{\circledR} 化学。该系统可与任何荧光团配合使用，包括但不限于与 Beacons 化学反应配合使用的荧光染料、与 Sunrise ${}^{\circledR }$${}^{\circledR }$^(®){ }^{\circledR} 化学反应配合使用的染料，以及诸如溴化乙锭之类的交联染料。用于标记反应混合物中的分析物的荧光染料和标记化学方法在本领域众所周知，在此无需进一步讨论。此外，尽管目前首选荧光检测，但本发明的检测系统并不局限于基于荧光标签的检测。该系统也可用于基于磷光标签、化学发光标签或电化学发光标签的检测。

As shown in FIG. 5A, the excitation spectra curves for FAM, TET, TAMRA, and ROX are typically very broad at the base, but sharper at the peaks. As shown in FIG. 5B, the relative emission spectra curves for the same dyes are also very broad at the base and sharper at the peaks. One serious problem is that these dyes have strongly overlapping characteristics in both their excitation and emission spectra. The overlapping characteristics have traditionally made it difficult to distinguish the fluorescent signal of one dye from another when multiple dyes are used to label different analytes in a reaction mixture.
如图 5A 所示，FAM、TET、TAMRA 和 ROX 的激发光谱曲线通常在底部非常宽，但在峰值处较尖锐。如图 5B 所示，相同染料的相对发射光谱曲线也是基底很宽，峰值较尖。一个严重的问题是，这些染料的激发光谱和发射光谱都有很强的重叠特性。传统上，当使用多种染料标记反应混合物中的不同分析物时，这种重叠特性会导致难以区分一种染料和另一种染料的荧光信号。

According to the present invention, multiple light sources are used to provide excitation beams to the dyes in multiple excitation wavelength ranges. Each light source provides excitation light in a wavelength range matched to the peak excitation range of a respective one of the dyes. In the preferred embodiment, the light sources are blue and green LEDs. FIG. 5C shows the effects of filtering the outputs of blue and green LEDs to provide substantially distinct exci-
根据本发明，使用多个光源在多个激发波长范围内为染料提供激发光束。每个光源提供的激发光波长范围与各自染料的峰值激发范围相匹配。在优选的实施方案中，光源为蓝色和绿色 LED。图 5C 显示了过滤蓝光和绿光 LED 的输出以提供基本不同的激发光的效果。
tation wavelength ranges. Typical blue and green LEDs have substantial overlap in the range of around 480 nm through 530 nm . By the filtering regime of the present invention, the blue LED light is filtered to a range of about 450 to 495 nm to match the relative excitation peak for FAM. The green LED light is filtered to a first range of 495 to 527 nm corresponding to the excitation peak for TET, a second range of 527 to 555 nm , corresponding to the excitation peak for TAMRA, and a third range of 555 to 593 nm corresponding to the excitation peak for ROX.
波长范围。典型的蓝光和绿光 LED 在大约 480 纳米到 530 纳米的范围内有大量重叠。根据本发明的滤波机制，蓝色 LED 光被滤波到约 450 至 495 nm 的范围，以匹配 FAM 的相对激发峰。绿色 LED 光被过滤到 495 至 527 nm 的第一范围，与 TET 的激发峰相对应；527 至 555 nm 的第二范围，与 TAMRA 的激发峰相对应；555 至 593 nm 的第三范围，与 ROX 的激发峰相对应。

