Not Applicable.
As multiplexed analytical systems continue to be miniaturized in size, expanded in scale, and increased in power, the need to develop improved systems capable of such functionality becomes more important. Furthermore, many analytical techniques are initially available only at high cost, and they can only be performed in controlled, laboratory settings by highly-trained laboratory technicians. For example, nucleic acid sequencing was originally possible only in research laboratories, using techniques and equipment that were expensive and complicated to perform. Advances in nucleic acid sequencing technologies have brought down the cost per unit sequenced and have therefore greatly expanded the availability of sequence data, but the sequencing reactions must still typically be performed in sophisticated laboratories with expensive equipment by highly trained individuals.
Many optical analytical techniques likewise rely on sophisticated equipment and expertise, and they are therefore also expensive and complicated to scale up. For example, conventional optical systems employ complex optical trains that direct, focus, filter, split, separate, and detect light to and from the sample materials. Such systems typically employ an assortment of different optical elements to direct, modify, and otherwise manipulate light entering and leaving a reaction site. Such systems are typically complex and costly and tend to have significant space requirements. For example, typical systems employ mirrors and prisms in directing light from its source to a desired destination. Additionally, such systems may include light-splitting optics such as beam-splitting prisms or diffraction gratings to generate two or more beams from a single original beam.
Integrated optical systems for nucleic acid sequencing have recently become available that enable large-scale, even genomic-scale, nucleic acid sequencing to be performed with standardized and commercially available laboratory equipment. See, for example, U.S. Patent Publication Nos. 2012/0014837, 2012/0021525, 2012/0019828, and 2016/0061740. Such equipment continues to remain relatively large and expensive, however, thus limiting the extent of adoption of the technology.
There is, therefore, a continuing need to decrease the size and cost of integrated devices and systems for nucleic acid sequencing, and thus to increase the availability of this technology on a wider scale and at lower cost.
The present disclosure addresses these and other needs by providing in one aspect integrated cartridges for nucleic acid sequencing, the cartridges comprising:
a multiplexed optical chip comprising;
In some embodiments, the cartridge further comprises a connector element in electronic contact with the optical detector, optionally wherein the protective enclosure comprises at least one aperture for access to the connector element. In some embodiments, the cartridge further comprises a thermal conductor in thermal contact with the multiplexed optical chip, optionally wherein the protective enclosure comprises at least one aperture for access to the thermal conductor. In some embodiments, the cartridge further comprises a flow cell in fluidic connection with the plurality of reaction regions on the multiplexed optical chip, optionally wherein the protective enclosure comprises at least one aperture for access to the flow cell. In any of these embodiments, the at least one aperture can be covered by a retractable protective shield.
In some of the above cartridge embodiments comprising a connector element, the cartridge further comprises a non-volatile, rewritable memory or a user-observable connection indicator in electronic contact with the connector element, optionally wherein the user-observable connection indicator comprises a light-emitting diode.
In some embodiments, the nucleic acid sequencing cartridge further comprises an electrostatic discharge protection element, optionally wherein the electrostatic discharge protection element comprises an electrostatic discharge dissipative plastic, a metallization, or a low-resistance foam. In some embodiments, the protective enclosure comprises an ejection pin on an external surface of the protective enclosure, wherein the ejection pin is configured for reversible association with an optical sequencing system. In some embodiments, the multiplexed optical chip is attached to a printed circuit board.
In some of the above cartridge embodiments comprising a flow cell, the flow cell comprises at least two fluidic ports, optionally wherein the flow cell comprises at least one input fluidic port and at least one output fluidic port, or at least four fluidic ports, optionally wherein the flow cell comprises at least two input fluidic ports and at least two output fluidic ports. In specific embodiments, the flow cell further comprises at least one trunk line, wherein the at least one trunk line is in fluidic connection with at least one input fluidic port, and wherein the at least one trunk line is configured to direct air bubbles away from the plurality of reaction regions.
In other specific embodiments, the at least two fluidic ports of the flow cell are independently controllable by fluidic valves, optionally wherein the flow cell further comprises at least one trunk line, wherein the at least one trunk line is in fluidic connection with at least one input fluidic port, and wherein the at least one trunk line is configured to direct air bubbles away from the plurality of reaction regions.
In some cartridge embodiments comprising a flow cell, the flow cell further comprises a physical alignment element, optionally wherein the physical alignment element comprises a hole, a slot, or a hole and a slot.
In some cartridge embodiments comprising a flow cell, the flow cell is fabricated from a material that is at least partly transparent to UV radiation, and optionally comprises a bottom surface in contact with the multiplexed chip, wherein the bottom surface is at least partially covered by a material that is at least partly opaque to visible light. In some embodiments, the flow cell is attached to the multiplexed optical chip by a UV-cure adhesive. In specific embodiments, the transparent material in the above flow cells can be a UV-transparent plastic, such as an acrylonitrile butadiene styrene plastic. In other specific embodiments, the material that is at least partly opaque to visible light is a paint, a laser engraved or embossed material, or an opaque plastic material.
In another aspect, the disclosure provides packaged nucleic acid sequencing devices comprising:
a multiplexed optical chip comprising;
In embodiments, the printed circuit board of the packaged nucleic acid sequencing devices comprise a connector element in electronic contact with the optical detector. In specific embodiments, the connector element is an edge connector, optionally further comprising a non-volatile rewritable memory or a user-observable connection indicator in electronic contact with the connector element.
In some embodiments, packaged nucleic acid sequencing device further comprises an electrostatic discharge protection element, a thermal conductor in thermal contact with the multiplexed optical chip, a flow cell in fluidic contact with the plurality of reaction regions on the multiplexed optical chip, or a combination of these features. More specifically, the electrostatic discharge protection element, the thermal conductor in thermal contact with the multiplexed optical chip, and the flow cell in fluidic contact with the plurality of reaction regions on the multiplexed optical chip can be any of the corresponding features described in the above nucleic acid sequencing cartridges.
In yet another aspect are provided packaged nucleic acid sequencing devices comprising:
a multiplexed optical chip comprising;
a flow cell in fluidic connection with the plurality of reaction regions on the multiplexed optical chip.
In specific embodiments, the flow cell in fluidic contact with the plurality of reaction regions on the multiplexed optical chip can be any of the corresponding features described in the above nucleic acid sequencing cartridges or packaged nucleic acid sequencing devices.
In still yet another aspect are provided systems for optical analysis comprising:
an optical source;
a nucleic acid sequencing cartridge comprising:
In some embodiments, the systems comprise the nucleic acid sequencing cartridges described above, the packaged nucleic acid sequencing devices described above, the flow cells described above, or a combination of these more specific components.
In some embodiments, the system further comprises a beam dump. In some embodiments, the system further comprises a fluidic clamp, optionally wherein the fluidic clamp comprises a plurality of clamping ports in fluidic connection with the flow cell, wherein the system further comprises a syringe pump in fluidic connection with the fluidic clamp, wherein the fluidic clamp is driven by a cam mechanism, or wherein the fluidic clamp comprises a beam dump.
In some system embodiments, the optical source is replaceable by a user.
In other system embodiments, the optical source is configured to emit an optical excitation beam, and the optical excitation beam is coupled to the optical coupler. More specifically, in some of these embodiments, the system is configured to move either the multiplexed optical chip or the optical excitation beam to maximize an optical alignment signal, the system does not include an alignment camera, or the multiplexed optical chip comprises at least one alignment feature at a defined location on the multiplexed optical chip.
In some embodiments, the system further comprises a cooling system in thermal contact with the multiplexed optical chip, optionally wherein the cooling system comprises an air blower or wherein the cooling system comprises a thermoelectric cooler.
In other system embodiments, the multiplexed optical chip comprises at least 2, at least 5, at least 10, at least 50, at least 100, at least 500, at least 1,000, at least 5,000, at least 10,000, or at least 50,000 optical waveguides, the multiplexed optical chip comprises no more than 100,000, no more than 50,000, no more than 10,000, no more than 5,000, no more than 1,000, no more than 500, or no more than 100 optical waveguides, or the multiplexed optical chip comprises from 1 to 100,000, from 100 to 10,000, or from 500 to 5,000 optical waveguides.
In some embodiments, the system further comprises a computer that receives at least one electronic signal from the optical detector and analyzes the at least one electronic signal, optionally wherein the analysis comprises obtaining nucleic acid sequencing information.
In some system embodiments, the optical source has a wavelength of excitation from about 450 nm to about 700 nm or from about 500 nm to about 650 nm, the multiplexed optical chip is fabricated on a silicon chip, the optical detector comprises a CMOS sensor, the plurality of reaction regions comprises a plurality of nucleic acid samples, the plurality of reaction regions comprises a plurality of nanoscale wells, or the plurality of reaction regions comprises a plurality of zero mode waveguides, in any combination.
