A real-time quantitative polymerase chain reaction (qPCR) reactor system for in-process sample testing

Abstract

The present invention provides a random-access PCR reactor for biological analysis, which includes a plurality of PCR reactors fixed on a platform and an optical system shared by all the PCR reactors on the platform. The optical system is fixed on a traverse mechanism, which can be moved to any PCR reactor ready for imaging. Other PCR reactors on the platform can be accessed for sample loading and replacement. The optical system has a light guide tube and a light guide plate, which distribute light evenly to all samples on the reactors. The light guide plate of the present optical system has a set of light reflecting structures strategically positioned to reflect incident light evenly to all samples under test in the PCR reactors.

Description

TECHNICAL FIELD

[0001] The present invention relates generally to light-based detection systems, such as automated systems for real-time quantitative polymerase chain reaction (hereinafter referred to as qPCR), digital PCR instruments, DNA sequencing instruments, and antigen-antibody ELISA instruments, and in particular, to illumination systems for the above instruments. BACKGROUND

[0002] Polymerase chain reaction (PCR) is a type of in vitro quantitative detection of nucleic acids. In medical and biological research laboratories, PCR is often used for various tasks, such as genetic disease detection, genetic fingerprinting, infectious disease diagnosis, gene cloning, paternity identification, and DNA computation. By using a thermostable DNA polymerase and a machine capable of rapidly heating and cooling the genetic sample (commonly referred to as a thermal cycler), the method can be automated.

[0003] In a typical PCR experiment, the target DNA is split into two strands and synthesized using primers to double the amount of target DNA. The above process is repeated until a large amount of DNA fragments is synthesized. This simple gene amplification technique can rapidly amplify DNA. In PCR technology, a small amount of target DNA can be amplified one million times in a short time, greatly improving the detection and analysis capabilities of DNA molecules.

[0004] In order to advance the PCR process, specific temperature changes must be applied to the solution containing the DNA to cause separation (melting), primer binding (annealing), and replication (elongation). Separation occurs at high temperatures, such as 95°C, and annealing occurs at low temperatures, such as 60°C. However, this process is very sensitive to sample volume and can result in large differences in the final amplification. The PCR reaction includes an early lag phase, an exponential growth phase, and a plateau phase. The sensitivity of the instrument mainly appears in the lag phase. When the exponential growth phase begins, a sufficient amount of product has accumulated to be detected by a specific instrument. In the last plateau phase, as the product more effectively resists the annealing primer, the number of enzymes becomes limited, and the amplification efficiency will decrease. Most of the quantitative information can be found in the exponential cycle period, but the exponential cycle period usually only contains 4 or 5 cycles out of 40 cycles.

[0005] Optical detection systems are commonly used to interrogate reactions in PCR, measuring the intensity of fluorescent emission from each sample tube in the reactor. To measure fluorescence, excitation light is directed at the sample in the sample vessel, and the light emitted by the fluorophore in the sample is detected. Efficient and effective transmission of light from the light source to the optically transparent well is often required. Standard techniques known to those skilled in the art are used, including rapid cycle PCR, for thermal cycling and fluorescence monitoring in each cycle of quantitative PCR. For traditional PCR methods, it is necessary to divide the reaction into multiple reaction tubes, and detect the PCR product after different cycle numbers. Optical systems for directing light to sample plates are known, for example US6942837B2, US7369227B2, US6852986B1, US7410793B2. While optical systems for directing light to sample vessels in a sample plate and detecting light from the sample vessels have been developed in the art. However, there is still a need to develop optical systems that more effectively distribute light to and receive light from sample vessels.

[0006] Real-time quantitative PCR (qPCR) is a method of quantifying fluorescence in a fluorescently labeled probe, which is based on conventional PCR methods or in real-time monitoring of the entire PCR process with fluorescent dyes, and quantitative analysis of the final standard curve of known templates.

[0007] There are other methods, such as digital PCR (dPCR), which divides one PCR reaction into many small independent PCR reactions, so that each small reaction contains an average of no more than one target nucleic acid molecule. Each small reaction contains about 1 or 0 target nucleic acid molecules, and gives a positive or negative binary readout at the end of PCR amplification. The absolute amount of the target gene is determined by counting the actual target molecules, which does not depend on the exponential amplification cycle and the reference gene used to quantify the initial amount. By using a large number of partitions, dPCR can be used to detect more subtle folding differences than qPCR.

