Nucleic acid detection system, device and method

Abstract

A method of analyzing nucleic acids using a device comprising a reaction chamber and a plurality of sensors located in the base of the chamber, and each sensor is preferably located within a respective well. The method comprises flowing a fluid containing the nucleic acid or fragment thereof into the reaction chamber. While the chamber is fully or at least partially sealed, amplification of the nucleic acid or the fragment is performed within the chamber using one or more amplification primers, while the production of amplicons is detected using the sensor. The amplicons are then sequenced or hybridized, and the sequencing or hybridization is detected using the sensor.

Description

[0001] This application is a divisional application of the application with international application number PCT / EP2015 / 059097, international application date April 27, 2015, application number 201580021281.1 in the Chinese national phase, and invention name “Nucleic Acid Detection System, Device and Method”. Technical Field

[0002] The present invention relates to systems, instruments, and methods for processing nucleic acids. The present invention facilitates the control and operation of workflows and components of instruments / systems. Processing can involve, for example, DNA / RNA sequencing and / or performing immunoassays. background

[0003] Significant breakthroughs have been made in the development of bioassays. Such assays include immunoassays, DNA sequencing, analytical chemistry, and coulometry. Some of these assays have been developed into instruments for diagnostics. Many of these assays are highly complex and require specialized knowledge to operate and interpret.

[0004] In the field of genetics, there are applications for whole genome sequencing, de novo sequencing, gene expression analysis and detection of single nucleotide polymorphisms (SNPs). Each of these detections provides the information of detection and the varying levels of complexity. In some cases, more than one of these detections will be needed and how to decide to proceed depends on the clinician.

[0005] Most detection workflows have three main aspects: sample preparation, detection, and interpretation of results. For each of these aspects, sequencing by synthesis technology used in genetics is an example of a complex process with many steps.

[0006] The preparation phase can begin with extraction of the template nucleic acid from a provided sample, followed by purification, fragmentation of the nucleic acid into workable lengths, clonally amplified template fragments, hybridization / ligation with primers / probes, and attachment / immobilization to a surface or bead. The sequencing by synthesis step itself requires the flow of different nucleotide types (dNTPs) throughout the template cloning process, where the nucleotides are bound to the template nucleic acid.

[0007] The detection stage can employ various techniques such as electrophoresis, chemiluminescence, and optical cameras, measuring changes in electrode impedance / capacitance, measuring changes in the intrinsic charge of the immobilized DNA, or monitoring proton concentration using ion-sensitive field-effect transistors (ISFETs).

[0008] The interpretation phase involves analyzing and extracting information from the test results. For example, this can include determining the sequence of the nucleotide incorporation detected from each fragment; determining their alignment / reassembly to form the whole genome; or comparing them to known sequences. The results can guide the processing or recommendations of complementary products, although the final results may be several days after the initial sample collection.

[0009] Among the detection processes identified above, one of the most exciting involves the use of ISFETs and other chemical FETs (ChemFETs). For example, the use of ISFETs for sequencing DNA and DNA fragments (as well as RNA and RNA fragments) is described in WO03 / 073088. This work has demonstrated that the incorporation of nucleotides (A, T, C, G) during DNA chain extension can be monitored using ISFETs to measure differences in ion concentrations as a byproduct of the reaction. As the nucleotides extend the DNA chain, they release pyrophosphate, which is hydrolyzed and produces H+ ions, lowering the pH. In a similar manner, ISFETs can be used to detect hybridization, where hybridization probes attach to matching sequences on a DNA chain.

[0010] An extension of this method using very large-scale FET arrays is described in US 2009 / 0026082 and provides massively parallel analysis. By analyzing a large number of DNA fragments in parallel, and then aligning and "stitching" the results together, long stretches of DNA can be sequenced in a relatively short period of time.

[0011] In the case of an ISFET-based assay, an ISFET chip is designed and manufactured to perform one or a set of predetermined assays. For example, an ISFET chip might be configured to detect the presence of a set of SNPs in a DNA sequence. A sample is first prepared on the bench. This can include enriching the sample to remove non-cellular material, lysing the enriched cells to release the DNA, and amplifying one or more DNA sequences. During or after this process, the amplified DNA sequences are attached to microbeads. These beads are then introduced to the chip. This can include placing the beads in wells formed above individual ISFETs, for example, using magnetic beads to introduce one bead into each well. The chip can then be inserted into an instrument where sequencing is performed. The instrument cyclically flows different nucleotides (A, T, C, G) through the chip, with wash steps between each nucleotide flow. An electrical signal representing chain extension is detected. The result provided by the instrument is the chain extension sequence for each ISFET. This data is then analyzed, for example, using a desktop PC connected to an analyzer, to measure the results based on their prevalence. Sequences with high prevalence will be recorded as valid sequences, while sequences with lower prevalence will be recorded as being attributed to noise. Valid sequences can then be used to determine the presence or absence of one or more SNPs in the sample being analyzed. Of course, other workflows and analysis procedures are possible.