An exemplary optical analytical system comprising an optical source and an integrated target waveguide device is illustrated in
The area of interest 150, which in the case of a nucleic acid sequencing device may also be referred to as a “sequencing area” or “sequencing region”, has a plurality of reaction regions 155, for example nanowells or zero mode waveguides (ZMWs). The optical waveguide 140 typically extends underneath the reaction regions 155, thereby illuminating the reaction regions from below by optical coupling with evanescent wave illumination. The reaction regions preferably contain fluorescent reactants, which, when excited by the evanescent wave illumination, emit fluorescent light 190, which can be detected in order to carry out the desired analysis (e.g., nucleic acid sequencing). In some cases, and as shown here, the target device also has an integrated sensor 180, also referred to as an optical detector. The emitted fluorescent light from the reaction regions is optically coupled through the device to be detected at a single pixel or group of pixels 185 within the optical detector. Such integrated target devices for fluorescence analysis are described, for example in U.S. Patent Publication Nos. 2008/0128627, 2012/0085894, 2016/0334334, 2016/0363728, 2016/0273034, 2016/0061740, and 2017/0145498 which are each incorporated herein by reference in their entireties. Target devices that include integrated optical detectors will also typically include electronic outputs 175. For example, the integrated optical detector detects and processes an optical emission signal, and then sends electronic data related to the detected signals out of the device through an electronic output or outputs. The electronic outputs can, for example, be bond pads on a silicon chip, which are typically wire bonded to a chip package, and the chip package will have electronic outputs for passing on the electronic signals from the chip. The electronic signals are typically sent to a computer (not shown), which processes the received signals to perform the desired analysis.
The optical waveguide on the target device can be any suitable waveguide including a fiber, a planar waveguide, or a channel waveguide. Typically channel waveguides are used. The waveguide is preferably a single mode waveguide, but it can be a multi-mode waveguide for some applications.
In
The present disclosure is generally directed to improved devices and systems for performing analytical operations, and particularly optical analysis of chemical, biochemical, and biological reactions for use in chemical, biological, medical, and other research and diagnostic applications. These devices and systems are particularly well suited for application in integrated analytical components, e.g., where multiple functional components of the overall analysis system are co-integrated within a single modular component. However, as will be clear upon reading the following disclosure, a number of aspects of the invention will have broad utility outside of such integrated devices and systems.
In general, the optical analyses that are subjects of the present disclosure seek to gather and detect one or more optical emission signals from a reaction of interest, the appearance or disappearance of which, or localization of which, is indicative of a given chemical or biological reaction and/or the presence or absence of a given substance within a sample material. In some cases, the reactants, their products, or other substance of interest (all of which are referred to as reactants herein) inherently present an optically detectable signal. In other cases, reactants are provided with exogenous labeling groups to facilitate their detection.
As is understood by those of ordinary skill in the art, fluorescently labeled nucleotides are used in a wide variety of different nucleic acid sequencing analyses. For example, in some cases such labels are used to monitor the polymerase-mediated, template-dependent incorporation of nucleotides in a primer extension reaction. In particular, a labeled nucleotide can be introduced to a primer template polymerase complex, and incorporation of the labeled nucleotide into the primer can be detected. If a particular type of nucleotide is incorporated at a given position, it is indicative of the underlying and complementary nucleotide in the sequence of the template molecule. In traditional Sanger sequencing processes, the detection of incorporated labeled nucleotides utilizes a termination reaction, where the labeled nucleotides carry a terminating group that blocks further extension of the primer. By mixing the labeled terminated nucleotides with unlabeled native nucleotides, nested sets of fragments are generated that terminate at different nucleotides. These fragments can then be separated by capillary electrophoresis, or other suitable technique, to distinguish those fragments that differ by a single nucleotide, and the labels for the fragments can be read in order of increasing fragment size to provide the sequence of the fragment (as indicated by the last added, labeled terminated nucleotide). By providing a different fluorescent label on each of the types of nucleotides that are added, the different nucleotides in the sequence can readily be differentiated (see, e.g., U.S. Pat. No. 5,821,058, which is incorporated herein by reference in its entirety for all purposes).
In some sequencing technologies, arrays of primer-template complexes are immobilized on surfaces of substrates such that individual molecules or individual and homogeneous groups of molecules (clonal populations) are spatially discrete from other individual molecules or groups of molecules, respectively. Labeled nucleotides are added in a manner that results in a single nucleotide being added to each individual molecule or group of molecules. Following the addition of the nucleotide, the labeled addition is detected and identified.
In some cases, the sequencing analyses utilize the addition of a single type of nucleotide at a time, followed by a washing step. The labeled nucleotides that are added are then detected, their labels removed, and the process repeated with a different nucleotide type. Sequences of individual template sequences are determined by the order of appearance of the labels at given locations on the substrate.
In other similar cases, the immobilized complexes are contacted with all four types of labeled nucleotides, where each type of nucleotide bears a distinguishable fluorescent label and a terminator group that prevents the addition of more than one nucleotide in a given step. Following the single incorporation in each individual template sequence (or group of template sequences), the unbound nucleotides are washed away, and the immobilized complexes are scanned to identify which nucleotide was added at each location. Repeating the process yields sequence information of each of the template sequences. In other cases, more than four types of labeled nucleotides are utilized.
In particularly elegant approaches, labeled nucleotides are detected during the incorporation process itself, in real time, by individual molecular complexes. Such methods are described, for example, in U.S. Pat. No. 7,056,661, which is incorporated herein by reference in its entirety for all purposes. In these processes, nucleotides are labeled on a terminal phosphate group that is released during the incorporation process, so as to avoid the accumulation of labels on the extension product, and accordingly to avoid any need for label removal processes that can potentially be deleterious to the complexes. Primer/template polymerase complexes are observed during the polymerization process, and nucleotides being added are detected by virtue of their associated labels.
In one particular example, labeled nucleotides can be observed using an optically confined structure, such as a zero mode waveguide (see, e.g., U.S. Pat. No. 6,917,726, which is incorporated herein by reference in its entirety for all purposes) that limits exposure of the excitation radiation to the volume immediately surrounding an individual primer/template polymerase complex. As a result, only labeled nucleotides that are retained by the polymerase during the process of being incorporated are exposed to excitation illumination for a time that is sufficient to generate fluorescence and thus to identify the incorporated nucleotide. Exemplary chips having arrays of nanoscale wells or zero mode waveguides and that are therefore considered suitable for these purposes include substrates having a metal or metal oxide layer on a silica-based layer, with nanoscale wells disposed through the metal or metal oxide layer to or into the silica-based layer (see, e.g., U.S. Pat. Nos. 6,917,726, 7,302,146, 7,907,800, 8,802,600, 8,906,670, 8,993,307, 8,994,946, 9,223,084, 9,372,308, and 9,624,540, which are each incorporated herein by reference in their entireties).
In another approach, the label on the nucleotide is configured to interact with a complementary group on or near the complex, e.g., attached to the polymerase, where the interaction provides a unique signal. For example, a polymerase may be provided with a donor fluorophore that is excited at a first wavelength and emits at a second wavelength, while the nucleotide to be added is labeled with a fluorophore that is excited at the second wavelength, but emits at a third wavelength (see, e.g., U.S. Pat. No. 7,056,661, previously incorporated herein). As a result, when the nucleotide and polymerase are sufficiently proximal to each other to permit energy transfer from the donor fluorophore to the label on the nucleotide, a distinctive signal is produced. Again, in these cases, the various types of nucleotides are provided with distinctive fluorescent labels that permit their identification by the spectroscopic or other optical signature of their labels.
In the various exemplary processes described above, detection of a signal event from a reaction region is indicative that a reaction has occurred. Further, with respect to many of the above processes, identification of the nature of the reaction, e.g., which nucleotide was added in a primer extension reaction at a given time or that is complementary to a given position in a template molecule, is also achieved by distinguishing the spectroscopic characteristics of the signal event.
The optical paths of the analytical systems of the disclosure serve one or more roles of delivering excitation radiation to the reaction region, e.g., to excite fluorescently-labeled molecules that then emit the relevant optical emission signal, conveying the optical signal emitted from the reaction region to the optical detector, and, for multispectral signals, i.e., multiple signals that may be distinguished by their emission spectrum, separating those signals so that they may be differentially detected, e.g., by directing different signals to different optical detectors or different regions on the same optical detector array. The differentially detected signals are then correlated with both the occurrence of the reaction, e.g., a nucleotide was added at a given position, and the determination of the nature of the reaction, e.g., the added nucleotide is identified as a particular nucleotide type, such as adenosine.