[0008] In general, qPCR instruments are real-time PCR detection methods, mainly with 2 functional modules: temperature control system and fluorescence detection / monitoring system. Such instruments are mainly composed of sample stage, gene amplification thermal cycling element, fluorescence detection optical system and microcircuit control system. Among them, the gene amplification thermal cycling element is basically similar in assembly. The fluorescence detection system includes fluorescence excitation system, emission system, optical system, fluorescence detection device and control system. The commonly used fluorescence excitation light source is halogen lamp, laser or LED. Fluorescence detection is usually achieved by photomultiplier tube, cooled CCD / CMOS camera or photodiode.

[0009] qPCR is one of the more common systems in use today, however, there are some problems with the current systems. One is obtaining thermal uniformity between the array of samples to be tested held in the reaction plate. Most PCR reactions are performed in multi-well reaction plates to test a large number of samples at one time. If the spacing between each well is large, it is more difficult to achieve temperature uniformity in all the wells for a multi-well reaction plate and even more difficult for a large array reaction plate in a PCR thermal cycler. To eliminate this problem, the wells must be brought closer to each other to accommodate more wells and samples in a smaller area. Currently, many PCR reactors use the bottom of the well for detection. This design does not allow the wells to be brought closer to each other, thus resulting in poor thermal uniformity between all the wells. To be able to bring the wells as close as possible, detection must be from the top of the reaction plate because the bottom of the reaction plate is on the thermal cycler. Some systems use multi-well semiconductor microchips, however, such systems are difficult to operate and expensive. In addition, the heating and cooling of multi-well semiconductor microchips results in uneven power and energy distribution and results in uneven and slow heating and cooling.

[0010] Depending on the type of detector used, qPCR instruments can be divided into point detectors (e.g. photomultiplier tubes or avalanche photodiodes) and two-dimensional planar array detectors (CCD cameras or CMOS). The two-dimensional scanning approach using a probe is slow but has good performance parameters such as high signal-to-noise ratio and large dynamic range. Two-dimensional array detectors are usually used without scanning, which are fast but have relatively poor performance.

[0011] Current PCR instruments come in two main forms: (i) 96-well or more, where multiple sample slots share one temperature control unit and a fixed light excitation detection system. In this case, the sample slots must be filled before starting. This limits the application and turnaround time. (ii) The other form is a single-well modular PCR instrument, where each sample slot has an independent temperature control unit and a light excitation detection system. This system has great flexibility. However, the single sample slot form is expensive to manufacture and has limited sample throughput expansion capability.

[0012] The present invention aims to design a new form of modular qPCR system where sample testing can be accessed randomly, thus significantly increasing the system throughput. The system uses a special type of illumination system and optical detection system. The high performance and movable optical detection system of the present invention provides a qPCR system that requires only one optical detection unit to simultaneously scan and monitor up to 10 and more thermal cycler modules. Therefore, it significantly reduces the manufacturing cost. SUMMARY

[0013] An automated sample-in-flight real-time qPCR instrument is disclosed. It consists of multiple PCR reactors, each with its own temperature control system, but sharing one optical system for detection. Because there is only one optical system, it can move quickly over a series of PCR reactors, and the automated sample-in-flight real-time qPCR reactor can run multiple qPCR reactors in short time intervals.

[0014] The sample-in-flight PCR reactor for biological analysis of the present invention consists of multiple PCR reactors fixed on a platform. Each PCR reactor has a number of reaction plates, each with a set of reaction wells for holding biological samples. The sample-in-flight PCR reactor has one optical system, which is shared by all PCR reactors on the platform. The optical system includes an illumination system and an imaging system. The illumination system includes (i) a light source with a set of lenses and filters; (ii) a light pipe, and (iii) a light guide plate. The light pipe consists of a series of light-conducting tubes that receive light from the light source and distribute it evenly into the light guide plate. The light guide plate is positioned on top of the PCR reactors on the platform so that it can illuminate all sample tubes of all reaction plates on that PCR reactor. The light pipe and light guide plate are configured in an L-shape, with the light guide plate forming the horizontal segment, above which there is an empty space. The imaging system is positioned in the empty space above the light guide plate to take images of the fluorescent light emitted by the light-emitting samples in the sample tubes of that reactor. The light guide plate is capable of evenly distributing light into all sample tubes in the PCR reactor being detected. Evenly distributed light is a key parameter for accurate sample analysis. The light guide plate is equipped with a set of light-reflecting structures to reflect a portion of each light ray into each biological sample in each sample holder, all of which can be illuminated simultaneously to produce emitted light.

[0015] The optical system is fixed on a horizontal translation system that can move and hold the optical system, moving and holding the optical system above any PCR reactor on the platform. A computer system coordinates the thermal cycling times of each reactor, the movement of the optical system, and the imaging of the emitted light. While the optical system is operating on a particular PCR reactor, other PCR reactors can be replaced with new PCR reactors with new samples, thereby providing a sample-in-flight PCR reactor.