FIG. 5D shows the effects of filtering light emitted (fluorescent output) from each of the four dyes to form distinct emission wavelength ranges. As shown previously in FIG. 5B, the fluorescent emissions of the dyes before filtering are spherically diffuse with overlapping spectral bandwidths, making it extremely difficult to distinguish the fluorescent output of one dye from another. As shown in FIG. 5D, by filtering the fluorescent outputs of the dyes into substantially distinct wavelength ranges, a series of relatively narrow peaks (detection windows) are obtained, making it possible to distinguish the fluorescent outputs of different dyes, thus enabling the detection of a number of different fluorescently-labeled analytes in a reaction mixture.
图 5D 显示了对四种染料各自发出的光（荧光输出）进行过滤以形成不同发射波长范围的效果。如图 5B 所示，过滤前染料的荧光发射呈球形扩散，光谱带宽重叠，因此极难区分一种染料和另一种染料的荧光输出。如图 5D 所示，通过将染料的荧光输出滤波到基本不同的波长范围内，可获得一系列相对较窄的峰值（检测窗口），从而可以区分不同染料的荧光输出，因此可以检测反应混合物中的多种不同荧光标记的分析物。
FIG. 6 is a schematic, plan view of the optical excitation assembly 46 of the heat-exchanging module. The assembly 46 is positioned adjacent the reaction vessel 2 to transmit excitation beams to the reaction mixture contained in the chamber 10. FIG. 7 is an exploded view of the excitation assembly 46. As shown in FIGS. 6-7, the assembly 46 includes a housing 219 for holding various components of the assembly. Housing 219 preferably comprises one or more molded pieces of plastic. In the preferred embodiment, the housing 219 is a multi-part housing comprised of three housing elements $220\mathrm{A},220\mathrm{B}$$220\mathrm{A},220\mathrm{B}$220A,220B220 \mathrm{~A}, 220 \mathrm{~B}, and 220 C . The upper and lower housing elements 220A and 220C are preferably complementary pieces that couple together and snap-fit into housing element 220B. In this embodiment, the housing elements 220A and 220C are held together by screws 214 . In alternative embodiments, the entire housing 219 may be a one-piece housing that holds a slide-in optics package.
图 6 是热交换模块的光学激发组件 46 的平面示意图。该组件 46 与反应容器 2 相邻，用于将激发光束传送到腔室 10 中的反应混合物。图 7 是激发组件 46 的剖视图。如图 6-7 所示，组件 46 包括一个外壳 219，用于容纳组件的各种部件。外壳 219 最好由一个或多个模制塑料件组成。在优选实施例中，外壳 219 是由三个外壳元件 $220\mathrm{A},220\mathrm{B}$$220\mathrm{A},220\mathrm{B}$220A,220B220 \mathrm{~A}, 220 \mathrm{~B} 和 220 C 组成的多部件外壳。上部和下部外壳元件 220A 和 220C 最好是互补件，它们耦合在一起并卡入外壳元件 220B。在本实施例中，外壳元件 220A 和 220C 通过螺钉 214 固定在一起。在其他实施例中，整个外壳 219 可以是一个容纳滑入式光学封装的一体式外壳。
The lower housing element 220C includes an optical window 235 into which is placed a cylindrical rod lens 215 for focusing excitation beams into the chamber 10. In general, the optical window 235 may simply comprise an opening in the housing through which excitation beams may be transmitted to the chamber $\mathbf{1}\mathbf{0}$$\mathbf{1}\mathbf{0}$10\mathbf{1 0}. The optical window may optionally include an optically transmissive or transparent piece of glass or plastic serving as a window pane, or as in the preferred embodiment, a lens for focusing excitation beams.
下部外壳元件 220C 包括一个光学窗口 235，其中放置了一个圆柱杆透镜 215，用于将激发光束聚焦到腔室 10 中。一般来说，光学窗口 235 可以简单地包括外壳上的一个开口，激发光束可以通过该开口传输到腔室 $\mathbf{1}\mathbf{0}$$\mathbf{1}\mathbf{0}$10\mathbf{1 0} 中。光学窗口可选择包括一块光学透射或透明的玻璃或塑料，用作窗格，或如优选实施例中那样，用于聚焦激发光束的透镜。
The optics assembly 46 also includes four light sources, preferably LEDs $100\mathrm{A},100\mathrm{B},100\mathrm{C}$$100\mathrm{A},100\mathrm{B},100\mathrm{C}$100A,100B,100C100 \mathrm{~A}, 100 \mathrm{~B}, 100 \mathrm{C}, and 100 D , for transmitting excitation beams through the window 235 to the reaction mixture contained in the chamber $\mathbf{1}\mathbf{0}$$\mathbf{1}\mathbf{0}$10\mathbf{1 0}. In general, each light source may comprise a laser, a light bulb, or an LED. In the preferred embodiment, each light source comprises a pair of directional LEDs. In particular, the four light sources shown in FIGS. 6-7 are preferably a first pair of green LEDs 100 A , a second pair of green LEDs 100 B , a pair of blue LEDs 100 C , and a third pair of green LEDs 100 D . The LEDs receive power through leads 201 which are connected to a power source (not shown in FIGS. 6-7). The LEDs are mounted to the optical circuit board $\mathbf{5}\mathbf{0}$$\mathbf{5}\mathbf{0}$50\mathbf{5 0} which is attached to the back of the housing element 220 B so that the LEDs are rigidly fixed in the housing. The optical circuit board $\mathbf{5}\mathbf{0}$$\mathbf{5}\mathbf{0}$50\mathbf{5 0} is connected to the main PC board of the heatexchanging module (shown in FIG. 4) via the flex cable 51. includes a 495 nm high pass reflector 208, a 527 nm high pass reflector 209, a mirror 210, a 555 nm , low pass reflector 211, and a 593 nm low pass reflector 212. The reflecting filters and mirrors 208-212 are angularly offset by ${30}^{\circ }$${30}^{\circ }$30^(@)30^{\circ} from the low pass filters 203-206.
光学组件 46 还包括四个光源，最好是 LED $100\mathrm{A},100\mathrm{B},100\mathrm{C}$$100\mathrm{A},100\mathrm{B},100\mathrm{C}$100A,100B,100C100 \mathrm{~A}, 100 \mathrm{~B}, 100 \mathrm{C} 和 100 D，用于通过窗口 235 向腔室 $\mathbf{1}\mathbf{0}$$\mathbf{1}\mathbf{0}$10\mathbf{1 0} 中的反应混合物传输激发光束。一般来说，每个光源可包括激光器、灯泡或 LED。在优选实施例中，每个光源包括一对定向 LED。具体来说，图 6-7 中所示的四个光源最好是第一对绿色 LED 100 A、第二对绿色 LED 100 B、一对蓝色 LED 100 C 和第三对绿色 LED 100 D。LED 通过连接到电源（图 6-7 中未显示）的引线 201 接收电源。LED 安装在光学电路板 $\mathbf{5}\mathbf{0}$$\mathbf{5}\mathbf{0}$50\mathbf{5 0} 上，光学电路板连接到外壳元件 220 B 的背面，这样 LED 就被刚性固定在外壳中。光电路板 $\mathbf{5}\mathbf{0}$$\mathbf{5}\mathbf{0}$50\mathbf{5 0} 通过柔性电缆 51 连接到热交换模块的主 PC 板（如图 4 所示）。反射滤波器和反射镜 208-212 与低通滤波器 203-206 的角度偏差为 ${30}^{\circ }$${30}^{\circ }$30^(@)30^{\circ} 。