[0012] In addition to these essentially laboratory-limited assay processes, point-of-care assay procedures and systems have been developed. For example, DNA Electronics (London, UK) has developed genetic testing kits that allow procedures (such as those described above) to be performed by essentially non-technical personnel at the point of care or point of sale. In many cases, testing using an ISFET-based assay will constitute only part of the workflow to be followed by a skilled person. Depending on the results of an ISFET-based assay, decisions regarding additional testing may need to be made, and these tests may need to be performed, for example, using additional ISFET-based assays.

[0013] WO2011 / 034790 and US2012 / 0109531 provide exemplary biological and physiological assay protocols and instruments that provide a degree of automation and flexibility. However, they are not directly applicable to the processing of nucleic acids. SUMMARY OF THE INVENTION

[0014] The inventors have devised a system that can flexibly perform assays on nucleic acid samples. Depending on the program being run, different functions can be performed. These can be used to optimize instrument results based on the data fed back.

[0015] According to a first aspect of the present invention, there is a method for analyzing nucleic acids using an apparatus comprising a reaction chamber and a plurality of wells located in the base of the chamber, and at least one sensor located in each well. The method comprises flowing a fluid containing the nucleic acid or a fragment thereof into the reaction chamber. While the chamber is completely or at least partially sealed, amplification of the nucleic acid or fragment is performed within the chamber using one or more amplification primers, and the production of amplicons can be detected using the sensor. The amplicons are then sequenced or hybridized, and the sequencing or hybridization is detected using the sensor.

[0016] Further aspects of the invention are set out in the dependent claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 An exemplary workflow using a system including an array of detection elements is illustrated;

[0018] Figure 2 The diagram shows a suitable Figure 1 ISFET-based chip modules used in the workflow;

[0019] Figure 3 Schematically illustrates the Figure 1 Various biological operations that are part of the workflow;

[0020] Figure 4 Schematically illustrates the process for implementing the workflow (e.g. Figure 1 (as shown in the figure);

[0021] Figure 5 yes Figure 4 A block diagram of the conceptual components of the instrument;

[0022] Figure 6 schematically illustrates the hardware and software components used to execute an operating system;

[0023] Figure 7 Schematic illustration of the components of a “biological” operating system and the application layer for implementing biological workflows.

[0024] Figure 8 is a flow chart illustrating a process for analyzing nucleic acids or fragments thereof; and

[0025] Figure 9 Schematically illustrates a method suitable for implementing Figure 7 The method of the box.

[0026] describe

[0027] By way of example, systems and methods are provided that allow an integrated array of detection elements (or "cells") to be configured to perform a variety of assays and even reconfigured during the performance of an assay that depends on an intermediate result. In other words, intermediate decision points are introduced into the workflow, the assay is then performed, and the decision made at each of the decision points determines how the workflow proceeds from that point, and the detection elements are configured accordingly. Such configuration can involve controlling the flow of fluid into the detection element, for example, to introduce suitable primers and hybridization probes and / or to activate primers or hybridization probes that have already been placed.

[0028] In a typical system, the chip includes an integrated array of detection elements, such as ISFETs, with different groups of detection elements configured to perform different tasks. For example, a first group of ISFETs can be configured to perform hybridization to measure the similarity of the sequence of the DNA being analyzed to a hybridization probe bound within the detection zone. Additional groups of ISFETs can be configured to sequence different segments of the DNA under analysis. Decision points incorporated into the workflow make decisions about which DNA segment to sequence based on the results of the genotyping process.

[0029] In an exemplary scenario, a hospitalized patient displays symptoms that could indicate trauma, a heart attack, a local infection, or sepsis. A physician must determine which of these is the real cause and administer the appropriate treatment. Figure 1 The horizontal axis represents an exemplary timeline of the workflow obtained for a specific sample. A blood sample is introduced into a sample inlet of a detection instrument incorporating an ISFET-based assay chip. The instrument may include pumps and valves connected to microfluidic flow channels and fluidic gates for directing the received sample to the appropriate portion of the chip.

[0030] As a first stage of the workflow, a diagnosis is made between conditions such as sepsis, local infection, trauma, or acute myocardial infarction (AMI). The outcome in this example is a diagnosis of sepsis.

[0031] It is now desirable to identify the family of pathogen causing the infection (to identify sepsis species and resistance genes). This is done by applying the extracted DNA to a set of ten PCR panels. These are selected as the results of the sepsis diagnosis. In some embodiments, all available modules are integrated onto a single chip, while in other embodiments, the modules are arranged on different chips. In any case, each of the ten PCR panels of the selected modules is configured to perform PCR on a different targeted DNA sequence corresponding to a different family of pathogen. Each PCR panel can include one or more wells, each located above an ISFET sensor. Amplification primers can be fixed at the base of each well or fixed in solution, and the amplification primers are different for each panel. The fluid control system is operated so that a fluid containing all four nucleotides (A, T, C, G) flows through each panel. Heating and temperature sensing elements are incorporated into the system to allow PCR amplification. In the illustrated example, a positive result is provided by panel 3 (i.e., only panel 3 produces an amplicon above some threshold level), thereby identifying the family of the pathogen. In an alternative arrangement, amplification primers may be introduced into the panel via a fluidics control system, for example along with the nucleotides, rather than being immobilized within the wells.