[0016] The follow-up PCR instrument includes multiple PCR reactors, each comprising an array of reaction plates thermally coupled to a temperature control element. The thermal cycling of each PCR reactor is independently controlled by its respective temperature control element. In one embodiment of the invention, the PCR reactors are arranged linearly. However, any other alignment, such as a row-column array distribution, is also feasible. The optical system, driven by an electrically driven beam (frame), can be moved onto the PCR reactors at different times to image the light emitted from the reaction plates above the PCR reactors. The optical system can move from one PCR reactor to another, simultaneously exciting and recording fluorescence on the reaction plates of each reactor. A computer processor controls the temperature of each PCR reactor and the movement of the optical system.

[0017] The optical system of this device is designed to provide uniform light to all reaction plates on each PCR reactor. Its compact structure allows for easy and precise movement. It uses a single light source, such as an LED or halogen lamp, to illuminate the entire PCR reactor through the optical system. A camera located above the sample area records the fluorescence emitted by the sample. Attached Figure Description

[0018] Embodiments of this application will now be described with reference to the accompanying drawings, but this does not constitute any limitation on the scope of the claims, wherein the same reference numerals denote the same parts.

[0019] Figure 1 The optical system of the present invention and the reaction plate disposed on top of the temperature-controlled heater are illustrated.

[0020] Figure 2 This illustration shows the irradiation system of the reaction tube of the present invention applied to a reaction plate;

[0021] Figure 3 The light guide tube of the present invention is illustrated;

[0022] Figure 4A A top view of the light guide plate in Embodiment 1 is shown;

[0023] Figure 4B A top view of the light guide plate in Embodiment 2 is shown, where the arrows indicate the direction of the light path;

[0024] Figure 5 The cross-section of a light guide plate with a parallel internal structure is shown.

[0025] Figure 6A This illustrates the light path in a light guide plate that has no internal reflective features.

[0026] Figure 6B A three-dimensional view of a light guide plate with a reflective structure on the top surface is shown.

[0027] Figure 6C A side view of a light guide plate with a reflective structure on the top surface is shown, along with the light path reflected into the reaction tube.

[0028] Figure 6D A three-dimensional view of a light guide plate with a reflective structure on the bottom surface is shown.

[0029] Figure 6E A side view of a light guide plate with a reflective structure on the bottom surface is shown, as well as the light path reflected into the reaction tube.

[0030] Figure 7A A three-dimensional view of a light guide plate with reflective structures on both the top and bottom surfaces is shown, along with the light path reflected into the reaction tube.

[0031] Figure 7B A side view of a light guide plate with reflective structures on both the top and bottom surfaces is shown.

[0032] Figure 8 It is a top view of a pre-formed heating cap that matches the holes in the reaction plate;

[0033] Figure 9A The PCR reactor of Example 1 is illustrated;

[0034] Figure 9B The PCR reactor of Example 2 is illustrated;

[0035] Figure 10 The diagram illustrates a linear array of 10 PCR reactors;

[0036] Figure 11 This illustrates the automated follow-up sample injection PCR system of the present invention. Detailed Implementation

[0037] Figure 1 The PCR reaction system of Example 1 is illustrated, which uses a single temperature control unit and four reaction plates. The system includes: a CCD camera 1, a camera lens 2, a camera filter wheel with a filter 3, a lightweight cooling fan 4 for cooling LEDs, an LED module 5, a lens 6, a light source filter 7, a light guide tube 8, a light guide plate 9, a radiator cooling fan 10, a heating module 11, a radiator 12, several reaction plates 13, each reaction plate having an array of sample holders or sample tubes 14 for holding biological samples, and a heated cover 15.

[0038] Figure 2The operation of this illumination system is illustrated. Light 200 from LED light source 210 (or any other light source) passes through light filtering system 220 and a lens system 225. The light then enters light guide 230, which includes multiple light guides. The light guides split the light into many independent rays. The light then enters light guide plate 240, where it is redirected horizontally along a reaction plate with sample reaction tubes 250. The light passing through light guide plate 240 is progressively reflected in a downward direction 212 toward sample reaction cell 250. Light guide plate 240 has a structure 245 to allow some light to pass through and some light to be reflected to the sample reaction tubes. Once light 212 enters sample tube 250, the luminescent material in the sample absorbs the excitation light and, in response, spontaneously generates and emits fluorescence. The fluorescence 214 emitted from sample tube 250 continues through light guide plate 240 after passing through heat cover 260. The emitted light is received by camera system 270 located above the chip. Each reaction tube is individually excited, and the emitted light it produces is recorded by the camera. The camera can be a charge-coupled device (CCD) detector array, a complementary metal-oxide-semiconductor (CMOS) detector array, or a photomultiplier tube detector. The camera's sensitivity should be high enough to capture the emitted light. Different camera resolutions can be used, such as a 4-megapixel camera. The light source filter 7 passes light of a specific spectrum, removing light with the same wavelength as the emitted light from the fluorescent dye. Typically, the wavelength of light passing through the light source filter is shorter than the wavelength of light passing through the camera filter wheel. The fluorescence emitted by the fluorescent dye passes through the filter wheel and is received by the digital camera. The optics are preferably designed to reflect short-wavelength excitation light and transmit long-wavelength emitted light. Any other combination is possible. The filter wheel is used to switch between different emission filters, selectively prioritizing the wavelength of light from the target fluorescent dye. Using the detection unit, a real-time full-field image of each reaction plate is captured during the PCR reaction.