The excitation assembly 46 transmits excitation beams to the chamber $\mathbf{1}\mathbf{0}$$\mathbf{1}\mathbf{0}$10\mathbf{1 0} in four distinct excitation wavelength ranges as follows. When the green LEDs 100A are activated, they generate an excitation beam that passes through the pair of 593 nm low pass filters 203 and through the lens 207. The excitation beam then reflects off of the 593 nm low pass reflector 212, passes through the 555 nm , low pass reflector 211, reflects off of the 527 nm high pass reflector 209, and passes through the lens 215 into the reaction chamber 10. The excitation beam from the LEDs 100 A is thus filtered to a wavelength range of 555 to 593 nm corresponding to the peak excitation range for ROX.
激发组件 46 向腔室 $\mathbf{1}\mathbf{0}$$\mathbf{1}\mathbf{0}$10\mathbf{1 0} 发射以下四个不同激发波长范围的激发光束。当绿色 LED 100A 启动时，它们会产生一束激发光束，该光束穿过一对 593 nm 低通滤波器 203 并穿过透镜 207。然后，激发光束从 593 纳米低通反射器 212 上反射，穿过 555 纳米低通反射器 211，从 527 纳米高通反射器 209 上反射，穿过透镜 215 进入反应室 10。这样，来自 LED 100 A 的激发光束被过滤到 555 至 593 nm 的波长范围，与 ROX 的峰值激发范围相对应。

When the green LEDs 100B are activated, they generate an excitation beam that passes through the pair of 555 nm , low pass filters 204, reflects off of the 555 nm , low pass reflector 211, reflects off of the 527 nm high pass reflector 209, and passes through the lens 215 into the reaction chamber 10. The excitation beam from LEDs 100B is thus filtered to a wavelength range of 527 to 555 nm , correspond60 ing to the peak excitation range for TAMRA.
当绿色 LED 100B 启动时，它们产生的激发光束穿过一对 555 纳米低通滤波器 204，从 555 纳米低通反射器 211 反射出来，从 527 纳米高通反射器 209 反射出来，并穿过透镜 215 进入反应室 10。这样，来自 LED 100B 的激发光束被过滤到 527 至 555 nm 的波长范围内，60 相当于 TAMRA 的峰值激发范围。