[0032] In an alternative configuration, rather than having multiple modules that can be used to perform different sets of PCR assays and selecting one of these based on a triage diagnosis, a universal module can be provided with a set of panels that are "programmed" to have specific amplification primers flow through them, resulting in the desired primers being immobilized within the panels (or more precisely, within the wells of the panels).

[0033] The third stage of the workflow is to identify the specific species of pathogen from the identified family. In this embodiment, it utilizes a set of hybridization detection performed by six different modules, although this could alternatively be implemented by detection amplification or sequencing, and the hybridization stage could be omitted. Each module includes one or more wells, and each well is located above the ISFET. Hybridization probes are fixed to the base of each well, and the probes are different for different modules. After preparation, the sample is delivered to the module by appropriate operation of the fluid control system. Heating and temperature sensing elements are incorporated into the system to allow repeated hybridization cycles. In this example, only species 2 detection produced a positive result. In an alternative arrangement, the fluid control system can be used to deliver the hybridization probes to the module.

[0034] As with the PCR modules, the hybridization modules can be selected from a larger number of available modules, or can be obtained by programming a set of universal modules to immobilize appropriate hybridization primers in appropriate wells, or can be pre-programmed.

[0035] As described, further consider the second (amplification / PCR) stage and the third (hybridization) stage, these can be carried out by the amplification and hybridization module that separate.Yet alternative approach is to carry out these stages in identical module.For example, this can be by carrying out and detecting amplification and afterwards activating or using the hybridization primer that has been fixed in the module to allow to carry out and detect hybridization and realize in given module.Activation process can comprise heating and removing the wax layer in the hole of module so that potential hybridization primer is exposed, or can carry out activation hybridization to promote hybridization by regulating temperature, or hybridization can occur during amplification stage.

[0036] The fourth and final stage of the workflow is to sequence one or more relevant segments of DNA, optionally again using a suitable chip module. Sequencing first requires that the one or more DNA segments to be sequenced have been amplified. Amplification can be performed in an antechamber in fluid communication with the sequencing chamber, for example using PCR. Alternatively, amplification can be performed in the sequencing chamber itself. Amplification is performed as described above for the second stage of the workflow. The sequencing chamber contains one or more wells above each ISFET, and sequencing primers are fixed to the base of the wells. Similarly, the fluid control system is operated to allow the prepared blood sample to flow into the module. The nucleotides (A, T, C, G) are then passed through the module in sequence. Sequencing of different segments of DNA can be performed in parallel, and the resulting data can be compared and spliced ​​together.

[0037] In some embodiments, sequencing can be performed within the same module used to perform Stage 2 amplification and Stage 3 hybridization.

[0038] One or more detected sequences can then be added to a database and compared to known pathogens to determine whether the pathogen is known or unknown. A treatment recommendation can be made upon completion, which may include the use of drugs with drug-gene relationships. An option can be obtained to perform genotyping testing for medication adjustments.

[0039] Identifying a sample at each stage triggers another assay with multiple additional possible identifications. After each stage in the workflow, subsequent workflow decisions are made. In addition, intermediate results can be used to inform treatment decisions. For example, if the results of the first stage indicate sepsis, initial treatment for it can be initiated. The second stage assay identifies what form of sepsis is present (viral, bacterial, fungal, etc.). If the second stage identifies the presence of bacteria, treatment with a broad-spectrum antibiotic can be recommended. Additional intermediate treatment decisions can be made as the workflow progresses.

[0040] Furthermore, it will be appreciated that during a particular assay phase, decisions relating to the assay may be made. For example, during the fourth phase of nucleic acid sequencing, each base identification may be used to determine whether to continue sequencing for further identifications, to stop sequencing, or to flow through a particular sequence of nucleotides.

[0041] In the case of a module that amplifies, by monitoring the entire reaction volume, any downstream process may be closed or ignored for any chamber that does not demonstrate amplification. For those chambers that do demonstrate amplification, it is necessary to decide whether to proceed to the next stage of the process. For example, if a resistance gene is identified, this can provide enough information to advise the physician to adjust the treatment specifically for the resistance gene, and therefore the workflow for the sample can end at this stage. However, if the gained amplification is still unclear or needs additional analysis, the decision to hybridize and order-check can be made. Similar to amplification mode, monitoring these modes allows the decision to proceed or not, to continue monitoring or not monitoring downstream events, including stopping detection completely and making the suggestion for treatment.

[0042] In the case of diagnosing trauma or AMI, there is an opportunity to screen the patient's drug interaction information, for example, to establish a genotype for drugs such as warfarin or PLAVIX. In both cases, the samples collected during the classification (which will contain a large number of patients' cells and DNA) can be genotyped. The first mode (PCR or isothermal amplification and detection) of the system can be used to determine the genotyping. It may not be necessary to proceed beyond this stage.