In conventional, fully free-space, analytical systems used for nucleic acid sequencing, the optical trains used to deliver excitation light to the reaction regions, and to convey optical signals from the reaction regions to the detector(s) can impart size, complexity, and cost aspects to the overall system that would preferably be reduced. For example, such optical trains may include collections of lenses, dispersion elements, beam splitters, beam expanders, collimators, spatial and spectral filters and dichroics, that are all assembled to deliver targeted and uniform illumination profiles to the different reactions regions. In large-scale systems, these components must be fabricated, assembled, and adjusted to ensure proper alignment, focus, and isolation from other light and vibration sources to optimize the transmission of excitation light to the reaction regions. As the number of addressed reaction regions, or the sensitivity of the system to variations in excitation light intensity is increased, addressing these and other issues becomes more important, and again typically involves the inclusion of additional componentry to the optical train, e.g., alignment and focusing mechanisms, isolation structures, and the like.
With respect to the collection and detection of optical emission signals, conventional systems typically employ optical trains that gather emitted optical signals from the reaction region, e.g., through an objective lens system, transmit the various different signals through one or more filter levels, typically configured from one or more dichroic mirrors that differentially transmit and reflect light of different wavelengths, in order to direct spectrally different optical signals to different detectors or regions on a given detector. These separated optical signals are then detected and used to identify the nature of the reaction that gave rise to such signals. As will be appreciated, the use of such differential direction optics imparts substantial space, size, and cost requirements on the overall system, in the form of multiple detectors, multiple lens and filter systems, and in many cases complex alignment and correlation issues. Many of these difficulties are further accentuated where the optical trains share one or more sub-paths with the excitation illumination, as signal processing will include the further requirement of separating out background excitation illumination from each of the detected signals.
Again, as with the excitation optical train, above, as the sensitivity and multiplex of the system is increased, it increases the issues that must be addressed in these systems, adding to the complexity of an already complex optical system. Further, the greater the number of optical components in the optical train, the greater the risk of introducing unwanted perturbations into that train and the resulting ability to detect signal. For example, optical aberrations in optical elements yield additional difficulties in signal detection, as do optical elements that may inject some level of autofluorescence into the optical train, which then must be distinguished from the signaling events.
In some embodiments, the systems of the instant disclosure further comprise a computer that receives at least one electronic signal from an optical detector, or region of an optical detector, for example the detected signals described above, and analyzes the at least one electronic signal. More specifically, the analysis performed by the computer can comprise obtaining nucleic acid sequencing information from the electronic signal, as would be understood by those of ordinary skill in the art.
The nucleic acid sequencing cartridges, packaged devices, and analytical systems of the instant disclosure typically comprise one or more small-scale integrated analytical devices that optionally also include one or more reaction regions, fluidic components, and excitation illumination paths and optionally excitation illumination sources. Integration of some or all of the above-described components into a single, miniaturized analytical device, also referred to as a multiplexed optical chip, addresses many of the problems facing larger, non-integrated analytical systems, such as size, cost, weight, inefficiencies associated with long path or free space optics, and the like. For example, highly multiplexed analytical systems comprising integrated waveguides for the illumination of nanoscale samples are described in U.S. Patent Publication Nos. 2008/0128627, 2012/0085894, 2016/0334334, 2016/0363728, 2016/0273034, 2016/0061740, and 2017/0145498, which are each incorporated herein by reference in their entireties. Additional nanoscale illumination systems for highly multiplexed analysis are described in U.S. Patent Publication Nos. 2014/0199016 and 2014/0287964, which are each incorporated herein by reference in their entireties.
Other examples of such integrated analytical systems are described, for example, in U.S. Patent Application Publication Nos. 2012/0014837, 2012/0019828, and 2012/0021525, which are each incorporated herein by reference in their entireties. By integrating the detection elements with the reaction regions, either directly or as a coupled part, the need for many of the various components required for free space optics systems, such as much of the conveying optics, lenses, mirrors, and the like, can be eliminated. Other optical components, such as various alignment functionalities, can also in many cases be eliminated, as alignment is achieved through the direct integration of the detection elements with the reaction regions. The cartridges, packaged devices, and systems of the present disclosure further improve the benefits afforded by such multiplexed devices by simplifying, to a greater extent, the optical, electronic, fluidic, mechanical, and thermal components of the analytical devices, thus further reducing the cost and complexity of such devices, and further improving the available signal in the process.
In an exemplary embodiment, the multiplexed optical chips of the instant cartridges, packaged devices, and systems include an array of analytical devices formed as a single integrated device that is typically configured for single use as a consumable device. In various embodiments, the integrated device includes other components including, but not limited to local fluidics, electronic connections, a power source, illumination elements, a detector, logic, and a processing circuit. Each analytical device in the array is preferably configured for performing an analytical operation, as described above.
While the components of each integrated device and the configuration of the devices in the system can vary, each analytical device within the system can comprise, at least in part, the general structure shown as a block diagram in
In various respects, “analytical device” or “integrated analytical device” refers to a reaction cell and associated components that are functionally connected. In various respects, “analytical system” refers to the larger system including the analytical device and other instruments for performing an analysis operation. For example, in some cases, the nucleic acid sequencing cartridges and packaged devices of the disclosure are part of an analytical instrument or analytical system. The nucleic acid sequencing cartridge or packaged device can be removably coupled into the instrument. Liquid samples and/or reagents can be brought into contact with the sequencing cartridge or packaged device before or after the sequencing cartridge or packaged device is coupled with the system. The system can provide electronic signals and/or illumination light to the sequencing cartridge or packaged device, and can receive electronic signals from the detectors or other electronic components in the sequencing cartridge or packaged device. The system can also provide mechanical support for and/or thermal exchange with the sequencing cartridge or packaged device. The instrument or system can have computers to manipulate, store, and analyze the data from the sequencing cartridge or packaged device. For example, the instrument can have the capability of identifying the order of added nucleotide analogs in a nucleic acid sequencing reaction. The identification can be carried out, for example, as described in U.S. Pat. No. 8,182,993, which is incorporated herein by reference for all purposes.
In some cases, one or more reactants involved in the reaction of interest can be immobilized, entrained or otherwise localized within a given reaction cell. A wide variety of techniques are available for localization and/or immobilization of reactants, including surface immobilization through covalent or non-covalent attachment, bead or particle based immobilization, followed by localization of the bead or particle, entrainment in a matrix at a given location, and the like. Reaction cells can include ensembles of molecules, such as solutions, or patches of molecules, or they can include individual molecular reaction complexes, e.g., one molecule of each molecule involved in the reaction of interest as a complex. Similarly, the sequencing cartridges and packaged devices of the disclosure can include individual reaction cells or can comprise collections, arrays, or other groupings of reaction cells in an integrated structure, e.g., a multiwall or multi-cell plate, chip, substrate, or system. Some examples of such arrayed reaction cells include nucleic acid array chips, e.g., GeneChip® arrays (Affymetrix, Inc.), zero mode waveguide arrays (as described elsewhere herein), microwell and nanowell plates, multichannel microfluidic devices, e.g., LabChip® devices (Caliper Life Sciences, Inc.), and any of a variety of other reaction cells. In various respects, the “reaction cell”, sequencing layer, and zero mode waveguides are similar to those described in U.S. Pat. No. 7,486,865, the entire contents of which is incorporated herein by reference for all purposes. In some cases, these arrayed devices can share optical components within a single integrated overall device, e.g., a single waveguide layer to deliver excitation light to each reaction region. Approaches to illuminating analytical devices with waveguides are provided in U.S. Pat. Nos. 8,207,509 and 8,274,040, which are each incorporated herein by reference for all purposes.
Although an analytical system may include an array of analytical devices having a single waveguide layer and reaction cell layer, it can be appreciated that a wide variety of layer compositions can be employed in the waveguide array substrate and cladding/reaction cell layer while still achieving the goals of the device (see, e.g., U.S. Pat. No. 7,820,983, incorporated herein by reference for all purposes).