When the blue LEDs 100 C are activated, they generate an excitation beam that passes through the pair of 495 nm low pass filters 205, through the 495 nm high pass reflector 208, through the 527 nm high pass reflector 209, and through the lens 215 into the reaction chamber 10 . The excitation beam from LEDs 100 C is thus filtered to a wavelength below 495 nm corresponding to the peak excitation range for FAM.
当蓝色 LED 100 C 启动时，它们产生的激发光束穿过一对 495 nm 低通滤波器 205，穿过 495 nm 高通反射器 208，穿过 527 nm 高通反射器 209，并穿过透镜 215 进入反应室 10。这样，来自 LED 100 C 的激发光束就被过滤到与 FAM 的峰值激发范围相对应的低于 495 nm 的波长。

When the green LEDs 100D are activated, they generate an excitation beam that passes through the pair of 527 nm low pass filters 206, reflects off of the mirror 210 , reflects off of the 495 nm high pass reflector 208, passes through the 527 nm high pass reflector 209, and passes through the lens 215 into the reaction chamber 10. The excitation beam from LEDs 100D is thus filtered to a wavelength range of 495 to 527 nm corresponding to the peak excitation range for TET. In operation, the LEDs $100\mathrm{A},100\mathrm{B},100\mathrm{C},100\mathrm{D}$$100\mathrm{A},100\mathrm{B},100\mathrm{C},100\mathrm{D}$100A,100B,100C,100D100 \mathrm{~A}, 100 \mathrm{~B}, 100 \mathrm{C}, 100 \mathrm{D} are sequentially activated to excite the different fluorescent dyes contained in the chamber 10 with excitation beams in substantially distinct wavelength ranges, as will be described in greater detail below.
当绿色 LED 100D 被激活时，它们会产生一束激发光束，该光束穿过一对 527 纳米低通滤波器 206，从反射镜 210 上反射，从 495 纳米高通反射器 208 上反射，穿过 527 纳米高通反射器 209，并穿过透镜 215 进入反应室 10。这样，来自 LED 100D 的激发光束被过滤到 495 至 527 nm 的波长范围，与 TET 的峰值激发范围相对应。在操作过程中，LED $100\mathrm{A},100\mathrm{B},100\mathrm{C},100\mathrm{D}$$100\mathrm{A},100\mathrm{B},100\mathrm{C},100\mathrm{D}$100A,100B,100C,100D100 \mathrm{~A}, 100 \mathrm{~B}, 100 \mathrm{C}, 100 \mathrm{D} 依次被激活，用基本上不同波长范围的激发光束激发反应室 10 中的不同荧光染料，这将在下文中详细说明。
FIG. 8 is a schematic, plan view of the optical detection assembly 48 of the heat-exchanging module. The assembly 48 is positioned adjacent the reaction vessel 2 to receive light emitted from the chamber 10. FIG. 9 is an exploded view of the detection assembly 48. As shown in FIGS. 8-9, the assembly 48 includes a housing 221 for holding various components of the assembly. The housing 221 preferably comprises one or more molded plastic pieces. In the preferred embodiment, the housing 221 is a multi-part housing comprised of upper and lower housing elements 234A and 234B. The housing elements 234A, 234B are complementary, mating pieces that are held together by screws 214. In alternative embodiments, the entire housing 221 may be a one-piece housing that holds a slide-in optics package.
图 8 是热交换模块的光学检测组件 48 的平面示意图。该组件 48 位于反应容器 2 旁，用于接收腔室 10 发出的光。图 9 是检测组件 48 的剖视图。如图 8-9 所示，组件 48 包括一个外壳 221，用于容纳组件的各种部件。外壳 221 最好由一个或多个模制塑料件组成。在优选实施例中，外壳 221 是由上下外壳元件 234A 和 234B 组成的多部分外壳。外壳元件 234A 和 234B 是互补的配合件，通过螺钉 214 固定在一起。在其他实施例中，整个外壳 221 可以是一个容纳滑入式光学组件的一体式外壳。

