Apparatus and method for measuring DNA / RNA production on biochips

Abstract

Microfluidic devices (e.g., systems, devices, etc.) and methods for microfluidics provide for the detection of polynucleotide concentrations. These devices may include one or more channels, chambers, and / or substrates for performing microfluidic operations, and may include removable, single-use, or reusable microfluidic components adapted for detecting polynucleotide concentrations. For example, the microfluidic components may be microfluidic cartridges that can be inserted, held, and / or installed within a microfluidic driver device that can monitor and control operations within one or more cartridges based in part on the detected concentration of polynucleotides.

Description

[Technical Field]

[0001] (Priority Claim) This application claims priority to U.S. Provisional Patent Application No. 63 / 396,765, filed August 10, 2022, entitled "Apparatuses and Methods for Measuring DNA / RNA Production in Biochips."

[0002] (Incorporated by reference) All publications and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. [Background technology]

[0003] "UV spectrophotometry" is a standard technique for quantifying nucleic acids. In UV spectrophotometry, a nucleic acid sample is placed in a chamber, typically a quartz cuvette, which is then placed inside a UV spectrophotometer. UV light passes through the sample over a specific path length, and the sample's absorbance at a specific wavelength is measured. Absorbance at 260 nm (A260) is often used to measure nucleic acids. Nucleic acids (e.g., DNA or RNA) contain conjugated double bonds in purine and pyrimidine rings, which have a specific absorption peak at approximately 260 nm. The maximum absorbance of nucleic acids occurs at a wavelength of 260 nm. The intensity of this absorbance is proportional to the concentration of the nucleic acid. From these physical characteristics, the nucleic acid concentration of a sample can be determined. The concentration of a sample can be determined based on the absorbance reading. The purity of a sample can also be estimated by comparing it with the absorbance at 280 nm or 230 nm.

[0004] However, such traditional UV spectrophotometric methods for quantifying polynucleotides typically require sample removal and transfer to a dedicated spectrophotometer device. Typically, a minimum sample volume of at least 1–75 μL is required to obtain an accurate instrument reading. This can be problematic when samples are highly diluted or when extraction techniques require elution of nucleic acids into small volumes. Contamination is also an issue. For example, when measuring DNA, biological molecules such as proteins, RNA, and chaotropic salts from the extraction procedure can falsely inflate nucleic acid concentration estimates. Buffer salts such as Tris, EDTA, and guanidine isothiocyanate strongly absorb at 230 nm and bleed into the 260 nm absorption range. Furthermore, free nucleotides present in the sample can affect UV quantification. Changes in sample pH also alter UV readings. An alternative to spectrophotometers is to add fluorescent probes and measure them with a fluorometer, but this requires further processing of the RNA / DNA sample and labeling such molecules with fluorescent molecules, increasing cost and complexity. All of these approaches have significant drawbacks and may require manual intervention and output measurements that are not tied to the microfluidic control of the system.

[0005] Described herein are devices, particularly microfluidic devices, that can address these issues. Summary of the Invention

[0006] Described herein are microfluidic devices (e.g., systems, devices, etc.) and methods for microfluidic detection of polynucleotide concentrations. These devices may include removable, single-use, or reusable microfluidic components, which may include one or more channels, chambers, and / or substrates for performing microfluidic operations. For example, the microfluidic components may be microfluidic cartridges, microfluidic chips ("biochips"), etc., sometimes referred to herein as "cartridges." Any of these cartridges may be generally flat, planar structures that can be inserted, held, and / or installed within a microfluidic driver device, which can monitor and control operations within one or more cartridges. The microfluidic driver device, sometimes referred to herein as a microfluidic pathway device, may include actuators, sensors, and a controller (including control circuitry, a processor, etc.). As described in detail below, the cartridges may be adapted to include one or more polynucleotide quantification regions adapted for optical sensing and quantification of polynucleotides, e.g., DNA, RNA (e.g., mRNA), etc. The polynucleotide quantification regions are configured for spectrophotometric reading by emitters and detectors that may be part of the microfluidic driver device. The microfluidic driver device may be further adapted to control activity of the microfluidic driver device and cartridge based on feedback from the spectrophotometric readings.