[0039] Figure 3 The diagram illustrates a light guide 8 that transmits light 310 from LED module 5 to light guide plate 9. Light guide 8 comprises several transparent materials 320 arranged in an overlapping manner. The shape and angle of each light guide are designed to guide light to light guide plate 9 with low loss and manage the percentage and angle of light output on each exit surface 330 to control uniformity and other illumination requirements. Light guide 8 includes an inlet 340 and multiple exit surfaces 330, the side surfaces of which have predetermined angles 350. Light guide 8 can be integrally formed or composed of several components. The transparent materials can be optical glass, PMMA, polyacrylic acid, polycarbonate, polyethylene, or other optically transparent materials. The number of transparent sheets depends on the size and number of reaction plates requiring illumination on each PCR reactor. For larger systems, 1-10 or even more glass sheets are preferred. The system can be modified to have different numbers of inlet and outlet surfaces.

[0040] Light guide tube 8 is connected to the attached Figure 4A The light guide plate 9 is shown. The light guide tube and the light guide plate form an L-shaped configuration, allowing light to enter from one side of the light guide plate, leaving an open space above the light guide plate for the location of the camera. The light guide plate is a substantially flat structure with length and width. The light guide plate has three main features: (i) an optical coupling or entrance feature 410, (ii) optical features or structures 420 for uniformity and efficiency, and (iii) a reflective surface 430. The entrance feature is configured to receive light from each light guide tube section with minimal loss. Light arriving in a substantially vertical direction is converted to a generally horizontal direction. The light guide plate guides and diffuses the light. The surface of the light guide plate is such that light is guided through the light guide plate by reflection from its top and bottom surfaces. The light guide plate conducts light throughout the sample area and has specific features and structures to illuminate each sample tube. In one embodiment, the entrance feature of the light guide plate is configured in the form of multiple steps 440, the size of which is set to match the exit surface 330 of the light guide tube. The light guide plate can be molded or made from any light-transmitting material. It conducts light and illuminates the entire sample area.

[0041] Figure 4B Another embodiment of the light guide plate inlet structure is shown, wherein the inlet portion of the light guide plate 410 has a structure 460 to focus light along the longitudinal axis 465 of the light guide plate and in a direction parallel to the surface of the reaction plate. The light guide plate has a surface coating on all surfaces. Figure 4B The distal end 470 of the light guide plate has a triangular structure to reflect light back into the light guide plate, thereby preventing light loss and generating a more uniform and diffused light source for PCR. The light guide plate also has [missing information - likely related to a specific orientation or feature] in a direction perpendicular to the longitudinal axis. Figure 4A The 420 array of cuts in the middle and Figure 4B The light guide plate has an array of 475 cutouts that reflect light downwards or upwards. It also has a triangular hole 480 along its longitudinal axis to diffuse light to the sides, resulting in a more uniform light distribution and minimal light loss.

[0042] Figure 5 Another embodiment of the entrance structure of the light guide plate 510 is illustrated, in which one step is polished flat, while the other step has a structure that causes the light to be columnar. The incident surface 520 is optically polished or has an OCA (optical contact angle) to fill the gap between the light guide plate exit surface and the light guide plate incident surface. Features on surface 530 are configured to guide the light rays 550 arriving at surface 530 to a predetermined direction, such as directions 556 and 557.

[0043] The surface of the light guide plate is reflective 560, or a reflective coating / material for TIR (total internal reflection) is applied to its surface to achieve reflection. For example, light rays 570 reaching the reflective surface 560 bounce off the reflective surface and travel along the direction of the light guide plate 575.

[0044] Figure 6A A side view of a simple light guide plate 600 without a reflective structure is shown. Due to the material properties of the reflective coating / material used for TIR (Total Internal Reflection), light 610 is reflected from the top surface 612 and the bottom surface 614. In a preferred embodiment, the light guide plate has a reflective structure, and the light guide plate can have different reflective structures to reflect light into the reaction chamber of the reaction plate. This light guide plate uniformly distributes the light across the entire PCR reactor being tested.