[0043] Figure 2 A module based on an ISFET chip suitable for use in the above process is shown. The module is suitable, at least in its physical structure, for use by an instrument (e.g., as described by reference to Figure 1 The present invention is universal across all different assay types (exemplified by the above discussion of the present invention), such as amplification, hybridization, and sequencing. In some cases, the module can be configured to perform two or more of amplification, sequencing, and hybridization. The left side of the figure illustrates a "pre-amplification" stage configured to prepare a sample for subsequent genetic analysis. This stage can, for example, separate cells in a blood sample and extract DNA from the cells. The pre-amplification stage then feeds the enriched sample to a reaction chamber comprising five wells, each of which is exposed to an ISFET sensor. The inset on the right side of the figure illustrates a side cross-sectional view through one of the rows of sensors. Each of the reaction chambers includes inlet and outlet fluid ports, as well as a central space for holding a volume of fluid. The ISFET sensor is disposed at the base of the well. In addition, the module can include a heater for heating the fluid in the chamber and a temperature sensor.

[0044] Depending on the assay to be performed, amplification and sequencing primers and hybridization probes can be fixed within the wells of the module. As a specific example, one might consider a scenario where the module is configured to perform amplification (PCR) followed by sequencing. In this case, a prepared sample containing extracted and fragmented DNA can be flowed into the chambers of the module along with amplification primers and nucleotides. The temperature is then cycled to perform PCR amplification. Sequencing primers are fixed within each well. The amplicons are then sequenced and the results read out from the ISFET.

[0045] One approach to achieving multimodal workflows is to assign each mode to a specific chip and assemble the chips into a cartridge that has all the required modes but does not have the ability or requirement to switch between modes for each chip. This design would overcome anticipated challenges such as thermal compatibility and functionalization, but would also result in more complex designs for fluid handling and control.

[0046] In alternative approaches, combinations of test kits with specific functions or target diseases are available, and these can be selected based on the results of earlier steps. The selection of downstream detection (box) can be an automated process or a process that includes user intervention (e.g., plugging two boxes together). Some examples of starting points for all-using classification systems with sample enrichment are as follows:

[0047] trauma :When wound diagnosis, suitable downstream detection can measure the pharmacogenomic characteristics (profile) of patient for drug interaction information.The classification box for carrying out wound diagnosis effectively becomes sample preparation (prep) module and can be plugged / inserted in the wound box that is suitable for, for carrying out subsequent genotyping reaction.

[0048] Sepsis Appropriate downstream testing can identify the specific species or strain and also identify any resistance genes present in the individual. The enriched sample from the sorting assembly can be inserted into the sepsis box and then subjected to downstream analysis. There is the possibility that the prescribed treatment has genetic factors to be considered, and there is the choice of individual genotype. Similarly, the sorted samples can be used to run genotyping tests (or these can be independent rapid tests).

[0049] Local infection The appropriate test will be determined using other provided information, including physician observations, to select the appropriate test panel. Because the location of the infection and / or the cause of the injury will affect the test to be performed, it is reasonable to have multiple different available cartridges (as opposed to having one cartridge that covers all potential infections within the hospital environment). To optimize the system, it may be necessary to plug the relevant test cartridges, which will be influenced by a variety of factors and data.

[0050] Now consider Figure 3 , which illustrates an example process for analyzing DNA samples. It is presumed that function has been assigned to the chip by placing specific primers into known areas / wells of the chip. Assuming that it is known what to place and where "a priori", it is possible to determine which amplifications have been made based on the sequence used for the primers. In this example, the primers used in the initial pattern of amplification are modified with the corresponding sequence from the sample and are ultimately used to produce a product that is sequenced later in the workflow.

[0051] The sample enters the reaction chamber, amplification occurs (real-time detection) and the primers printed are modified to reflect the target sequence. After completing the amplification mode, the solution phase primer is rinsed off and replaced with a sequencing primer. Being able to detect hybridization events can provide additional sequence information. After sequencing, one or more Nucleotide can be made to flow in the whole reaction chamber.

[0052] The system will require less complex fluid handling and control systems than other approaches, but may require additional mechanical drives to allow the different modes to follow each other.

[0053] In principle, the system has the ability to proceed to the amplification mode, detect any amplification that occurs, and decide whether to proceed to the next stage. In instances where there is no amplification, the system can decide not to monitor any downstream reactions, or stop any further reactions from occurring. Similarly, for subsequent modes, failure to detect hybridization may shut down the downstream sequencing mode, whereas detection of hybridization can allow a go-or-no-go decision to be made.

[0054] In certain embodiments, the instrument that comprises above-mentioned chip / module or is used for using together with above-mentioned chip / module comprises the operating system for being connected with the component of instrument, and is set to receive data to configure or reconfigure instrument.In certain embodiments, instrument is set to receive the real-time data of the biological data representing about sample, and operating system can reconfigure the workflow of the detection of sample or reprogram the component of instrument.In this sense, there is the feedback of detection data to instrument thereby to operate instrument in an optimal manner.In certain embodiments, instrument is set to receive the data from external source or the application running on operating system, and operating system can configure instrument thereby to run biological detection in a customized manner.