The multiplexed optical chips of the instant cartridges, packaged devices, and systems typically include a plurality of analytical devices 200 as illustrated in
In various embodiments, the reaction cell 202 and detector element 220 are provided along with one or more optical elements in an integrated device structure. By integrating these elements into a single device architecture, the efficiency of the optical coupling between the reaction cell and the detector can be improved. As used herein, the term integrated, when referring to different components of an analytical device typically refers to two or more components that are coupled to each other so as to be immobile relative to each other. As such, integrated components can be irreversibly or permanently integrated, meaning that separation would damage or destroy one or both elements, or they can be removably integrated, where one component can be detached from the other component, provided that when they are integrated, they are maintained substantially immobile relative to one another. In some cases, the components are integrated together, for example as a single fabricated device, such as in a single silicon chip. In some cases, the detector portion is part of a separate instrument, and the reaction cell component is part of a detachable device, such as a detachable chip. In the case where the reaction cell component is in a chip separate from the detector component, optical element components for directing the optical emission signal from the reaction cell to the detector can be in either the reaction cell component, in the detector component, or a combination in which some components are in the reaction cell component and others are in the detector component.
In conventional optical analysis systems, discrete reaction vessels are typically placed into optical instruments that utilize only free-space optics to convey the optical signals to and from the reaction vessel and to the detector. These free space optics tend to include higher mass and volume components, and have free space interfaces that contribute to a number of weaknesses for such systems. For example, such systems have a propensity for greater losses of light given the introduction of unwanted leakage paths from these higher mass components. They also typically introduce higher levels of auto-fluorescence. All of these inherent weaknesses reduce the signal-to-noise ratio (SNR) of the system and reduce its overall sensitivity, which, in turn can impact the speed, accuracy, and throughput of the system. Additionally, in multiplexed applications, signals from multiple reaction regions (i.e., multiple reaction cells, or multiple reaction locations within individual cells), are typically passed through a common optical train, or common portions of an optical train, using the full volume of the optical elements in that train to be imaged onto the detector plane. As a result, the presence of optical aberrations in these optical components, such as diffraction, scattering, astigmatism, and coma, degrade the signal in both amplitude and across the field of view, resulting in greater noise contributions and cross talk among detected signals.
In some cases, the reaction region of the instant multiplexed optical chips comprises a nanoscale well, for example, a nanoscale well having no linear dimension of greater than 500 nm A nanoscale well of the optical chips of the disclosure can, for example, be cylindrical with a base diameter between about 50 nm and 200 nm. The depth of the well can, for example, be from about 50 nm to about 400 nm In some cases, the reaction regions can comprise zero mode waveguides (ZMWs). Zero mode waveguides are described, for example in U.S. Pat. Nos. 7,170,050, 7,486,865, and 8,501,406 which are each incorporated herein by reference in their entireties.
Such devices have sought to take advantage of the proximity of the reaction region or vessel in which signal producing reactions are occurring, to the detector or detector element(s) that sense those signals, in order to take advantage of benefits presented by that proximity. As alluded to above, such benefits include the reduction of size, weight, and complexity of the optical train, and as a result, increase the potential multiplex of a system, e.g., the number of different reaction regions that can be integrated and detected in a single cartridge, packaged device, or system. Additionally, such proximity potentially provides benefits of reduced losses during signal transmission, reduced signal cross-talk from neighboring reaction regions, and reduced costs of overall systems that utilize such integrated devices, as compared to systems that utilize large free space optics and multiple cameras in signal collection and detection.
In the multiplexed optical chips of the present disclosure, there are a number of design criteria that can benefit from optimization. For example, in these optical chips, an over-arching goal is in the minimization of intervening optical elements that could interfere with the efficient conveyance of optical emission signals from the reaction region to the detector, as well as contribute to increased costs and space requirements for the device, by increasing the complexity of the optical elements between the reaction regions and the sensors.
Additionally, and with added importance for single molecule detection systems, it is also important to maximize the amount of optical emission signal that is detected for any given reaction event. In particular, in optical detection of individual molecular events, a relatively small number of photons corresponding to the event of interest are typically relied on in the measurements. While high quantum yield labeling groups, such as fluorescent dyes, can improve detectability, such systems still operate at the lower end of detectability of optical systems. Fluorescent dyes finding utility in the analytical reactions performed using the instant systems are well known. Any suitable fluorescent dye can be used, for example, as described in PCT International Publication No. WO2013/173844A1 and U.S. Patent Application Publication Nos. 2009/0208957A1, 2010/0255488A1, 2012/0052506A1, 2012/0058469A1, 2012/0058473A1, 2012/0058482A1, and 2012/0077189A1.
In the context of the cartridges, packaged devices, and systems of the present disclosure, the size and complexity of the optical pathways poses a greater difficulty, as there is less available space in which to accomplish the goals of separation of excitation and signal, or separation of one signal from the next. Accordingly, the multiplexed optical chips of the instant cartridges, packaged devices, and systems take advantage of simplified optical paths associated with the analyses being carried out, in order to optimize those analyses for the integrated nature of those optical chips.
Excitation illumination is delivered to the reaction region from an excitation light source (not shown) that may be separate from or may be integrated into the optical device. As shown, an optical waveguide (or waveguide layer) 306 is used to convey excitation light (shown by arrows) to the vicinity of reaction region 302, where an evanescent field emanating from the waveguide 306 illuminates reactants within the reaction region 302. Use of optical waveguides to illuminate reaction regions is described in e.g., U.S. Pat. Nos. 7,820,983, 8,207,509, and 8,274,040, which are each incorporated herein by reference for all purposes.
The integrated device 300 optionally includes light channeling components 308 to efficiently direct emitted light from the reaction regions to a detector layer 312 disposed beneath the reaction region. The detector layer will typically comprise multiple detector elements, for example the four illustrated detector elements 312a-d that are optically coupled to a given reaction region 302. For DNA sequencing applications, it is often desirable to monitor four different signals in real time, each signal corresponding to one of the nucleobases. The different signals can be distinguishable, for example, by wavelength, intensity, or any other suitable distinction, or combination of distinctions. Although illustrated as a linear arrangement of pixels 312a-d, it will be appreciated that the detector elements can be arranged in a grid, n by n square, annular array, or any other convenient orientation or arrangement. In some cases, each of the detector elements or channels will have a single pixel per reaction region, wherein the different analytical signals may be distinguishable by, for example, their different intensities. In some cases, the detector elements will each comprise multiple pixels, for example two, three, four, or even more pixels per reaction region. The detector elements are connected electronically to conductors that extend out of the chip for providing electronic signals to the detector elements and for sending out signals from the detector elements, for example to an attached processor. In some embodiments, the detector layer is a CMOS wafer or the like, i.e., a wafer made up of CMOS sensors or CCD arrays. See, for example, CMOS Imagers From Phototransduction to Image Processing (2004) Yadid-Pecht and Etienne-Cummings, eds.; Springer; CMOS/CCD Sensors and Camera Systems (2007) Holst and Lomheim; SPIE Press.
Emitted signals from the reaction region 302 that impinge on these detector elements are then detected and recorded. As illustrated in the integrated device of
In some cases, optical elements are provided to selectively direct light from given sets of wavelengths to given detector elements. Typically, no specific light re-direction is used, such that the light reaching each region of the filter layer is substantially the same.
The detector layer is operably coupled to an appropriate circuitry, typically integrated into the substrate, for providing a signal response to a processor that is optionally integrated within the same device structure or is separate from but electronically coupled to the detector layer and associated circuitry. Examples of the types of circuitry useful in such devices are described in U.S. Patent Application Publication No. 2012/0019828, previously incorporated by reference herein.
The multiplexed optical chips of the instant disclosure, which may also be referred to herein as target waveguide devices, target devices, or integrated analytical devices, typically have at least one optical coupler and an integrated waveguide that is optically coupled to the optical coupler and that delivers an input optical signal to the plurality of reaction regions. In some embodiments, the optical coupler of the instant devices is a low numerical aperture coupler. In some embodiments, the optical coupler is a diffraction grating coupler. In specific embodiments, the optical coupler is a diffraction grating coupler with low numerical aperture. In some cases, an optical source is directed onto a single coupler, while in other cases, the optical source is directed onto multiple couplers, for example from 2 to 16 couplers. In some cases, each coupler receives substantially the same power. In some cases, different power levels are directed to different couplers on the target device. While this description may refer to “the coupler” on the device, it is understood that in some cases there can be a single coupler, and that in other cases, there will be a plurality of couplers on a given device. Target waveguide devices having suitable couplers are described, for example, in U.S. Patent Application Publication No. 2016/0363728, which is incorporated herein by reference in its entirety.