[0007] For example, the devices and methods described herein may be configured to quantify RNA and / or DNA produced within a microfluidic device, e.g., a cartridge driven by a microfluidic driver device, via an exponential biochemical reaction. These devices can modify or control and adjust output to compensate for variations due to imperfections in reagents, biochips, or temperature. In particular, the methods and devices described herein can microfluidically measure the concentration of RNA and / or DNA produced as the device operates and control the operation of the device when producing therapeutic mRNA, including, but not limited to, mRNA vaccines. These methods and devices may enable confirming the success of various amplification and synthesis steps, pooling multiple batches, and normalizing output. This may also enable cartridge reuse and optimization. Repeating synthesis steps within a cartridge can generate individual outputs that previously required extensive quality analysis for release. Alternatively, the methods and devices described herein may enable pooling of output into a single batch, requiring only a single quality analysis process and simplifying manual sample collection and sorting. If the measured concentration of RNA / DNA product per replicate is within nominal parameters, the device can automatically add it to the pool, or if the concentration is outside the parameters, it can be microfluidically diverted to a "further test" or "waste" container. This measurement-driven diversion can be driven by an operator or automatically determined by the microfluidic system.

[0008] These methods and devices may also allow for standardization of output: the complex biochemical processes for producing RNA and DNA can be sensitive to parameters such as reagent supply, temperature, and biomolecule stability, and production yields can vary.

[0009] Once the output has stabilized, measurement and dilution / concentration steps between DNA / RNA production and the next step in the therapeutic production process can be eliminated. To stabilize the output, the methods and devices described herein may select a lower target concentration and dilute the output of the system, so that the output is within a more controlled range. To perform this dilution automatically, the pre-dilution concentration of the DNA / RNA must be known and used to determine the amount of diluent to add.

[0010] The methods and devices described herein may also enable automated microfluidic RNA / DNA production to be iteratively improved for greater control. For example, once it is demonstrated that the process being performed by the device (e.g., within a cartridge) is tightly controlled with regular and consistent output concentrations, the product can be released without the need for detailed analysis. Instead, a "parametric release" may be performed, using metrics such as temperature, reagent consumption, and intermediate RNA / DNA concentrations to demonstrate that the process proceeded nominally.

[0011] Generally, described herein are cartridges ("biochips") that are specifically adapted to enable an automated or semi-automated system, e.g., a microfluidic driver device, to determine the concentration of a therapeutic polynucleotide being processed (e.g., formed, mixed, compounded) within the cartridge and / or to modify the operation of the microfluidic driver device on the cartridge based on the determined concentration. For example, the present specification describes a cartridge (e.g., a cartridge device) for processing polynucleotides, the cartridge comprising: a first layer having a first thickness; a second layer having a second thickness; an elastic material extending between the first layer and the second layer and separating the first layer from the second layer by a separation thickness; and a UV measurement region formed through the first layer and the second layer, wherein a region of the first layer in the UV measurement region has a thickness less than the first thickness, and a region of the second layer in the UV measurement region has a thickness less than the second thickness, and the elastic material is not present between the region of the first layer in the UV measurement region and the region of the second layer in the UV measurement region, forming a UV measurement chamber having the separation thickness. A therapeutic polynucleotide may also refer to a solution (e.g., an aqueous solution) of polynucleotides.

[0012] Any of these cartridges can be formed from a polymeric material, such as a cyclic olefin copolymer (COC) material or a cycloolefin polymer (COP). While these materials are not very UV-transparent and (as surprisingly found herein) can change their UV transmission characteristics with UV exposure, the methods and apparatus described herein can be adapted or tailored to enable sensitive and accurate estimation of UV absorption and, therefore, polynucleotide (e.g., therapeutic polynucleotide) concentration. The first and second layers can be formed from the same or different materials. For example, the second layer can include a COC material and / or a COP material.