[0055] Figure 4An instrument including a chip as described above is illustrated at a very high level, including both an operating system and an application layer (including one or more applications). The instrument receives input information from various sources (which can be automatic or manual input information). The figure shows three sources: applied medical databases, hospital rules, and input information generated by clinicians. As output information, the instrument provides a diagnosis and / or a recommended treatment plan.

[0056] The physical components of the bioinstrument will depend on the assay type and technology employed. Figure 5 is a block diagram of the conceptual elements of an apparatus according to a preferred embodiment.

[0057] • Sample preparation equipment;

[0058] • The cartridge includes means for combining the sample and reagents for a biochemical reaction to form a complex to be detected;

[0059] •Microfluidic networks ensure that samples and reagents are directed from component to component in a controlled manner;

[0060] •Storing reagents in instruments connected to tubing and pumps, where valves and solenoids control the flow of reagents;

[0061] • A sensor or sensor array adjacent to or integrated with the cartridge detects a signal from the complex or a property of the complex.

[0062] •Interface circuitry is connected to one or more sensors to read the electrical signals from the sensors.

[0063] •Control panel includes

[0064] •Signal processor

[0065] •CPU / controller

[0066] • Memory

[0067] •OS

[0068] • Application layer

[0069] While some embodiments of the OS can be used with suitable complex assays such as ELISA, preferred embodiments are used with complex instruments that have longer, flexible workflows and provide greater data depth. Complex instruments and assays, such as gene sequencers, provide large data streams that can be fed back into the OS and provide multiple decision points to change the workflow or select a different assay.

[0070] In an exemplary apparatus:

[0071] •Determination of DNA sequencing;

[0072] •The sensor cartridge is a semiconductor chip with a large array of ISFETs;

[0073] • Sample preparation involves emulsion PCR of the template and binding to separate beads presented to a well near the ISFET;

[0074] • Reagents dATP, dTTP, dCTP, dGTP and wash solution are supplied to the wells via pumps;

[0075] • For a multiplexed array of analog-to-digital converters (ADCs), the ISFET signals are read one row at a time;

[0076] • The signal processor filters and identifies nucleotide incorporation events, which are stored in a large-scale memory.

[0077] The operating system controls these components and workflows.

[0078] Preferred embodiments of the instrument can be designed to receive specific biological samples from a patient, such as blood, saliva, stool, mucus, etc.

[0079] The operating system provides an interface between the instrument hardware and the application layer, which contains a set of instructions for running the instrument. The OS can be thought of as similar to a combination of the BIOS (Basic Input Output System) and a conventional operating system for a personal computer. The OS accesses and controls the components of the instrument, allowing it to turn them on and off or set their properties. The OS defines how instructions affect the instrument and is responsible for translating high-level instructions into low-level component controls. The OS can also be designed to protect the instrument, for example by ensuring that the sequence of instructions is logical and that the required parameters are within design limits.

[0080] The OS includes both hardware and software aspects. The hardware may include a control board with a processor, memory, a data bus, and component drivers. The control board may be electrically connected to another hardware component of the instrument, such as a sensor chip, or it may share the board with such another hardware component. The software aspects are preferably encoded as non-volatile memory on the control board and determine the operation of the OS hardware. Although various OS systems can be designed using known structures for receiving and processing signals and instructions to control the instrument, in Figure 6 Exemplary embodiments are illustrated in and described below.

[0081] The non-volatile memory of OS code programming forms OS firmware 30. When the code is read by CPU 31, the control board can operate the instrument. One or more applications can be stored on memory 32 to provide machine instructions for controlling the instrument in a customized manner. The CPU reads the code from the application memory 32, which is interpreted by the OS to form instructions for controlling the instrument. The CPU receives data from an external source on a data bus 36. The data can be sensor data about biological samples, data about instrument hardware, and control feedback data. The data can be digital data or analog data. In the case of analog data, the control chip will also include an analog-to-digital converter. The data can be stored in the data memory 33. The processor 38 is connected to a plurality of drivers 34 that provide signals to the operating components. For example, the driver can open or close a reagent valve (digital control) or control the temperature of a heater (analog or PWM control). The control board can also include a transceiver and a data port for communicating with an external device.

[0082] In computer science terms, an OS can include dynamic link libraries (DLLs) that provide code blocks that can be called from applications. They are shared, reusable resources. DLLs offer the advantage of providing commonly used code, saving reprogramming with each application and ensuring that certain functions are robust. For example, in a genetic instrument, a DLL might perform PCR on a sample in a cartridge. In pseudocode, an application might simply indicate:

[0083] PCR (number_cycles)

[0084] The OS performs PCR using a set of code that turns on the heaters to set the temperature of the cartridge to the denaturation temperature, hybridization temperature, and extension temperature with appropriate delays between steps and repeats the cycle based on the parameter "number_cycles." The application is saved from rewriting this code, and the temperature will not exceed the design limits.