Grating couplers and their use in coupling light, typically light from optical fibers, to waveguide devices are known in the art. For example, U.S. Pat. No. 3,674,335 discloses reflection and transmission grating couplers suitable for routing light into a thin film waveguide. In addition, U.S. Pat. No. 7,245,803 discloses improved grating couplers comprising a plurality of elongate scattering elements. The couplers preferably have a flared structure with a narrow end and a wide end. The structures are said to provide enhanced efficiency in coupling optical signals in and out of planar waveguide structures. U.S. Pat. No. 7,194,166 discloses waveguide grating couplers suitable for coupling wavelength division multiplexed light to and from single mode and multimode optical fibers. The disclosed devices include a group of waveguide grating couplers disposed on a surface that are all illuminated by a spot of light from the fiber. At least one grating coupler within the group of couplers is tuned to each channel in the light beam, and the group of couplers thus demultiplexes the channels propagating in the fiber. Additional examples of grating couplers are disclosed in U.S. Pat. No. 7,792,402 and PCT International Publication Nos. WO 2011/126718 and WO 2013/037900. A combination of prism coupling and grating coupling into an integrated waveguide device is disclosed in U.S. Pat. No. 7,058,261. In the multiplexed optical chips of the instant cartridges, packaged devices, and systems, optical energy can be provided from fibers, lenses, prisms, mirrors, or any other suitable optical source.
In the multiplexed optical chips of the instant cartridges and packaged devices, there can be a significant distance between the coupler and the area of interest, e.g., the reaction regions, as described above. The distance that the light travels in the waveguide from coupler to an area of interest can be, for example, several centimeters, for example from 1 cm to 10 cm. The distance referred to herein is the distance the light travels within the waveguide, e.g. the routing distance of the light through the waveguide or waveguides. Typically, where light is routed from a coupler over relatively long distances to an area of interest, a single waveguide is used to route the light from the coupler to a region close to the area of interest, where splitting of the routing waveguide into multiple waveguides can occur. Where multiple waveguide branches are desired within the area of interest, the splitting from a routing waveguide to waveguide branches in the area of interest is typically carried out near the area of interest rather than near the coupler, although in some embodiments, it can be advantageous for the splitting to occur nearer to the coupler, in particular where link efficiency variation is a problem, for example as described in U.S. Patent Application Publication No. 2016/0216538. One routing waveguide per coupler is typically the most efficient approach for routing over relatively long distances. Using one routing waveguide involves fewer elements and typically uses less space on the device than when multiple routing waveguides per coupler are used.
As just mentioned, the multiplexed optical chips of the instant cartridges, packaged devices, and systems advantageously comprise a plurality of optical waveguides, the optical waveguides configured to receive the optical excitation beam from the at least one optical coupler. For example, a multiplexed optical chip can comprise at least 2, at least 5, at least 10, at least 50, at least 100, at least 500, at least 1,000, at least 5,000, at least 10,000, or at least 50,000 optical waveguides. In some embodiments, the chip can comprise no more than 100,000, no more than 50,000, no more than 10,000, no more than 5,000, no more than 1,000, no more than 500, or no more than 100 optical waveguides. In other embodiments, the chip can comprise from 1 to 100,000, from 100 to 10,000, or from 500 to 5,000 optical waveguides.
In some embodiments, the multiplexed optical chip of the disclosed cartridges, packaged devices, and systems comprises at least one optical splitter, wherein the at least one optical splitter comprises an optical input and a plurality of optical outputs, and wherein the optical input of the at least one optical splitter is configured to receive the optical excitation beam from the optical coupler. Such devices also typically comprise a plurality of optical waveguides, the optical waveguides configured to receive the optical excitation beam from the plurality of optical outputs of the at least one optical splitter.
In specific embodiments, the multiplexed optical chip of the instant cartridges, packaged devices, and systems comprises no more than one optical coupler for providing illumination light to reaction regions. In other specific embodiments, the at least one optical splitter comprises 2 to 512 optical outputs.
In addition to the number of waveguides, the number of analytical regions per waveguide can be varied in order to obtain the desired level of multiplexing and performance. For example, the number of analytical regions per waveguide, e.g. nanoscale wells, can be, for example, from 1 to 100,000 analytical regions, from 100 to 10,000 analytical regions, or from 500 to 5,000 analytical regions on each waveguide of the chip. Those of skill in the art will understand how to set these numbers in order to obtain the desired performance and level of multiplex.
Integrated chip devices for use in nucleic acid sequencing, for example the integrated optical chips described in the previous section, are traditionally bonded to ceramic substrates. Although such packaging provides a rigid and highly stable platform for the integrated device, it can be expensive to produce and inflexible, particularly where the optical chip is part of a consumer product, such as a table-top nucleic acid sequencing system. In such systems, the integrated chip is ideally designed to be readily and reliably removable and replaceable by an end user. For example, the sockets typically used in the computer chip industry for connection of integrated circuits to computer boards are not generally designed to allow rapid and convenient exchange of chips on a circuit board. Integrated chip devices are also typically quite small, which makes them relatively difficult to handle by an end user. The use of such chips in larger analytical systems, such as systems for nucleic acid sequencing, thus typically requires that the system includes a robotic handling system, or the like, which greatly increases cost and complexity of the systems.
The instant disclosure addresses these issues by providing, in some aspects, packaged nucleic acid sequencing devices comprising a multiplexed optical chip, for example any of the integrated waveguide devices described above, wherein the multiplexed optical chip is attached to a printed circuit board (PCB).
Suitable PCBs for use in the instant packaged nucleic acid sequencing devices are well known in the art. PCBs typically provide mechanical support for an attached chip device or devices. They also typically provide one or more electronic connections for the attached devices using, for example, conductive tracks, pads and/or other features etched from one or more sheet layers of copper laminated onto and/or between sheet layers of a non-conductive substrate. The individual chip devices, and any other components used in the packaged device, are generally soldered or wire bonded to the PCB to provide both an electronic connection and a solid mechanical site of attachment. In some embodiments, however, the optical chip is attached to the PCB using a silver-doped epoxy or other suitable method, for example, any “die attach” process for mechanical attachment of the chip to the PCB, as would be understood by those of ordinary skill in the art.
In the packaged nucleic acid sequencing devices of the instant disclosure, the multiplexed optical chip, including an associated optical detector, is preferably attached to a standard printed circuit board assembly that preferably also comprises an electronically-connected card-edge connector to facilitate the reversible connection of the packaged nucleic acid sequencing device with an analytical system. Analytical systems suitable for use with the packaged nucleic acid sequencing devices, which preferably also comprise an optical source and electronic controls, will be further described below. The printed circuit board assembly additionally optionally contains a non-volatile rewritable memory, for example an electrically erasable programmable read-only memory (EEPROM), or other comparable component, to store unique identifiers associated with the various components of the packaged device, including, for example, serial numbers, usage information, laser-to-chip alignment data, and the like. The printed circuit board assembly can likewise also optionally contain an LED, or other optical, audio, or tactile signal, to give an end user rapid feedback that an electronic connection between the cartridge and the analytical system has been formed.
The instant packaged devices also preferably comprise a rigid protective cartridge that encloses the multiplexed optical chip and the attached printed circuit board. Cartridge enclosures for electronic microcircuits and other types of electronic devices have been disclosed previously, in particular, in the video game industry (see, e.g., U.S. Pat. Nos. 4,095,791, 4,149,027, and 4,763,300, which are each incorporated herein by reference in their entireties). Such cartridge enclosures can advantageously protect the enclosed electronic and other sensitive components from electric discharge, in particular, where the cartridge will be handled by an end user. More details regarding suitable features to protect against electrostatic discharge are described below. Cartridge-type enclosures also provide an ergonomic gripping surface, also referred to as a finger grip, where the user can handle the cartridge without causing damage to mechanically or electronically fragile internal components. The enclosures can further provide an electronic connector, for example a card-edge connector, where the electronic components of the device, in particular the outputs from the CMOS sensor, can be reliably and reversibly connected to the electronic components of an analytical system. Cartridge-type enclosures can also provide retractable covers over apertures in the cartridge enclosure to reversibly expose electronic, optical, fluidic, and thermal connectors, while also protecting those connectors from physical damage or exposure prior to insertion of the cartridge into an analytical system. In some embodiments, the cartridges can include an inexpensive foil covering over one or more of the connection ports that can be removed by the end user prior to use. The foil can protect the optics and fluidics ports from dust and other types of contamination.