[0013] For example, the first thickness may be about 0.1 mm to about 5 mm, and the second thickness may be about 0.1 mm to about 5 mm. The first layer and the second layer may have the same thickness or different thicknesses.

[0014] The elastic material or pieces of elastic material may be sandwiched between a first layer and a second layer (with additional layers being included above or below the first and second layers). The elastic material may form a layer. The elastic material may be held directly between these two layers, or may be adhesively and / or mechanically held between the first and second layers. The separation thickness may be between 1 mm and 0.1 mm. The elastic layer may be uniform or non-uniform in thickness.

[0015] Because the UV sensing chamber can be formed in the space between the first and second layers where no elastic material is present, the path length of the UV sensing region can be relatively short and can be approximately the same as the separation thickness. Thus, the elastic material can surround the UV sensing chamber (on the sides of the UV sensing chamber). A cartridge can have one or more UV sensing chambers. In some examples, the UV sensing chamber is in a consistent location across various different cartridge designs or configurations.

[0016] Generally, any of these cartridges may include one or more fluid channels in the second layer that are in fluid communication with the UV measurement chamber. The fluid channels may be formed by milling, cutting, etc., from the top surface of the second layer and may be at least partially covered by an elastic material / layer. In some examples, the fluid channels (sometimes referred to herein as fluid channels) are disposed within the second layer. The second layer may be formed by multiple sublayers.

[0017] The cartridge device may also include multiple pneumatic valves arranged to control flow into and out of the UV measurement chamber. Each of the multiple pneumatic valves may be formed by an air pressure chamber in the first layer and a fluid chamber in the second layer, and a portion or region of elastic material separating the air pressure chamber from the fluid chamber may move up and down to open / close (or remain open in a neutral position) the fluid chamber, which may be in fluid communication with fluid channels and / or other fluid chambers in the cartridge. The air pressure chamber of each valve may be in fluid communication with an air pressure channel in the first layer, which is configured to communicate with a pressure port in an outer region of the device to actuate the pneumatic valve (e.g., move a region of elastic material between the air pressure chamber and the fluid chamber). The fluid chamber may be in fluid communication with the UV measurement chamber through a fluid channel in the second layer (e.g., so that when the air pressure valve is opened, fluid can flow between the fluid chamber and the UV measurement chamber).

[0018] Any of these cartridges may include an in vitro transcription (IVT) chamber formed at least partially within the second layer and in fluid communication with the UV measurement chamber. The cartridge may also include one or more mixing chambers (mixers), reaction chambers, etc. Generally, the microfluidic driver device may include one or more thermal control elements (e.g., Peltier elements, heaters / coolers, etc.) for controlling the temperature of one or more regions of the cartridge.

[0019] Generally, cartridge devices may include one or more vacuum ports on an exterior region of the device configured to connect to a negative pressure source for drawing fluid into and / or from the UV measurement chamber (e.g., into a waste port and / or waste chamber). For example, the apparatus may be configured to maintain a vacuum within the UV measurement chamber such that when a valve is opened to a chamber or channel holding a therapeutic polynucleotide (e.g., generally a solution containing a therapeutic polynucleotide), the resulting negative pressure may draw the therapeutic polynucleotide into the UV measurement chamber.

[0020] Any of these cartridge devices may include a first inlet channel in the second layer that is in fluid communication with a first chamber configured to hold a polynucleotide sample fluid, and a second inlet channel in the second layer that is in fluid communication with a second chamber configured to hold a blank sample fluid.

[0021] Any of these cartridges may be configured for continuous mixing of solutions, for example, any of these cartridges may include an additional dilution and mixing chamber in fluid communication with a UV measurement chamber configured to dilute the fluid sample.