The lower housing element 234B includes an optical window 237 into which is placed a cylindrical rod lens 232 for collimating light emitted from the chamber 10. In general, the optical window may simply comprise an opening in the housing through which the emitted light may be received. The optical window may optionally include an optically transmissive or transparent piece of glass or plastic serving as a window pane, or as in the preferred embodiment, the lens $\mathbf{2}\mathbf{3}\mathbf{2}$$\mathbf{2}\mathbf{3}\mathbf{2}$232\mathbf{2 3 2} for collimating light emitted from the chamber 10 .
下部外壳元件 234B 包括一个光学窗口 237，窗口内放置了一个圆柱杆透镜 232，用于准直从腔室 10 发射的光线。一般来说，光学窗口可能只是外壳上的一个开口，通过它可以接收发射的光线。光学窗口可选择包括一块光学透射或透明的玻璃或塑料，用作窗格，或如优选实施例中的透镜 $\mathbf{2}\mathbf{3}\mathbf{2}$$\mathbf{2}\mathbf{3}\mathbf{2}$232\mathbf{2 3 2} ，用于准直从腔室 10 发射的光线。
The optics assembly 48 also includes four detectors 102A, 102B. 102C, and 102D for detecting light emitted from the chamber 10 and received through the window 237 . In general, each detector may be a photomultiplier tube, CCD, SMOS detector, photodiode, or other solid-state detector. In the preferred embodiment, each detector is a PIN photodiode. The detectors 102A, 102B. 102C, and 102D are preferably rigidly fixed in recesses formed in the lower housing element 234B. The detectors are electrically connected by leads 245 to the optical circuit board 52 (see FIG. 4) which is preferably mounted to the underside of the lower housing element 234B.
光学组件 48 还包括四个探测器 102A、102B.102C 和 102D，用于检测从腔室 10 发射并通过窗口 237 接收的光。一般来说，每个探测器都可以是光电倍增管、CCD、SMOS 探测器、光电二极管或其他固态探测器。在优选实施例中，每个检测器都是 PIN 光电二极管。探测器 102A、102B.102C 和 102D 最好刚性固定在下壳体元件 234B 中形成的凹槽中。探测器通过引线 245 与光电路板 52（见图 4）电连接，光电路板 52 最好安装在下壳体元件 234B 的底部。

The optics assembly 48 further includes a set of filters and lenses arranged in the housing 221 for separating light emitted from the chamber 10 into different emission wavelength ranges and for directing the light in each of the emission wavelength ranges to a respective one of the detectors. As shown in FIG. 9, the lower housing element 234B preferably includes walls 247 that create separate detection channels in the housing, with one of the detectors positioned at the end of each channel. The walls 247 preferably include slots for receiving and rigidly holding the filters and lenses. The filters and lenses may also be rigidly fixed in the housing 221 by an adhesive used alone, or more preferably, with an adhesive used in combination with slots in the housing.
光学组件 48 还包括一组布置在外壳 221 中的滤光片和透镜，用于将从腔室 10 发射的光分离成不同的发射波长范围，并将每个发射波长范围内的光导向各自的一个检测器。如图 9 所示，下壳体元件 234B 最好包括壁 247，在壳体中形成独立的检测通道，每个通道的末端都有一个检测器。壁 247 最好包括用于接收和固定滤光片和透镜的槽。滤光片和透镜也可以通过粘合剂单独固定在外壳 221 中，或更优选地通过粘合剂与外壳中的槽相结合固定在外壳 221 中。
In general, the filters in the optics assembly 48 may be selected to block light emitted from the chamber 10 outside of any desired emission wavelength ranges. The optics 102 C , and 102D.
一般来说，可以选择光学组件 48 中的滤光片来阻挡腔室 10 发射的任何所需发射波长范围之外的光。光学元件 102 C 和 102 D。

The detection assembly 48 detects light emitted from the chamber 10 in four emission wavelength ranges as follows. As shown in FIG. 8, the emitted light passes through the lens 232 and strikes the 565 nm low pass reflector 229. The portion of the light having a wavelength in the range of about 505 to 537 nm (corresponding to the peak emission wavelength range of FAM) reflects from the 565 nm low pass reflector 229 , passes through the 537 nm high pass reflector 230, reflects from the 505 nm high pass reflector 231, passes through the pair of 505 nm high pass filters 223, through the lens 242, through the 515 nm Schott Glass ${}^{(1)}$${}^{(1)}$^((1)){ }^{(1)} filter 222 A , and is detected by the first detector 102 A .
检测组件 48 在以下四个发射波长范围内检测从腔室 10 发射的光。如图 8 所示，发射光穿过透镜 232，照射到 565 纳米低通反射器 229 上。波长在约 505 至 537 nm 范围内（对应于 FAM 的峰值发射波长范围）的部分光从 565 nm 低通反射器 229 反射，穿过 537 nm 高通反射器 230，从 505 nm 高通反射器 231 反射，穿过一对 505 nm 高通滤波器 223，穿过透镜 242，穿过 515 nm 肖特玻璃 ${}^{(1)}$${}^{(1)}$^((1)){ }^{(1)} 滤波器 222 A，并被第一检测器 102 A 检测到。