[0045] Figure 6B and 6C The diagram illustrates a perspective view and a side view of a light guide plate, showing a reflective structure on its top surface. In a preferred embodiment, the reflective structure is a set of parallel cuts made along the width of the light guide plate, these cuts being substantially perpendicular to the longitudinal axis of the light guide plate. In one embodiment, the cuts are triangular cross-sections with a predetermined height and angle. However, the reflective structures 631-633 can have different shapes, such as triangular / rectangular / circular. The angles and shapes are based on the incident light angle and the reflected light direction. These angles can range from 10 to 90 degrees. The reflective structures (optical features) are strategically positioned to reflect light onto the reflective plate. These features are designed not only to provide a uniform light distribution but also to improve the overall efficiency of light use. The structures and cuts on the light guide plate can be fabricated by laser etching, injection molding, embossing, etc. Figure 6C The diagram illustrates light 641, which is reflected from the bottom surface along direction 642 and then reflected from structure 633 along direction 643 into the reaction tank 650.

[0046] Figure 6D and 6E Another embodiment of the light guide plate is illustrated, wherein a reflective structure 661 is located on the bottom surface of the light guide plate. Light is reflected from the lower structure and then from the reflective surface 650 on the top surface toward the reaction tank 670. Figure 7A and 7B An embodiment of a light guide plate with reflective structures (cutouts) on both the top and bottom surfaces is illustrated. In a preferred embodiment, the structure on the top surface 681 is a right-angled triangle with a 45-degree angle 682, wherein its hypotenuse faces the incident light source (to the right). The bottom triangle 683 has a 41-degree angle 684 at the bottom surface, and its hypotenuse faces the incident light source (to the right).

[0047] The number and spacing 685 of the reflective structures are the same as the number and spacing of the reaction cells on each PCR reactor. For example, if the spacing 685 between rows of reaction cells is 4 mm, the reflective structures will be spaced 4 mm apart. To obtain a uniform light distribution, the height of the triangles (the depth of the cuts) gradually increases along the light guide plate. In one embodiment where the light guide plate has 20 cuts, starting from the inlet side, there are four cuts with a height of 0.06 mm, four cuts with a height of 0.08 mm, four cuts with a height of 0.18 mm, and eight cuts with a height of 0.4 mm. The reflective structures help improve the uniformity of the light guide plate ends. Therefore, there are fewer cuts on the bottom surface than on the top surface. In one embodiment, for a light guide plate with 20 cuts on the top surface, there are nine cuts on the bottom surface with a height of 0.4 mm. The cuts on the bottom surface are offset 686 relative to the top surface to prevent them from blocking light emitted from the reaction tubes located below the light guide plate.

[0048] Figure 8 The diagram illustrates a heated cap located on top of a reaction plate. The heated cap has an array of holes aligned with the top surface of the reaction tubes within the reaction plate. Figure 9A An embodiment of the PCR apparatus of this application is illustrated. The PCR apparatus includes a heat sink 910, which can be cooled by a heat sink cooling fan 920. A heating module 930 is disposed on top of the heat sink to support reaction plates 940. A heat cover 950 is disposed on top of the reaction plates. In this embodiment, a PCR apparatus houses four reaction plates, which are disposed in the heating module. The PCR apparatus is designed to heat and cool samples to precise temperatures to facilitate nucleotide denaturation, annealing, and then polymerase extension in each round of DNA amplification. In one embodiment, a Peltier thermal cycler is used. It uses a solid-state active heat pump to transfer heat from one side to the other against a temperature gradient by consuming electrical energy. A very useful feature of the Peltier module is that a thermal gradient can be established, allowing for optimization of the annealing step of the analysis in a single run. Figure 9B This is another implementation of the PCR reactor. A heated lid 950 is positioned above the heating module 930 and the reaction plate 940. The heated lid has openings to allow fluorescence to pass through. Determining the optimal primer annealing temperature is crucial for efficient and specific amplification of the product. This PCR system can achieve any desired temperature gradient for each reactor.

[0049] The reaction plates can have different rows and columns of reaction tubes. In one embodiment of this system, each microarray has a 4×8 array of sample tubes (sample racks). Each reaction tube may have a different volume, ranging from 1 μl to 125 μl. Any other format, smaller or larger, can also be used. In one embodiment of this PCR instrument, each unit has four reaction plates, each plate has 32 reaction tubes, for a total of 128 reaction tubes, with annealing, polymerization, or denaturation temperatures tested in a single run. The thermal gradient can be adjusted to optimize reaction conditions in a single run, determine the optimal annealing temperature for multiple primer sets, and perform reactions requiring different annealing temperatures simultaneously. Therefore, each photograph taken from each PCR reactor contains 128 images of each reaction tube. Placing all images of the reaction tubes in a single image makes image analysis and comparison much easier than with existing techniques. Fluorescent reporter genes, such as DNA-binding dyes or labeled probes, can be used to measure the fluorescence intensity of each PCR reaction, thus determining the presence of the target in the experimental sample.