[0022] In any of these examples, the cartridge may be configured to prevent air bubbles from being present in the sensing region of the UV measurement chamber (so that the air bubbles do not interfere with UV detection of the therapeutic nucleotide absorption signal). In some examples, the UV measurement chamber may include an undercut region surrounded by a resilient material, e.g., below the thicker regions of the first and second layers (outside the thinned regions of the first and second layers). Thus, the diameter of the UV measurement chamber may be larger than the diameter of the region of the first layer in the UV measurement region (and larger than the diameter of the region of the second layer in the UV measurement region) to prevent air bubbles in the central region of the UV measurement chamber.

[0023] For example, a cartridge device for processing polynucleotides may include a first layer including a polymeric material having a first thickness, a second layer including a polymeric material having a second thickness, an elastic material extending between the first layer and the second layer, an ultraviolet (UV) measurement region formed through the first layer and the second layer, a region of the first layer in the UV measurement region having a thickness equal to or less than the first thickness, a region of the second layer in the UV measurement region having a thickness equal to or less than the second thickness, and the elastic material removed from between the region of the first layer and the region of the second layer in the UV measurement region to form a UV measurement chamber, and an in vitro transcription (IVT) chamber formed at least partially in the second layer and in fluid communication with the UV measurement chamber.

[0024] Also described herein are methods of using any of these cartridges with any of the microfluidic driver devices described herein. Generally, these methods involve on-cartridge ("closed-circuit") detection and determination of therapeutic polynucleotide concentration. Any of these methods may include techniques for optimizing or improving detection, including adjusting one or more parameters of the microfluidic driver device based on feedback from the detected concentration of therapeutic polynucleotide (e.g., from a detected UV absorption signal) or feedback from a difference signal between the UV absorption signal of the therapeutic polynucleotide signal and one or more blanks.

[0025] For example, described herein is a method of manufacturing polynucleotides using a microfluidic driver device operating on a cartridge, the method including: forming a therapeutic polynucleotide in the cartridge; driving, with the microfluidic driver device, a first blank solution into an ultraviolet (UV) measurement chamber of the cartridge and obtaining a first absorption measurement through the cartridge; driving a sample solution of the therapeutic polynucleotide into the UV measurement chamber of the cartridge and obtaining a second absorption measurement through the cartridge; driving a second blank solution into the UV measurement chamber of the cartridge and obtaining a third absorption measurement through the cartridge; and determining the concentration of the therapeutic polynucleotide by extrapolating the concentration of the therapeutic polynucleotide from the first absorption measurement, the second absorption measurement, and the third absorption measurement.

[0026] As described above, any of these methods may include adjusting, in a processor of the microfluidic driver device, the operation of the microfluidic driver device based on the estimated concentration of the therapeutic polynucleotide. For example, adjusting may include directing the resulting therapeutic polynucleotide to a pool for additional therapeutic polynucleotides. Additional therapeutic polynucleotides may also be formed within the cartridge. In some examples, adjusting the operation of the microfluidic driver device may include modifying the synthesis (formation) of the therapeutic polynucleotide, including during an amplification step during which samples may be removed and tested.

[0027] For example, any of these methods may include comparing an estimated concentration of a therapeutic polynucleotide to a concentration range by a processor of the microfluidic driver device, and, based on this comparison, directing the therapeutic polynucleotide to one of pooling with one or more additional batches of therapeutic polynucleotide formed in the cartridge, discarding, or further analysis.

[0028] In some examples, the method can include diluting the therapeutic polynucleotide to a standard concentration for output under control of a processor of the microfluidic driver device. Thus, the microfluidic driver device (e.g., a controller of the microfluidic driver device) can add fluid to achieve the target concentration using an estimate of the determined concentration and / or volume (e.g., which may be optically sensed and / or based on the known volumes of the cartridge chambers and / or channels).

[0029] In some examples, the method may include automatically (or semi-automatically, e.g., with user confirmation, input, or modification) adjusting, by the microfluidic driver device, one or more parameters of the therapeutic polynucleotide formation based on the estimated concentration of the therapeutic polynucleotide. Examples of the one or more parameters may include one or more of temperature, reagent volume, reagent concentration and / or amount, reaction time, mixing volume, and / or time, etc.