The instant inventors have designed cartridge enclosures for the above-described multiplexed optical nucleic acid sequencing chips that provide all of the above advantages. Various views of an exemplary nucleic acid sequencing cartridge comprising such a protective enclosure are shown in
As just described, the nucleic acid sequencing cartridges of the instant disclosure preferably comprise a flow cell in fluidic connection with the plurality of reaction regions on the multiplexed optical chip. More specifically, the flow cell, which is preferably bonded to the optical chip, enables reagent solutions to be provided to the reaction regions in a controlled manner. The flow cell comprises at least one, but preferably a plurality of, input and output ports that are ducted to fluid ports on top of the cartridge, such that liquid reagents can be introduced into the reaction regions of the multiplexed optical chip from outside the cartridge and optionally even from outside the analytical system. In one embodiment, the flow cell of the cartridge includes an additional port into which an end user could pipette a sample, thus decreasing dead volume and minimizing the possibility of sample cross-contamination within an instrument.
In some embodiments, the instant nucleic acid sequencing cartridges comprise features to minimize and/or protect the components from electrostatic discharge (ESD), which can arise from the handling of an electronic device, such as a nucleic acid sequencing cartridge comprising a multiplexed optical chip, by an end user. ESD can be controlled in a variety of ways, as is understood in the art. For example, the chip can be enclosed within an ESD-dissipative plastic. Such enclosures are well known in the art of video game cartridge manufacture. Alternatively, the inside of a cartridge surrounding the packaged device can be metallized, thus creating a Faraday cage or shield to protect the enclosed components. In yet another alternative, all of the cartridge pins can be shorted together via a low-resistance foam that is removable upon insertion of the cartridge into the analytical system.
It is understood that the nucleic acid sequencing cartridges of the instant disclosure will also include an optical coupling interface to inject optical energy into the waveguides of the multiplexed optical chip. An exemplary optical port 460 is illustrated in the device of
The instant nucleic acid sequencing cartridges are preferably designed so that any excitation light not launched into the waveguides of the multiplexed optical chip is efficiently captured by a beam dump associated with the analytical instrument or the cartridge. Such excess optical energy is ideally converted to heat by the beam dump. The analytical instrument may also include an optical pathway, for example fiber optic cables, to direct an optical alignment signal from the multiplexed optical chip to an alignment detector. For example, a fiber optic cable can route some of the diffracted beam to a photodiode for use in inferring the position of the beam relative to the optical chip.
The above-described nucleic acid sequencing cartridges enable single-molecule, real-time (“SMRT”) sequencing with a number of advantages over existing devices and systems. First, because the packaging in these devices is self-contained, there is accordingly no need for a separate cell tray for the multiplexed optical chip. Second, the enclosed devices are safe for an end user to handle directly, without concern for damage from electrostatic discharge or chemical contamination. Third, the flow cell architecture of the device eliminates the need to cap the reagents in the reaction regions with mineral oil or any other protective liquid, thus enabling the possible reuse of the multiplexed optical chips and thus further decreasing the cost of nucleic acid sequencing in these systems. Fourth, inclusion of an optional onboard non-volatile rewritable memory (e.g., an EEPROM chip) in each cartridge device allows cell-based data to be securely maintained without the complexity and lack of reliability of alternative methods for storing such information. Fifth, the design of the flow cell significantly reduces the amount of sample required per sequencing run and further provides for more even, and thus less variable, loading of the sample. Finally, the simplified design and function of the cartridge devices eliminates the need for robotic components in analytical systems relying on these devices, thus reducing the cost and complexity of the systems.
In another aspect, the instant disclosure provides novel flow cells for the delivery of nucleic acid sequencing samples and reagents to the plurality of reaction regions in the active sequencing area of a multiplexed optical chip. Traditional chip-loading methods can be inefficient and uneven. Although flow cells for loading analytical devices, including multiplexed optical chip devices, are known, where these devices have square or rectangular shapes, loading at the corners of the devices can be especially inefficient and uneven.
The instant inventors have addressed at least some of the inadequacies of current flow cell performance by creating the novel designs described herein. In these flow cells, a flow cell chamber covers the sequencing region of the multiplexed optical chip, thus delivering liquid samples and reagents from an input port or ports on the flow cell to the plurality of reaction regions on the chip. The flow cell optionally includes at least one larger-bore pathway, also called a trunk line, to facilitate removal of air bubbles from the flow cell. The exact dimensions of the trunk line can be adjusted as desired to maximize the likelihood that any air bubbles in the liquid sample or reagent will be diverted to the trunk line rather than to the sequencing region of the chip. The dimensions of the trunk line may depend, for example, on the specific composition of the liquids used in the flow cell, as well as on the materials used to fabricate the flow cell and the chip. In specific embodiments, the flow cell includes at least two larger-bore pathways or trunk lines. In even more specific embodiments, the flow cell can include three, four, or even more larger-bore pathways or trunk lines.
As illustrated in the exemplary drawings of
It should be understood that fluidic ports 420 are preferably associated with rubber O-rings, or another suitable sealing element, to provide a significantly leak-free fluidic connection between the nucleic acid sequencing cartridge and the fluidic delivery components of the analytical instrument. The O-rings are not shown in the fluidic ports 420 of
The bottom surface of another exemplary flow cell is illustrated in
An exemplary filling sequence for a flow cell with two input ports and two output ports is illustrated in
The flow cells of the instant disclosure can be fabricated from any suitable material, provided that the material is compatible with the liquid reagents used in the nucleic acid sequencing reactions and that the material displays other suitable chemical, physical, and optical properties. In some embodiments, the material can be glass or crystalline silicon, although the brittleness of these materials may be considered disadvantageous in some situations. In addition, the opacity of crystalline silicon can preclude the bonding of such a flow cell to the optical device using a UV-curable adhesive. In some embodiments, the flow cells can be fabricated from a clear material, such as a clear glass or a clear plastic material. In specific embodiments, the material is a plastic material, for example a flexible clear plastic material. In preferred embodiments, the flow cells can be fabricated from an acrylonitrile butadiene styrene (ABS) plastic, preferably a UV-clear ABS plastic. Alternatively, the material can be polystyrene, acrylic, glass, polyether ether ketone (PEEK), or the like. In some embodiments, the material is a coated material, such as a parylene-coated ABS, or another suitable coated material.
The flow cells can preferably be bonded to the detector layer, typically a CMOS sensor layer, of the multiplexed optical chip. As will be described in more detail in a later section, the flow cells are most preferably bonded to the detector layer using a UV-cure adhesive. Such an adhesive is advantageous for these purposes, because the curing can be performed at a relatively low temperature, where the potential damage to heat-sensitive components in the plurality of reaction regions (e.g., biotin) is minimized. A UV-cure adhesive also minimizes the need for solvents or other noxious agents that may inhibit or inactive reagents used in the sequencing reactions. When a UV-cure adhesive is used for the bonding, it is generally preferable that the flow cells be fabricated from a UV-transparent material.
The just-described flow cells offer a number of advantages in the loading of multiplexed optical chips for nucleic acid sequencing compared to existing technologies. For example, they enable a simpler instrument interface and workflow than current approaches with open wells, which require a pipetting robot to fill the reaction regions of an optical chip. In addition, flow cells require reduced overall sample volumes, including a reduced input of sample nucleic acids and reduced volumes of other reagents, thus resulting in a lower cost per sequencing run. Importantly, they improve uniformity in loading of an optical chip and, because they do not require an overlay of oil, they will facilitate reuse of expensive sequencing chips.
As mentioned above, the top surface of the flow cell is preferably designed to engage with a fluidic manifold, which may also be referred to as a fluidic bulkhead or fluidic clamper. The fluidic manifold can be associated with the analytical instrument that is used for nucleic acid sequencing, or it can be part of a separate fluidics system that is used more specifically to load liquid reagents into the optical sequencing devices prior to insertion of the devices into the analytical instrument. As mentioned above, the engagement between the fluidic manifold and the flow cell creates a fluidic connection that enables delivery of liquid reagents from the instrument to the active sequencing region on the multiplexed optical chip.
An exemplary fluidic manifold 900 is illustrated in
It should also be understood that in preferred embodiments, the fluidic manifold has two main functional pieces that are movable relative to one another. In the exemplary fluidic manifold shown in
The optional optical fiber (or fibers) 925 shown in
In some embodiments, the instant nucleic acid sequencing cartridges, packaged devices, or analytical systems comprising these cartridges or devices, additionally comprise features to dissipate heat. Heat is generated in the analytical systems comprising the instant cartridges or packaged devices, both from the optical source, for example a laser optical source, and also from the CMOS sensors used in these systems. Since the reagents used in nucleic acid sequencing are typically sensitive to high temperatures, it can be important to provide for the dissipation of heat from the multiplexed optical chips of the instant packaged devices and from the analytical systems more generally.