[0050] Figure 10 The automated follow-up sample delivery PCR reactor system of the present invention is illustrated, comprising 10 PCR reactors arranged in a linear fashion. Each PCR reactor has its own thermal controller, but the start-up time of each PCR reactor is independently controlled.

[0051] Figure 11 An embodiment of a complete PCR reactor of this system 1100 is illustrated. The system has 10 independent PCR units on platform 1110. An optical system 1120 is mounted on a traversing system 1130. The traversing system moves the optical system onto any PCR reactor ready for imaging. Simultaneously, the reaction plates in any other PCR reactor can be replaced with a new set of reaction plates containing new samples. In this system, the PCR reactors are linearly arranged, thus using linear motion. However, the system can be designed to have any type of PCR arrangement, such as row and column arrangements, and can be configured for two-dimensional or three-dimensional traversing to move the optical system onto any desired PCR reactor in the system.

[0052] The computer sets the thermal cycling parameters for each PCR reactor and moves the optical system via a control traverse system, controlling the detection unit to take pictures and store the data acquired from the detection camera. Each PCR unit has a different start time, thermal cycling temperature, and heating time. The movement of the optical system and imaging is set to match the movement of each reactor. For example, after illuminating and imaging the fluorescence emitted by one PCR reaction for a few seconds, the traverse system moves the optical element to another PCR unit ready for illumination and imaging, and so on. The PCR reactor that has finished operating is replaced with a new reaction plate and detected. This method allows for in-feed PCR units.

[0053] The computing unit includes a system control heater, a traverse system, a camera, and switches. The heater control system controls the heater, cooling fan, and corresponding sensors. The thermal cycling parameters of each temperature control element can be individually set and configured in the software before starting any program. The motors can be configured to move the detection unit to the desired reactor location for image capture at any predefined time point. The initial conditions of the PCR program for each microreactor do not need to be identical. To capture images at the same time point during the thermal cycle of each microreactor, it is preferable to start the PCR thermal cycle in a sequentially delayed manner. The camera is configured using software to set the image capture time, exposure time, camera gain, target area, and frame rate, etc. A filter wheel is used to obtain high-quality images by setting the desired filter combination.

[0054] The software also provides a complete set of tools for image processing and data analysis. Various methods can be implemented within the software to calibrate full-field images and reduce imaging noise, including but not limited to flat-field calibration, color filter calibration, dark frame subtraction, median averaging of multiple images, and background subtraction.

[0055] This optical system can be used in many instruments besides PCR and qPCR, such as fluorescence microscopes, flow cytometers, and microfluidic chip devices used in drug discovery and other life science research. Of course, any system needs to obtain continuous, reproducible, robust, and uniform light distributed across the measurement area. The analyzed sample can be part of a reaction involving species, including biopolymers such as oligonucleotides (DNA, RNA, iRNA, siRNA), proteins (including antibodies, enzymes, agonists, antigens, hormones, toxins), oligosaccharides, and non-polymer species such as steroids, lipids, phospholipids, small organic molecules (e.g., retinoic acid), pesticides and non-peptide toxins, hormones, and antigens. Due to the interaction of light with chemicals present in the sample solution, the cold light emitted by all samples (fluorescence or phosphorescence) is then recorded on a camera or similar system. The recorded image contains images from all wells on the microarray chip, making the system compact, easy to use, and inexpensive. This optical system can be used in many other light-based detection systems, such as droplet digital PCR. The wells can contain biological samples such as oligonucleotides, DNA molecules, RNA molecules, chromosomes, or protein molecules. This illumination system can be used with a variety of bioanalytical tools, such as microtiter plate readers, DNA sequencers, PCR instruments, q-PCR instruments, microscopes, flow cytometers, microfluidic chips, diagnostic medical devices, and therapeutic medical devices.

[0056] The optical system of this device provides sensitive detection for accurate quantification and target identification. Scanning directly above the reaction plate, the device individually illuminates and detects fluorescence from each reaction cell with high sensitivity and without crosstalk. During data acquisition, the optical system automatically collects data from all reaction tubes, allowing you to input or edit relevant information for each tube according to your schedule.

[0057] The foregoing description is intended only to illustrate the principles of the invention. Furthermore, since many modifications and variations will readily occur to those skilled in the art, it is not intended to limit the invention to the exact structures and operations shown and described; therefore, all suitable modifications and equivalents fall within the scope of this invention.