[0085] and Figure 6 It is equivalent to a control board in the manufacturing process, which can combine memory, processor, drive and data input / output connections into a single large-scale integrated semiconductor chip.

[0086] Alternatively, the instrument may have a data port connected to the instrument assembly and configured to receive control signals and send data to a remote computer via the data port. The remote computer would include a processor and an OS and be configured to receive data from the instrument, run applications, and send control signals to the instrument. In this case, the instrument system would include the instrument assembly and the remote computer.

[0087] Conceptually, the instructions may be provided to the OS through a separate application layer or housed in a memory portion of the control chip. The present invention is not intended to be limited by the structure for providing instructions to the OS.

[0088] The approach presented here allows for different levels at which the system can be configured and / or reconfigured between the user's request for a test and the generation of test results. Based on the requested test and the data received, the operating system can alter the workflow or operation of hardware components. The instrument can thus optimize each test, and it is conceivable that no two tests will be run identically.

[0089] If a single instrument is referred to as a system, a group of instruments or an instrument and external hardware can be referred to as a supersystem. Components of an instrument can be referred to as subsystems. Examples of external hardware include sample preparation devices, bioinformatics computers, etc. Examples of instrument components include reagent delivery units, sensors, cartridges, etc.

[0090] At the super-system level, a combination of instruments and / or external devices is connectable so that samples can be set to flow to multiple instruments or external devices for processing. For example, the OS can receive data about the sample type (blood, saliva, etc.) and select specific sample preparation equipment and operating conditions. In another example, the OS can decide to test samples in parallel on multiple instruments to obtain faster and more complete results.

[0091] At the system level, the OS can enter detection mode or select a specific detection cartridge type, (select a sepsis / tumor chip with pathways for Phase I or Phase II detection).

[0092] At the subsystem level, on-chip circuitry can be configured to turn sensors, heaters, noise filters, and data acquisition elements on or off.

[0093] Additional examples of reconfigurability are provided below.

[0094] At the super-system level, OS receives previous test data and determines the presence of endemic hospital-acquired infections and arranges all instruments to screen all samples using infectious disease assays. In addition to the assays selected by the user, OS will select these assays. By assuming that all patients have the same local disease and stopping deep DNA sequencing once OS has confirmed the presence of infection, OS can change the workflow, often requiring the identification of infectious variants for subsequent testing. OS can then communicate with external devices to report endemic infections to hospitals or government health agencies.

[0095] At the sensor chip level, data processing can be improved by changing or selecting different circuit parameters based on initial sensor signal quality that indicates high background noise. The OS can redirect the raw sensor signal through filters that reject some noise to allow real-time signal improvement without discarding all data at a later date.

[0096] At the GSIC level, the OS can direct one or more GSICs to amplify DNA in their reaction wells. Each GSIC will set the heater temperature, reference electrode voltage, sensor voltage, and receive reagents as needed to meet requirements. If the OS receives sensor data indicating low copy number in a particular well, it can re-instruct the respective GSIC to further amplify DNA in that well.

[0097] OS control of instrument components can be simple or complex. For example, at a high level, the OS may simply instruct a component to perform a function or implement a setting, with local circuitry ensuring compliance. At a more complex level, the OS may continuously monitor and control the component to implement the function or setting.

[0098] To facilitate reconfiguration of instrument components or workflows during an assay, the OS is configured to receive real-time data from the instrument. The data may be sensor data about a biological sample. The data is real-time in the sense that it is generated by the sensor during an assay run and is not limited to data generated in real time at a given instant.

[0099] The CPU can have a dedicated port to capture events or important signals. This allows the OS to be event-driven, making decisions related to reconfiguring the instrument when events occur. Alternatively, at points determined by the OS or application, such as when a decision point is reached in the workflow, the CPU can retrieve data from memory 33. The latter is suitable in the presence of large data streams, and the OS determines the best time to make a decision. For example, for a genetic assay, the OS can wait until an event indicating that gene-specific amplification has been completed is received before deciding whether to sequence. The OS can then periodically retrieve data about the sequence of a specific well from memory to decide when to stop sequencing.

[0100] The application layer can be provided to an interface with the OS layer. One or more applications can be loaded onto the memory to provide machine instructions to the OS to operate the instrument in a specific manner. An application is a set of machine instructions that can be read by the operating system. The application can be pre-installed on the instrument or loaded by the user. The program can be customized for a specific user (such as a hospital) to run a specific genetic test (such as detecting infectious diseases from a blood sample) according to a specific workflow (such as repeating a process or skipping another process).

[0101] In a preferred embodiment, the application is responsible for configuring or reconfiguring the instrument hardware and workflow. For example, if it detects certain viruses that have already been detected, an application installed by a specific hospital can guide deeper sample sequencing. The hospital may have a policy to test all samples for the specific virus strain that is now endemic to the ward, thereby reconfiguring future tests except those requested by the user.