Thermal control within a packaged device can be provided in several ways. In some embodiments, a low-cost thermoelectric cooler (TEC) and heatsink can be included in a cartridge surrounding the packaged device. In other embodiments, the TEC is included in the analytical instrument, at a remote location from the packaged device, and thermal contact is established between the TEC and the multiplexed optical chip via an Indium pad or the like. Use of a remote TEC may be advantageous from a cost perspective, but such a configuration can depend on the accurate and reproducible measurement of temperature at an area of interest on the optical chip. In preferred embodiments, an impinging jet of cooled air is blown in from a blower fan associated with the analytical instrument and is used to cool the CMOS sensor. The cool air can enter the cartridge or packaged device at an entry port, for example aperture 445, as shown in the cartridge of
An exemplary cooling system for the cartridges and packaged devices of the instant disclosure is illustrated in
In another aspect, the disclosure provides complete analytical systems for use in automated nucleic acid sequencing, in particular single molecule, real-time sequencing, that comprise an analytical instrument and any of the nucleic acid sequencing cartridges or packaged devices described above. The cartridges and packaged devices used in these systems preferably comprise a multiplexed optical chip that is attached to a printed circuit board, as previously described. Even more preferably, the multiplexed optical chip and the printed circuit board are surrounded by a protective enclosure, for example the above-described cartridge enclosures.
As described above, the nucleic acid sequencing cartridges and packaged devices can, in preferred embodiments, be removably inserted into the analytical instrument, and the analytical instrument can include other desired optical, electronic, fluidic, mechanical, or thermal components. Liquid sequencing reagents can be brought into contact with the cartridges and packaged devices, either before or after the cartridge or packaged device has been inserted into the instrument. Where liquid reagents are delivered to the cartridge or packaged device after it has been inserted into the analytical instrument, the instrument preferably includes pumping and other fluidic components to direct the liquids to the reaction regions on the multiplexed optical chip in a controllable manner. For example, the instrument can include a syringe pump, or the like, to deliver liquid reagents to the reaction regions.
The analytical instrument can provide electronic signals to an associated cartridge or packaged sequencing device and can receive electronic signals from detectors or other electronic components within the cartridge or device. The instrument typically includes one or more computers to manipulate, store, and analyze data obtained from the device. For example, the instrument can have the capability to identify the order of added nucleotide analogs for the purpose of nucleic acid sequencing. The identification can be carried out, for example, as described in U.S. Pat. No. 8,182,993, and U.S. Patent Application Publication Nos. 2010/0169026 and 2011/0183320 which are each incorporated herein by reference for all purposes in their entireties.
In preferred embodiments, the analytical systems of the disclosure comprise any suitable cartridge or packaged nucleic acid sequencing device, as described herein, and at least one optical source for providing illumination light to the one or more waveguides of the packaged device or devices. More preferably, the analytical systems further comprise an electronic system for providing voltage and current to the detector and for receiving signals from the detector and/or a computer system for analyzing the signals from the detector to monitor the analytical reaction, for example, to obtain sequence information about a template nucleic acid. In other preferred embodiments, the analytical systems of the instant disclosure comprise a cooling system, for example, any of the cooling systems described above, that removes heat from the multiplexed optical chip and/or from other components of the system. In some embodiments, the cooling system comprises a blower fan. In some embodiments, the cooling system comprises a thermoelectric cooler.
An exemplary analytical system comprising the above features is illustrated in
Also shown in
As also shown in
The optical source used in the instant analytical systems can be any suitable optical source, as would be understood by those of ordinary skill in the relevant art. Optical sources that emit in the visible wavelength range are particularly useful for the analysis systems of the present disclosure, for example optical sources that emit between 450 nm and 700 nm or from 500 nm to 650 nm In some embodiments, the instant systems can include more than one optical source.
In preferred embodiments, the optical source is a laser source. Any suitable type of laser can be used for the instant systems. In some cases, solid state lasers are used, for example, III-V semiconductor lasers. Recently, progress has been made in producing solid state lasers that emit in the desired wavelength range. Particularly useful lasers are GaInN solid state lasers. Lasers suitable for use in the disclosed systems, including GaInN lasers, are described, for example in Sizov et al., “Gallium Indium Nitride-Based Green Lasers,” J. Lightwave Technol., 30, 679-699 (Mar. 1, 2012), Nakamura, et al. “Current Status and Future Prospects of InGaN-Based Laser Diodes”, JSAP Int. No. 1, January, 2000, Jeong et al. Nature, Scientific Reports, “Indium gallium nitride-based ultraviolet, blue, and green light emitting diodes functionalized with shallow periodic hole patterns”, DOI: 10.1038, and Tagaki et al., “High-Power and High-Efficiency True Green Laser Diodes”, SEI Tech Rev, No. 77, October 2013; which are each incorporated by reference herein for all purposes in their entireties.
In some embodiments, the optical source is a light emitting diode, for example a superluminescent light emitting diode. In some embodiments, the optical source is a vertical-cavity surface-emitting laser, or other comparable optical device.
In specific embodiments of the analytical instrument, the optical source can be configured to be replaceable by an end user, thus decreasing upkeep, maintenance, and repair costs for the user. More particularly, all of the optics in these sequencing systems, including the laser(s) and the entire beam train, can be encapsulated into a single optics box or module. This box can be removable and replaceable directly by an end user to facilitate inexpensive, rapid self-servicing of the instrument.
In one embodiment of such a system, the user lifts a cover on the instrument, disconnects a single cable, and then removes the optics module from the system. By reversing the previous steps, the user can replace the optics module with a new or rebuilt unit, thus placing the instrument back into service. The defective optics module can be shipped back to the manufacturer for refurbishment or disposal. In some embodiments, the user releases a locking mechanism, for example a turnable knob or twistable cam, on top of the optics module prior to removing the module from the system. In some embodiments, a dovetail connector is used to connect the module to the system instead of, or in addition to, a cable.
In specific embodiments, the optics cartridge can be registered to the instrument by a number of methods, including via a hole and slot or other similar kinematic mounting.
The invention thus makes it practical for an end user to service any and all optical problems that may arise in their own instruments, much in the same way that an end user is able to replace toner and ink cartridges in desktop printing systems. Instrument downtime and costs are accordingly minimized in these systems.
In another aspect are provided novel procedures and structures for minimizing the bleaching of sequencing reagents on a packaged device comprising a flow cell. As described above, the flow cells used in the packaged nucleic acid sequencing devices of the instant disclosure are preferably plastic, for example a flexible plastic, and are more preferably a UV-clear plastic, such as ABS plastic. Use of a UV-clear plastic allows the flow cell to be bonded to the detector layer using a UV-cure adhesive, thus enabling the cure to be performed quickly and at a relatively low temperatures, thereby avoiding degradation of temperature-sensitive reagents in the reaction regions of the optical chip. ABS plastic also has advantages in being chemically compatible with the reagents used in nucleic acid sequencing reactions and in being non-brittle. Alternative exemplary materials for the instant flow cells include polyether ether ketone (PEEK), polyethylene terephthalate (PET), Glass Filled PET, and the like.
Although the use of a UV-clear plastic is advantageous from a bonding, chemical, and physical perspective, it can be disadvantageous when an optical chip having an attached flow cell is illuminated, since routing waveguides on the optical chip can release optical energy above the chip, either through scattering or as an evanescent wave, and this released light can result in the photobleaching of fluorescent reagents in the flow cell, as well as increased background fluorescence, for example if excitation optical energy reaches the fluorescent reagents above the chip. In particular, where the routing waveguides pass underneath attachment sites for the flow cell, the clear-plastic material can provide a pathway for the released light to reach fluorescent reagents within the flow cell above the chip and thus photobleach the reagents and/or cause background fluorescence.
The inventors of the instant disclosure have recognized this problem and have designed novel bonding procedures and bonded flow cell structures to avoid these problems. Specifically, the inventors have designed flow cell structures that can block released light from reaching the fluorescent reagents in the flow cell while at the same time allowing sufficient light to pass through the flow cell to cure the adhesive used to bond the flow cell to the multiplexed optical chip.
As shown in
Other variants of the above structures would be understood to solve this problem by those of ordinary skill in the art.
In another aspect, the instant disclosure provides novel methods that improve the efficiency and extent of loading of a nucleic acid analytical sample onto a multiplexed optical chip. Whereas nucleic acid samples are typically loaded onto such devices using static loading techniques (e.g., by applying the nucleic acid sample to the device and incubating without further mixing or circulation), these approaches can be inadequate as the size and multiplex of an analytical device increases.