[0030] Generally, the devices and methods described herein can be configured to automatically adjust to determine accurate concentrations. For example, the methods and devices described herein can be configured to prevent saturation, e.g., when minimal UV light is absorbed by the therapeutic polynucleotide, to improve detection limits when concentrations are too high, to reduce sensitivity, or to reduce background noise, e.g., in absorption by the walls (first and / or second layers) of the UV measurement chamber. For example, the light output, detector sensitivity, or, in some methods and devices, serial dilutions of the therapeutic polynucleotide can be performed to generate a graph of absorption by dilution. Thus, any of these methods can include determining the concentration of a therapeutic polynucleotide, which can include performing serial dilutions in one or more chambers of a cartridge under the control of a microfluidic driver device and repeatedly driving a sample solution of the therapeutic polynucleotide for each of the one or more dilutions formed by the serial dilutions to generate a dilution curve. Furthermore, estimating the concentration of a therapeutic polynucleotide can include estimating the concentration of the therapeutic polynucleotide from the dilution curve.

[0031] In any of these methods, estimating the concentration of the therapeutic polynucleotide may include altering one or more of the sensitivity of a UV detector and the intensity of a UV emitter (e.g., a UV light source) of the microfluidic driver device in response to the second absorption measurement through the cartridge. The method or device may include automatically adjusting either or both of the sensitivity of the UV detector and the intensity of the UV emitter based on the detected absorption measurement of the sample containing the therapeutic polynucleotide. Thus, any of these devices and methods may automatically, semi-automatically (e.g., by user input), and / or manually adjust the LED intensity based on the detector reading. In some examples, the device of the method may adjust the LED intensity and / or detector sensitivity during measurement (on the fly), thereby increasing the dynamic range.

[0032] Any of the methods described herein may include pretreating the UV measurement chamber with UV light before obtaining a first absorption measurement through the cartridge. As described herein, it has been surprisingly found that the absorption profile of the material forming the chamber changes over time with the amount of UV light applied. Surprisingly, the absorption of cartridge materials (e.g., COC, COP, and other polymers) can actually decrease initially (e.g., within the first 60, 55, 50, 45, 40, 35, 30 minutes, etc.), resulting in an increased detection signal. Because readings from a particular cartridge may be short (e.g., within 5, 4, 3, 2, 1, 50, 45, 30, 20 seconds, etc.), it may be beneficial to pretreat the material to provide a reading with a greater detection signal. Also, the absorption of a material may increase with further gamma irradiation, and therefore the methods and apparatus described herein may include features that improve or increase transmission through the material, for example, thinning the material in the UV measurement region to improve transmission of UV light.

[0033] Any of these methods may also include performing an in vitro transcription (IVT) reaction to form a therapeutic polynucleotide and / or creating a template for the IVT reaction within the cartridge. In any of these methods, the therapeutic polynucleotide comprises a therapeutic mRNA. Any of these methods may also include encapsulating the therapeutic mRNA with a delivery vehicle in the cartridge.

[0034] Generally, the microfluidic driver device may monitor the steps of driving a first sample (e.g., a blank), driving a second sample (e.g., a sample of interest), and driving a third sample (e.g., a second blank) by pneumatically deflecting one or more regions of a membrane of the cartridge to open and / or close a valve of the cartridge due to higher pressure on the blank or sample fluid, and / or by creating a vacuum in the UV measurement chamber before opening the valve. For example, the method may include causing the microfluidic driver device to pneumatically deflect one or more regions of a membrane of the cartridge to drive a first blank solution, a second blank solution, and / or a sample solution into the UV measurement chamber.