[0102] Applications provide the user with an interface to the instrument. Different applications can offer different options to their users, from a single start button to a complex array of options that provide flexibility. The OS interprets the application's instructions but maintains overall control and responsibility for the instrument. For example, a program can direct the flow of reagents, but the OS has more direct control over pumps and valves and can override a request to pump a reagent once it has been consumed.

[0103] In another embodiment, the application is loaded and can be operated from outside the computer to the instrument. The remote computer can be a server, tablet computer (tablet), cloud computer or smart phone, and can be connectable to other computers. The instrument is connectable to the remote computer, preferably via the port connection on the control panel. The connection allows the remote computer to receive the biological detection data from the instrument and send instructions to the OS. The remote computer can contain or be able to access the database of the medical data of the patient providing the sample. The remote application can implement case-based reasoning (case-base-reasoning), the inspiration of medical decision-making, or machine learning, so that the application can use medical data to make a wise decision about how to configure the instrument. It can include indicating that the instrument OS uses a specific workflow, setting component settings and / or operating a specific assay. The remote application can receive real-time data from the OS to revise its decision and reconfigure the instrument.

[0104] For example, a doctor using a remote application requests testing for a patient. The remote application retrieves the patient's medical history and physiological results and compares them with similar medical cases to recommend genetic testing to detect the presence of known cancer biomarkers. The application instructs the OS to load the tumor assay kit. The application can optimize the sequencing workflow to know which bases are important for these genes. After receiving real-time data about certain genes from the instrument, the application instructs the instrument to stop further sequencing. The application then instructs the instrument to load assays to detect the patient's response to certain treatments. The application receives the final genetic results, adds them to the medical database, and outputs treatment recommendations to the doctor. The application continues to build relationships between the genetic results and the medical database to upgrade its case-based reasoning so that similar cases in the future can be detected on the instrument in a more refined manner.

[0105] In order to make the instrument particularly flexible and customizable, the instrument is set to accept one or more application programs and is installed in the memory. These application programs can be written by a third party who wishes to optimize the instrument operation or to perform a specific scheme. This allows a third party to utilize the workflow and components of the instrument to experiment and to optimize it. Alternatively, some users may have specific needs, provide some preferred sample types, or need to perform specific medical schemes or medical insurance policies. Application programs provide instructions to configure the instrument before detection begins and / or reconfigure the instrument when receiving real-time data from OS.

[0106] For example, a specific hospital only uses blood samples and hopes to detect Escherichia coli (E. coli) for each sample in addition to any requested detection, and obtain a 90% confidence interval for certain biomarkers. The application guides the instrument to purify the sample in a specific manner optimized for obtaining the patient and microbial components, and then run both the bacterial assay box and the human genotype box. For the bacterial box, only a simple presence / absence test is requested, but for the human genotype box, a deeper sequencing is requested. The OS receives real-time data and sends it to the application, which requests the OS to further sequence the DNA, thereby determining antibiotic resistance if specific bacteria are present and the patient data indicates that the antibiotic is unfavorable, or returning the test if the result is identified by the OS as less than 90% credible.

[0107] The OS is designed to receive "machine code" and translate it into output signals to control the measurement. Machine code is typically a binary string compiled from code written in a high-level programming language such as C++. Depending on the implementation of the OS, the machine code can provide simplified, high-level instructions or more customizable low-level instructions. For example, the instructions in pseudocode may include the following.

[0108] Advanced instructions:

[0109] Run sample preparation

[0110] Purification of samples

[0111] Dissolution operation

[0112] Run tumor screening tests

[0113] Run sepsis test

[0114] Run a Lifestyle Check

[0115] Send data to insurance companies

[0116] Read patient profile

[0117] Run sequencing assays

[0118] Reading genotype

[0119] Export genotypes to the cloud

[0120] Reassembly sequence

[0121] medical_association(genotype)

[0122] drug_association(genotype)

[0123] Quantifying DNA

[0124] Low-level instructions:

[0125] Make nucleotides (A, T, C, G) flow

[0126] Run PCR (Temperature 1, Period 1, Temperature 2, Period 2, Temperature 3, Period 3, Cycles)

[0127] Washing assay

[0128] Load beads (z) into wells (x, y)

[0129] Read the result of GSIC (x, y)

[0130] Synthetic primer (ATCC GGTTA)

[0131] Heating hole (54℃)

[0132] filter_data(filter type, parameters)

[0133] ADC (parameters)

[0134] Application program can include or have the access to the database of the biological numerical value (such as genotype, antibody, SNP, microorganism species) that can be compared with the biological data output from mensuration.Each data entry in the database can have one or more relevant diagnosis, medical treatment or drug parameters.Each data entry in the database can be associated with one group of instructions.Application program receives the data from mensuration and compares it with the database to determine species or variant, finds the medicine or therapy of recommendation, or runs described one group of instructions.This accelerates the process from receiving data to providing meaningful output information.For example, in the DNA sequencing determination that data output is the base flow on template nucleic acid therein, application program queries each base or the partial sequence of base in the database.Some of these will not return the association that application program does not perform any operation.Some of these bases will differentiate the species of microorganism in sample and database can contain instruction to continue sequencing thereby determine concrete variant.