[0058] Regarding the foregoing description, it should be understood that the optimal relationships between the components of the present invention in terms of size, shape, form, material, function and operation, assembly and use are considered obvious and will be obvious to those skilled in the art, and equivalent relationships to those shown in the drawings and described in the specification are intended to be covered by the present invention.

Claims

1. A follow-up PCR reactor system for bioanalysis, characterized in that, include: a) Multiple PCR reactors, each PCR reactor comprising multiple reaction plates configured with multiple sample tubes for placing biological samples, and a thermal cycler with temperature control elements for controlling the temperature of multiple biological samples. b) An optical system comprising an illumination system and an imaging system for receiving and recording emitted light from each biological sample; The irradiation system includes: i) A light guide array for splitting light from a light source into an array of light rays, wherein each light guide has an exit surface; each light guide has a trapezoidal cross-sectional shape with a short side, a long side, and a thickness; wherein the short side of each trapezoidal shape is exposed to the light source, and the long side is configured to connect to the light guide and guide light rays into the light guide; ii) A light guide plate for receiving and redirecting each ray of light along the top surface of the reaction plate, wherein the light guide plate is provided with an incident surface that contacts the exit surface of the light guide tube; the light guide plate is substantially rectangular and has a top surface, a bottom surface and a thickness; the light guide plate has a reflective coating or a material for total internal reflection and has an uncoated area at a predetermined position to allow the emitted light to pass through; iii) A light reflection structure array configured on the light guide plate is used to reflect each beam of light into each biological sample in each sample tube. At this time, all biological samples are illuminated at the same time, thereby generating multiple beams of emitted light. The light reflection structure of the light reflection structure array is disposed on the top surface, bottom surface, or both the top and bottom surfaces of the light guide plate; the reflected light of the light reflection structure array is disposed at a position that allows the emitted light to pass through the light guide plate; c) A transverse system for fixing an optical system, wherein an imaging system is disposed on the transverse system to be exposed on the top surface of multiple reaction plates to receive multiple emitted lights from multiple biological samples, and the transverse system is configured to move the optical system at predefined time intervals to precisely capture each PCR reaction and take images of multiple emitted lights. d) A computer system is used to coordinate the thermal cycling time of each reactor, the movement of the transverse system, and the image acquisition of multiple emission lights. As a result, the in-feed PCR reactor can perform multiple PCR experiments and allows any other PCR reactor to be replaced at any time while the optical system is on top of one PCR reactor.

2. The PCR reactor system according to claim 1, characterized in that, in, The traverse system is a linear traverse system that moves the optical system linearly on a fixed row of PCR reactors.

3. The PCR reactor system according to claim 1, characterized in that, in, The lateral movement system is a two-dimensional lateral movement device that moves the optical system in a planar motion on multiple rows of fixed PCR reactors.

4. The PCR reactor system according to claim 1, characterized in that, It also includes a plate with multiple windows located on top of the reaction plate to allow light of the desired wavelength to pass through each window.

5. The PCR reactor system as described in claim 1, characterized in that, It also includes a heating control unit configured to provide a circulating temperature in a manner suitable for polymerase chain reaction of biological samples, employing a direct contact thermal cycler, a metal block, or a Peltier-based thermal control system.

6. The PCR reactor system as described in claim 1, characterized in that, It also includes a heating block and an electronic controller, both configured together to control the thermal environment of discrete biological samples.

7. The PCR reactor system as described in claim 1, characterized in that, It also includes a heat cap, which is used to control the thermal environment of multiple biological samples.

8. The PCR reactor system according to claim 1, characterized in that, in, The sample-in-process PCR reactor is a real-time polymerase chain reaction instrument, a DNA sequencing instrument, an antigen-antibody ELISA instrument, or a digital PCR instrument.

9. An optical system, characterized in that, It includes an illumination system and an imaging system for receiving and recording multiple beams of emitted light from biological samples with exposed surfaces in multiple reaction plates; The irradiation system includes: i) A light guide array for splitting light from a light source into an array of light beams, wherein each light guide has an exit surface; each light guide has a trapezoidal cross-sectional shape with a short side, a long side, and a thickness; wherein the short side of each trapezoidal shape is exposed to the light source, and the long side is configured to connect to the light guide and guide light into the light guide; ii) A light guide plate for receiving and redirecting each light beam along the exposed surfaces of a plurality of reaction plates, wherein the light guide plate is provided with an incident surface in contact with the exit surface of each light guide tube; the light guide plate is substantially rectangular and has a top surface, a bottom surface and a thickness; the light guide plate has a reflective coating or a material for total internal reflection and has uncoated areas at predetermined locations to allow emitted light to pass through; iii) An array of light-reflecting structures configured on a light guide plate to partially reflect each light beam into each biological sample in each sample tube, wherein all biological samples are simultaneously illuminated to cause multiple beams of emitted light; an imaging system is located above the exposed surface to receive and record the light emitted from the array of biological samples; The light reflection structure of the light reflection structure array is disposed on the top surface, bottom surface, or both the top and bottom surfaces of the light guide plate; the reflected light of the light reflection structure array is disposed at a position that allows the emitted light to pass through the light guide plate.