Meanwhile, the portion of the light having a wavelength 5 in the range of about 537 to 565 nm (corresponding to the peak emission wavelength range of TET) reflects from the 565 nm low pass reflector $\mathbf{2}\mathbf{2}\mathbf{9}$$\mathbf{2}\mathbf{2}\mathbf{9}$229\mathbf{2 2 9}, reflects from the 537 nm high pass reflector 230, passes through the pair of 537 nm high pass filters 224 , through the lens 242, through the 550 nm Schott Glass ${}^8$${}^8$^(8){ }^{8} filter 222 B, and is detected by the second detector 102B.
同时，波长 5 在约 537 至 565 nm 范围内（对应于 TET 的峰值发射波长范围）的那部分光从 565 nm 低通反射器 $\mathbf{2}\mathbf{2}\mathbf{9}$$\mathbf{2}\mathbf{2}\mathbf{9}$229\mathbf{2 2 9} 反射，从 537 nm 高通反射器 230 反射，通过一对 537 nm 高通滤波器 224，通过透镜 242，通过 550 nm 肖特玻璃 ${}^8$${}^8$^(8){ }^{8} 滤波器 222 B，并被第二检测器 102B 检测到。

Similarly, the portion of the light having a wavelength in the range of about 565 to 605 nm (corresponding to the peak emission wavelength range of TAMRA) passes through the 565 nm low pass reflector 229, through the 605 nm high pass reflector 227 , through the pair of 565 nm high pass filters 225, through the lens 242, through the 570 nm Schott
同样，波长在约 565 至 605 纳米范围内（与 TAMRA 的峰值发射波长范围相对应）的那部分光穿过 565 纳米低通反射器 229，穿过 605 纳米高通反射器 227，穿过一对 565 纳米高通滤光片 225，穿过透镜 242，穿过 570 纳米肖特滤光片 225，穿过 570 纳米高通滤光片 227，穿过 565 纳米低通反射器 229，穿过 605 纳米高通反射器 227。

Glass® filter 222C, and is detected by the third detector $\mathbf{1}\mathbf{0}\mathbf{2}\mathbf{C}$$\mathbf{1}\mathbf{0}\mathbf{2}\mathbf{C}$102C\mathbf{1 0 2 C}. The portion of the light having a wavelength over 605 nm (corresponding to the peak emission wavelength range of ROX) passes through the 565 nm low pass reflector $\mathbf{2}\mathbf{2}\mathbf{9}$$\mathbf{2}\mathbf{2}\mathbf{9}$229\mathbf{2 2 9}, reflects from the 605 nm high pass reflector 227 , reflects from the mirror 228, passes through the pair of 605 nm high pass filters 226, through the lens 242, through the 620 nm Schott Glass ${}^{(1)}$${}^{(1)}$^((1)){ }^{(1)} filter 222D, and is detected by the fourth detector 102D. In operation, the outputs of detectors 102A, $102\mathrm{B},102\mathrm{C}$$102\mathrm{B},102\mathrm{C}$102B,102C102 \mathrm{~B}, 102 \mathrm{C}, and 102 D are analyzed to determine the concentrations of each of the different dyes contained in the chamber 10, as will be described in greater detail below.
Glass® 滤光片 222C，并被第三检测器 $\mathbf{1}\mathbf{0}\mathbf{2}\mathbf{C}$$\mathbf{1}\mathbf{0}\mathbf{2}\mathbf{C}$102C\mathbf{1 0 2 C} 检测到。波长超过 605 nm（对应于 ROX 的峰值发射波长范围）的部分光穿过 565 nm 低通反射器 $\mathbf{2}\mathbf{2}\mathbf{9}$$\mathbf{2}\mathbf{2}\mathbf{9}$229\mathbf{2 2 9} ，从 605 nm 高通反射器 227 反射，从反射镜 228 反射，穿过一对 605 nm 高通滤波器 226，穿过透镜 242，穿过 620 nm 肖特玻璃 ${}^{(1)}$${}^{(1)}$^((1)){ }^{(1)} 滤波器 222D，并被第四探测器 102D 检测到。在操作过程中，对检测器 102A、 $102\mathrm{B},102\mathrm{C}$$102\mathrm{B},102\mathrm{C}$102B,102C102 \mathrm{~B}, 102 \mathrm{C} 和 102 D 的输出进行分析，以确定腔室 10 中包含的每种不同染料的浓度，下文将对此进行更详细的描述。