[0135] Figure 7 Schematically illustrates the functional and structural features of another exemplary instrument, which includes an application layer, an operating system, and a set of primitives and resources. The figure illustrates, in particular, that the operating system makes a set of assay processes, including amplification, sample preparation, etc., available to one or more application programs.

[0136] Figure 8 A flow chart illustrating a process for analyzing nucleic acids or fragments thereof is provided. At various points in the process, output information detected is provided to inform workflow decisions, and workflow control decisions are received. Figure 9 A cartridge 1 suitable for performing the assay is schematically illustrated. The cartridge can be configured for use with an instrument that provides liquid to the cartridge via a fluid interface 2 and exchanges data with the cartridge via a data interface 3. The cartridge includes a controller 4 that controls a liquid delivery system 5 that is fluidically connected to the fluid interface 2. The controller also controls a set of reaction chambers 6, each of which includes a set of sensors 7.

Claims

1. A device for analyzing nucleic acids, comprising: A cartridge (1) having a plurality of reaction chambers (6) each having a sensor (7) configured or configurable to detect amplification, sequencing, and hybridization in the reaction chambers (6); a fluid delivery system (5) for controlling the flow of fluids, including fluids containing the nucleic acid or fragments thereof, to the reaction chamber (6), thereby facilitating a multimodal workflow comprising amplification, sequencing, and hybridization of the nucleic acid and / or fragments thereof, wherein the hybridization is directed to a hybridization probe; and A controller (4) is connected to the sensor (7) and to the fluid delivery system (5), for obtaining the detection result and for controlling the fluid delivery system (5) to dynamically guide the workflow for analysis, characterized in that the device is configured to stop any further reaction from occurring in the absence of amplification and to turn off the downstream sequencing mode of the reaction chamber when hybridization cannot be detected.

2. The apparatus of claim 1 , wherein the apparatus is further configured to make decisions during sequencing whereby each base call is used to determine whether to continue sequencing for further calls, stop sequencing, or flow through a particular order of nucleotides.

3. A device according to claim 1, wherein at least a part of the fluid delivery system (5) is arranged on the box (1), and the box (1) comprises a fluid inlet and an outlet for connecting the fluid delivery system (5) or the part of the fluid delivery system (5) to an external fluid source and a fluid discharge system.

4. The device according to claim 1, wherein the sensors (7) are all of the same sensor type.

5. The device according to claim 4, wherein the sensor (7) is a ChemFET, more preferably an ISFET.

6. The device according to any one of claims 1 to 4, wherein at least some of the reaction chambers (6) and associated sensors (7) are configured to implement an assay panel group, the reaction chambers (6) and sensors (7) of a given panel of the panel group being configured to implement and detect the same reaction type.

7. The apparatus according to claim 6, wherein the controller (4) is configured to operate the assay panels of the panel set in parallel.

8. An apparatus as claimed in claim 6 or 7 and comprising a plurality of panel groups.

9. The device according to any one of claims 1 to 8, wherein the fluid delivery system (5) comprises one or more fluid flow control gates under the control of the controller (4) for directing the fluid to the desired reaction chamber (6).

10. The device according to any one of claims 1 to 9, wherein each reaction chamber (6) comprises a plurality of identical sensors (7).

11. A method for analyzing nucleic acid, comprising: Providing an apparatus having a plurality of reaction chambers each having a sensor; controlling and / or directing fluid delivery, including fluid containing the nucleic acid or fragments of the nucleic acid, to the reaction chamber to facilitate a multimodal workflow including amplification, sequencing, and hybridization of the nucleic acid and / or fragments thereof, the hybridization being to a hybridization probe; detecting any amplification, sequencing, and hybridization in the reaction chamber using the sensor; and applying the detection results in the step of controlling and / or directing fluid delivery to dynamically direct the workflow, and Characterized in that the control includes stopping any further reaction in the absence of amplification and shutting down the downstream sequencing mode in the absence of detectable hybridization.

12. The method of claim 11, wherein the step of controlling and / or directing fluid delivery comprises providing instructions to a user to perform a manual fluid delivery control step.

13. The method of claim 11 or 12, wherein the sensors are all of the same sensor type.

14. The method according to any one of claims 11 to 13, wherein the step of controlling and / or directing fluid delivery comprises controlling and / or directing fluid delivery so as to cause two or more of amplification, sequencing, and hybridization to be performed sequentially in the same reaction chamber.

15. The method of any one of claims 11 to 14, wherein the step of using the sensor comprises detecting amplification by detecting the production of amplicons above a threshold level.

16. The method according to any one of claims 11 to 15, wherein the step of using the sensor comprises detecting the sequence of nucleic acid fragments.

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