10. The system according to claim 9, characterized in that, in, The light guide tube includes a set of light-transmitting materials with a rectangular cross-section or other shapes for guiding light to the light guide plate with low loss and managing the percentage and angle of light output at each exit surface in the light guide tube to control uniformity and other lighting requirements.

11. The system according to claim 9, characterized in that, in, The slots or cut structures on each light guide plate are light-reflecting structures. Each slot has a height and the position of each slot reflects light into a row of sample tubes.

12. The system according to claim 9, characterized in that, in, The light reflection structure is a triangular structure used to reflect light back to the light guide plate, preventing light loss and generating a more uniform and diffused light source for each PCR reactor.

13. The system according to claim 9, characterized in that, in, The light-reflecting structure is disposed on the top surface of the light guide plate and has a right-angled triangular cross-section with a 45-degree angle, wherein the hypotenuse of each right-angled triangular cross-section faces the incident light from the incident surface of the light guide plate.

14. The system according to claim 9, characterized in that, in, The light-reflecting structure is located on the bottom surface of the light guide plate and has a right-angled triangular cross-section at a 41-degree angle to the bottom surface, with the hypotenuse of each right-angled triangular cross-section facing the incident light from the incident surface of the light guide plate.

15. The system according to claim 9, characterized in that, in, The top surface of the light guide plate has 20 reflective structures, of which the first 8 structures have a height of 0.4 mm, the next 4 structures have a height of 0.18 mm, the next 4 structures have a height of 0.08 mm, and the last 4 structures have a height of 0.06 mm.

16. The system as described in claim 9, characterized in that, in, The bottom surface of the light guide plate has 9 reflective structures, each with a height of 0.4 mm and a spacing of 4 mm.

17. The system as claimed in claim 9, characterized in that, The light guide tube is either integrally molded or composed of multiple transparent material components to divide light, wherein the transparent material is optical glass, PMMA, polyacrylic acid, polycarbonate, polyethylene or other optically transparent materials.

18. The system according to claim 9, characterized in that, Suitable for simultaneously irradiating and recording emitted light from each reaction plate, including arrays of 1×1 to 32×48 or more.

19. The system as claimed in claim 9, characterized in that, in, The light source includes light-emitting diodes (LEDs) or LED arrays or halogen lamps or mercury lamps or lasers.

20. The system according to claim 9, characterized in that, in, The light source includes an LED light source capable of generating a total output optical power of at least 10 milliwatts to 100 watts for all radiation wavelengths.

21. The system as described in claim 9, characterized in that, The wavelength of light generated by the light source can cover the entire visible light range, or cover UV, or cover IR, or cover from 450 nanometers to 700 nanometers.

22. The system as described in claim 9, characterized in that, in, The fluorescent dyes used are capable of generating a spectrum of light that can be excited and emitted by mixing each biological sample.

23. The system as described in claim 9, characterized in that, It also has an optical filter between the light source and the light guide tube to control the spectral content of the light emitted by the light source.

24. The system according to claim 9, characterized in that, It also includes: an imaging sensor, a microarray reactive chip, and an emission filter located between the imaging sensor and the microarray reactive chip. The emission filter allows light signals of a selected wavelength to reach the sensor, and the emission filter filters the spectrum of the light to obtain the desired wavelength.

25. The system according to claim 9, characterized in that, in, The imaging system includes an imaging sensor, a microarray reactive chip, and a set of optical lenses located between the imaging sensor and the microarray reactive chip to image the entire surface of the chip and filter the spectrum of light to the desired wavelength.

26. The system as described in claim 9, characterized in that, Also includes: The camera is a charge-coupled device detector array, a complementary metal-oxide-semiconductor detector array, a photomultiplier tube detector, a photodiode, or an array of photodiodes.

27. The system according to claim 9, characterized in that, in, The system is configured to receive and generate multiple discrete sample images from each sample tube, the sample tube having a volume of 1 to 100 microliters.

28. The system as described in claim 9, characterized in that, in, The light source is configured to generate an excitation beam, the excitation beam being approximately 0.1 cm. 2 Up to 1000cm 2 The excitation is basically uniform within the range.

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