The instant inventors have identified the inadequacy of traditional loading methods and have developed novel approaches for addressing this issue. In particular, one approach has already been described above with respect to novel analytical devices comprising a flow cell feature for delivering samples and reagents to a sequencing chip. As shown above, the use of a flow cell to load a sample chip results in the more efficient loading than is possible using a traditional open-well loading process with a pipette. See, e.g.,
The loading methods are further improved by solution reflow or recirculation over the active area of an analytical chip device, for example using the flow cell device. Specifically, the loading solution can, for example, be flowed back and forth over the device (i.e., “reflowed”), resulting in a 2× improvement on template loading at very low picomolar concentrations. Furthermore, fully recirculating the sample across the active area surface of the analytical chip, for example by the recovery of sample at an outlet port of the flow cell, and by the subsequent reintroduction of the sample at an inlet port in the flow cell, ideally at a second inlet port in the flow cell, can significantly improve loading of the sample on the analytical chip device.
In some embodiments, the methods of loading may include the step of replenishing the supply of samples and/or reagents either before or during a sequencing run. Such replenishment can be particularly advantageous during long sequencing runs, where the supply of reagents can be depleted during the course of a run.
An exemplary loading process in accordance with these aspects of the disclosure is illustrated in
The above flow methods can additionally serve as a method to concentrate nucleic acid samples on an analytical device. Specifically, nucleic acid sample material can be concentrated over a surface of the optical device under flow conditions. Such approaches can be particularly useful in systems that require large sample volumes. For example, the same molar amount of a nucleic acid sample material can be diluted over a large volume and then be re-concentrated over the surface as it is immobilized in the reaction regions of the optical device.
Accordingly, in some embodiments, the methods of loading can comprise the steps described in the following numbered paragraphs:
1. A method for loading an analytical device comprising the steps of:
In some embodiments, the packaged devices and systems of the instant disclosure, including the cartridge-enclosed packaged devices described above, can be loaded with a nucleic acid sample by the end user using improved sample delivery devices, systems, and methods. In particular, these devices, systems, and methods allow for a nucleic acid sample to be delivered directly to the optical chip by the user, thereby minimizing the overall volume of nucleic acid used in an analytical method. The devices, systems, and methods find utility in a variety of applications, including DNA sequencing, RNA sequencing, on-chip PCR, and the like.
In a typical automated nucleic acid sequencing system, the nucleic acid sample is either placed directly into an open well fluid chamber or a flow cell chamber by a user or a robot as part of the instrument workflow prior to a sequencing run. The sample thereby sits on either the user bench or is placed by the user onto the instrument. Such approaches can, however, require relatively large volumes of sample and can result in the relatively inefficient delivery of the nucleic acid sample to the active sequencing region of the analytical device.
The sample delivery approaches disclosed herein allow for overall lower sample volume by being incorporated directly onto the optical chip. The devices and methods thereby additionally enable lower overall system costs (both capital and operating). A general background summary of on-chip microfluidic systems is provided by Rolland et al. (2004) J. Am. Chem. Soc. 126, 2322, which is incorporated by reference herein for all purposes.
In step 1 of the work flow, an end user, or a robotic equivalent, retrieves a fresh optical chip device 1400 from a suitable storage location or shipping box, and the device is placed on a surface, or other suitable location, for loading. In step 2, the foil seal is removed from the device, and a nucleic acid sample 1424 is placed into sample capsule 1422. As will be described in more detail below, the sample capsule is nested within a sample reservoir housing that is attached to, or fabricated in, a flow cell on the device. By including the sample capsule as part of the analytical device itself, the total volume of sample required for an analysis can be extremely low. For example, a volume of between 10-100 μL can be used for loading such devices, compared to standard volumes of 150-300 μL in systems where the sample compartment is not part of the analytical chip. In step 3 of the work flow, a coverslip, gasket, or other such fluid separation interface 1425 can be added to the top of the sample capsule, and the loaded chip device can then be placed into the instrument, either by the user or by a robotic mechanism. The cover slip feature creates a small barrier between the instrument's pneumatic engagement mechanism and the nucleic acid sample. The function of the cover slip can alternatively be provided by the instrument itself, for example as the loaded chip is inserted into the instrument.
As illustrated in the drawings of
It should be understood that the fluidic openings of the sample capsule and the sample reservoir housing can be aligned by alternative designs and/or mechanisms, for example by a “push-push” mechanism, wherein in a first push, the holes are not aligned, but wherein in a second push, the holes of the sample capsule and the sample reservoir housing become aligned, and thereby enable the sample to flow from the sample capsule to the active sequencing region/ZMW array on the optical chip device.
An alternative structural design for the delivery of a nucleic acid sample from the sample capsule onto the active sequencing region/ZMW array of a chip device is illustrated in
It should be understood that in any of the above low-volume sample loading devices, a controllable fluidic connection between the nucleic acid sample in the sample capsule and the plurality of reaction regions on the optical device can be achieved in a variety of ways by the moveable positioning of the sample capsule within the sample reservoir housing. In particular, when the sample capsule and the sample reservoir housing each has a fluidic opening (or “hole”) of similar size and appropriate orientation, positioning of the sample capsule so that the fluidic openings are not aligned prevents a fluidic connection of the two spaces, and a movement of the sample capsule that sufficiently aligns the fluidic openings results in a fluidic connection. As illustrated in the examples of
It should also be understood that even when an open fluidic connection has been established between the sample capsule and the active sequencing region/ZMW array of the optical device, flow of the nucleic acid sample may require either an increased pressure from the sample side, or a decreased pressure from the device side. In specific embodiments, the sample is drawn from the sample capsule to the active sequencing region/ZMW array by the opening of an outlet port in the flow cell and the removing of gas or liquid from the system to draw the sample into the flow cell. In some embodiments, pressure in the system is further controlled by a valve or a vent.
In some embodiments of the above-described sample-delivery devices, at least some of the reagents necessary for an analysis are provided together with the chip cartridge. In the case of a DNA sequencing reaction, for example, the sequencing enzyme and other necessary components can be provided in a “binding kit”. These components can be configured to react with an end user's DNA sample to form a polymerase-template complex, which is subsequently contacted with the reaction regions on the optical chip to immobilize the complex within those regions.
In some embodiments, the above-described devices comprise the features described in the following numbered paragraphs:
1. A packaged nucleic acid sequencing device comprising:
In another aspect, the disclosure provides alternative improved fluidic devices and methods for sample delivery to an analytical device, such as an optical chip device for nucleic acid sequencing. Unlike the just-described sample-delivery devices, where a nucleic acid sample is added to a low-volume sample capsule directly associated with the flow cell on the surface of the optical chip device, these devices are designed to allow a user to load a sample into a port that is accessible from the exterior of a cartridge that comprises the optical chip device, for example any of the cartridge designs described above. Specifically, in these device embodiments, the user loads a sample through the sample port into a sample reservoir located within the cartridge, and the cartridge is then inserted into the analytical instrument. A pumping system, and interior fluidic connectors, transport the sample from the sample reservoir through the flow cell to the active sequencing region/ZMW array on the optical chip device prior to the sequencing run.
An exemplary cartridge device 1700 with a separate sample reservoir associated with the cartridge is illustrated in
In some of the just-described cartridge device embodiments, the device can include a check valve between the sample reservoir and the fluidic port on the flow cell to prevent backflow of reagents into the sample reservoir. In some embodiments, the flow cell can include an additional dedicated port within the flow cell that is separate from the inlet and outlet ports shown in the above flow cell devices and that enables the sample to be loaded directly from the sample reservoir onto the active sequencing region/ZMW array. In some embodiments, the sample reservoir is connected to one of the flow cell inlet or outlet ports through a T-type connection. In any of the above embodiments, flow of sample from the sample reservoir to the active sequencing region/ZMW array on the optical chip device can be driven either by pressurizing the sample reservoir or by depressurizing an outlet port on the flow cell.
It will be readily apparent to one of ordinary skill in the relevant arts that other suitable modifications and adaptations to the analytical devices and systems described herein can be made without departing from the scope of the invention or any embodiment thereof.
All patents, patent publications, and other published references mentioned herein are hereby incorporated by reference in their entireties as if each had been individually and specifically incorporated by reference herein.
While specific examples have been provided, the above description is illustrative and not restrictive. Any one or more of the features of the previously described embodiments can be combined in any manner with one or more features of any other embodiments in the present invention. Furthermore, many variations of the invention will become apparent to those skilled in the art upon review of the specification. The scope of the invention should, therefore, be determined by reference to the appended claims, along with their full scope of equivalents.
This application claims the benefit of U.S. Provisional Application No. 62/961,175, filed on Jan. 14, 2020, the disclosure of which is incorporated herein by reference in its entirety.
Number | Date | Country | |
---|---|---|---|
62961175 | Jan 2020 | US |