FIG. 10 is a perspective view of a multi-site reactor system 60 according to the present invention. The reactor system 60 comprises a thermal cycler 62 and a controller, such as a personal computer 64 . The thermal cycler 62 comprises a base instrument 66 and multiple heatexchanging modules 37 (described with reference to FIG. 4). The base instrument 66 has a main logic board with edge connectors 68 for receiving the modules 37 . The base instrument 66 also preferably includes a fan 70 for cooling its electronic components. The base instrument 66 may be connected to the controller 64 using any suitable data connection, such as a universal serial bus (USB), ethernet connection, or serial line. It is presently preferred to use a USB that connects to the serial port of computer 64. Although a laptop computer is shown in FIG. 10, the controller may comprise any type of device having a processor. Further, the thermal cycler may be linked to a computer network rather than to a single computer.
图 10 是根据本发明设计的多点反应器系统 60 的透视图。反应器系统 60 包括一个热循环器 62 和一个控制器，如个人计算机 64。热循环仪 62 包括一个基础仪器 66 和多个热交换模块 37（参见图 4）。基础仪器 66 有一个主逻辑板，板上有用于接收模块 37 的边缘连接器 68。基础仪器 66 最好还包括一个风扇 70，用于冷却其电子元件。基础仪器 66 可以通过任何合适的数据连接方式连接到控制器 64，例如通用串行总线 （USB）、以太网连接或串行线路。目前最好使用 USB 连接到计算机 64 的串行端口。虽然图 10 中显示的是笔记本电脑，但控制器可以是任何类型的具有处理器的设备。此外，热循环仪还可以连接到计算机网络，而不是一台计算机。
The term “thermal cycling” is herein intended to mean at least one change of temperature, i.e. increase or decrease of temperature, in a reaction mixture. Therefore, chemicals undergoing thermal cycling may shift from one temperature to another and then stabilize at that temperature, transition to a second temperature or return to the starting temperature. The temperature cycle may be performed only once or may be repeated as many times as required to study or complete the particular chemical reaction of interest.
术语 "热循环 "在此是指反应混合物中至少有一次温度变化，即温度的升高或降低。因此，进行热循环的化学品可以从一个温度转移到另一个温度，然后稳定在该温度，过渡到第二个温度或返回到起始温度。温度循环可以只进行一次，也可以根据研究或完成特定化学反应的需要多次重复。

In the specific embodiment of FIG. 10, the thermal cycler 62 includes sixteen independently-controllable heatexchanging modules 37 arranged in two rows of eight modules each. It is to be understood, however, that the thermal cycler can range from a one to four-site hand-held instrument to a multi-hundred site clinical and research instrument. Common to all these embodiments are one or more independently-controllable modules $\mathbf{3}\mathbf{7}$$\mathbf{3}\mathbf{7}$37\mathbf{3 7}, and a controller for operating individually programmed independent temperature/time profiles for each module. The thermal time-courses for nucleic acid amplifications or other reactions can be fine tuned to a particular target, and independent control of individual modules $\mathbf{3}\mathbf{7}$$\mathbf{3}\mathbf{7}$37\mathbf{3 7} permits simultaneous reactions to be run at different thermal profiles.
在图 10 的具体实施例中，热循环仪 62 包括十六个可独立控制的热交换模块 37，每 个模块八个，分两排排列。不过，可以理解的是，热循环仪的范围可以从一到四个部位的手持式仪器到多达上百个部位的临床和研究仪器。所有这些实施方案的共同点是一个或多个可独立控制的模块 $\mathbf{3}\mathbf{7}$$\mathbf{3}\mathbf{7}$37\mathbf{3 7} ，以及一个控制器，用于操作每个模块单独编程的独立温度/时间曲线。核酸扩增或其他反应的热时间曲线可根据特定目标进行微调，独立控制单个模块 $\mathbf{3}\mathbf{7}$$\mathbf{3}\mathbf{7}$37\mathbf{3 7} 可在不同的热曲线下同时运行反应。

The thermal cycler 62 also provides for independent loading, cycling, and unloading of individual sites at different times allowing for optimal use and throughput. The thermal cycler 62 is also modular, in that each heatexchanging module 37 can be individually removed from the base instrument 66 for servicing, repair, or replacement. This modularity reduces downtime since all the modules 37 are not off line to repair one, and the instrument 66 can be upgraded and enlarged to add more sites as needed. The modularity of the thermal cycler 62 also means that individual modules 37 can be precisely calibrated, and modulespecific schedules or corrections can be included in the control programs, e.g., as a series of module-specific calibration or adjustment charts.
热循环仪 62 还可以在不同时间对各个部位进行独立的加载、循环和卸载，从而达到最佳的使用效果和吞吐量。热循环仪 62 也是模块化的，每个热交换模块 37 都可以单独从基础仪器 66 上拆下，以进行保养、维修或更换。这种模块化设计减少了停机时间，因为所有模块 37 都不会因为维修一个模块而停机，而且仪器 66 可以根据需要升级和扩大，以增加更多的站点。热循环仪 62 的模块化还意味着可以对单个模块 37 进行精确校准，并将特定模块的时间表或校正纳入控制程序，例如作为一系列特定模块校准或调整图表。

The thermal cycling system 60 of the invention also has significant advantages in terms of power management. The controller 64 can interleave the thermal profiles of each independent module 37 to save power as compared to a single block heater. For example, current can be reduced by half by control of one module to heat (high power) while a second module is cooling (low power). Thus, by interleaving of pulse power to only so many modules 37 as have reactants in them, the instantaneous current requirements for the base instrument 66 can be minimized, permitting more modules 37 per instrument that can still be powered from a standard $110\mathrm{V},15$$110\mathrm{V},15$110V,15110 \mathrm{~V}, 15 ampere circuit. Because of this sophisticated power management system, which is made possible by the independent control of the modules 37 , the instrument 66 may also be configured into a hand-held, battery operated device.
本发明的热循环系统 60 在电源管理方面也有显著优势。与单块加热器相比，控制器 64 可以交错控制每个独立模块 37 的热曲线，从而节省电能。例如，通过控制一个模块加热（高功率），同时控制第二个模块冷却（低功率），可以将电流减少一半。因此，通过交错向多个模块 37 提供脉冲功率，使其中只含有反应物，就可以最大限度地降低基本仪器 66 的瞬时电流要求，从而使每个仪器可以使用标准 $110\mathrm{V},15$$110\mathrm{V},15$110V,15110 \mathrm{~V}, 15 安培电路为更多模块 37 供电。由于模块 37 的独立控制使这一复杂的电源管理系统成为可能，因此仪器 66 也可以配置成手持式电池供电设备。

In embodiments in which the base instrument 66 operates on external power, e.g. 110 V AC , the instrument preferably includes two power connections 76, 78. Power is received though the first connection 76 and output through the second connection 78. Similarly, the instrument 66 preferably includes network interface inlet and outlet ports 72, 74 for receiving a data connection through inlet port 72 and outputting data to another base instrument through outlet port 74. As shown schematically in FIG. 11, this arrangement permits multiple thermal cyclers $\mathbf{6}\mathbf{2}\mathrm{A},\mathbf{6}\mathbf{2}\mathbf{B},\mathbf{6}\mathbf{2}\mathrm{C},\mathbf{6}\mathbf{2}$$\mathbf{6}\mathbf{2}\mathrm{A},\mathbf{6}\mathbf{2}\mathbf{B},\mathbf{6}\mathbf{2}\mathrm{C},\mathbf{6}\mathbf{2}$62A,62B,62C,62\mathbf{6 2} \mathrm{A}, \mathbf{6 2 B}, \mathbf{6 2} \mathrm{C}, \mathbf{6 2} to be daisy-chained from one controller 64 and one external power source $\mathbf{8}\mathbf{0}$$\mathbf{8}\mathbf{0}$80\mathbf{8 0}. Using a USB, it is theoretically possible to daisy-chain $\mathbf{1}\mathbf{2}\mathbf{7}$$\mathbf{1}\mathbf{2}\mathbf{7}$127\mathbf{1 2 7} thermal cycler instruments to a single controller, although due to limits of computing power, one should use several computers for controlling 127 instruments.
在基础仪器 66 通过外部电源（如 110 伏交流电）运行的实施例中，仪器最好包括两个电源连接 76 和 78。电源通过第一连接 76 接收，通过第二连接 78 输出。同样，仪器 66 最好还包括网络接口入口和出口端口 72、74，用于通过入口端口 72 接收数据连接，并通过出口端口 74 将数据输出到另一个基础仪器。如图 11 中的示意图所示，这种安排允许多个热循环仪 $\mathbf{6}\mathbf{2}\mathrm{A},\mathbf{6}\mathbf{2}\mathbf{B},\mathbf{6}\mathbf{2}\mathrm{C},\mathbf{6}\mathbf{2}$$\mathbf{6}\mathbf{2}\mathrm{A},\mathbf{6}\mathbf{2}\mathbf{B},\mathbf{6}\mathbf{2}\mathrm{C},\mathbf{6}\mathbf{2}$62A,62B,62C,62\mathbf{6 2} \mathrm{A}, \mathbf{6 2 B}, \mathbf{6 2} \mathrm{C}, \mathbf{6 2}