[0035] For example, a method of manufacturing polynucleotides using a microfluidic driver device operated with a cartridge may include: forming a therapeutic polynucleotide in a cartridge, the therapeutic polynucleotide comprising a therapeutic mRNA; determining the concentration of the therapeutic mRNA by driving, with the microfluidic driver device, a first blank solution into an ultraviolet (UV) measurement chamber of the cartridge and obtaining a first absorption measurement through the cartridge, driving a sample solution of the therapeutic polynucleotide into the UV measurement chamber of the cartridge and obtaining a second absorption measurement through the cartridge, driving a second blank solution into the UV measurement chamber of the cartridge and obtaining a first absorption measurement through the cartridge, and extrapolating the concentration of the therapeutic polynucleotide from the first, second, and third absorption measurements; and adjusting, in a processor of the microfluidic driver device, the operation of the microfluidic driver device based on the estimated concentration of the therapeutic polynucleotide.

[0036] Also described herein are devices (e.g., systems) configured to perform these methods, which may include a microfluidic driver device. Any of these systems may include software, hardware, and / or firmware for performing these methods. In particular, any of these systems may include a controller having control circuitry including one or more processors for coordinating the acts (and performing these methods) described herein, including, in particular, estimating the concentration of a therapeutic polynucleotide. Any of these controllers may include a non-transitory computer-readable storage medium that stores a set of instructions that may be executed by the system.

[0037] For example, systems for forming and / or manufacturing polynucleotides, or in some examples, systems for forming therapeutic polynucleotides, such as (but not limited to) therapeutic mRNA, are described herein. Any of these systems may include a cartridge mount; a plurality of pressure lines; a plurality of fluid lines each coupled or configured to couple to a fluid source, where each fluid line and at least a subset of the pressure lines are configured to be secured to an inlet or outlet port on a cartridge held within a mounting mount; an ultraviolet (UV) light source; a UV photodetector; and a controller configured to control the application of pressure through the pressure lines to drive fluid through the cartridge, the controller directing the formation of therapeutic polynucleotides in the cartridge and measuring a first blank through a UV measurement chamber of the cartridge. and estimating the concentration of the therapeutic polynucleotide by driving a blank solution through the UV measurement chamber of the cartridge and taking a first absorption measurement using the UV light source and UV light receiver, driving a sample solution containing the therapeutic polynucleotide through the UV measurement chamber of the cartridge and taking a second absorption measurement using the UV light source and UV light receiver, and driving a second blank solution through the UV measurement chamber of the cartridge and taking a third absorption measurement using the UV light source and UV light receiver, and the controller is further configured to estimate the concentration of the therapeutic polynucleotide from the first absorption measurement, the second absorption measurement, and the third absorption measurement.

[0038] The controller may be further configured to modify the operation of the system based on the estimated concentration of the therapeutic polynucleotide. For example, the controller may be further configured to compare the estimated concentration of the therapeutic polynucleotide with a concentration range and, based on the comparison, direct the therapeutic polynucleotide to one of pooling with one or more additional batches of therapeutic polynucleotide formed in the cartridge, discarding, or further analysis. The controller may be configured to dilute the therapeutic polynucleotide to a standard concentration for output. In some examples, the controller is configured to automatically adjust one or more parameters of the formation of the therapeutic polynucleotide based on the estimated concentration of the therapeutic polynucleotide. The one or more parameters may include one or more of temperature, reagent volume, reagent concentration, time, and mixing.

[0039] The controller may be further configured to perform a serial dilution in one or more chambers of the cartridge and repeat the step of driving the sample solution of the therapeutic polynucleotide through each of one or more dilutions of the sample solution of the therapeutic polynucleotide formed by the serial dilution to create a dilution curve. The controller may be configured to estimate the concentration of the therapeutic polynucleotide from the dilution curve. Alternatively, or in addition, the controller may be configured to alter one or more of the sensitivity of the UV detector and the intensity of the UV light source in response to the second absorption measurement through the cartridge.

[0040] The controller can be configured to pre-treat the UV measurement chamber with UV light before taking the first absorption measurement through the cartridge.

[0041] The controller may be further configured to form a therapeutic polynucleotide, including performing an in vitro transcription (IVT) reaction to form the therapeutic polynucleotide. In some examples, the controller is further configured to generate a template for the IVT reaction within the cartridge. Generally, the therapeutic polynucleotide may be a therapeutic mRNA. The controller may be configured to encapsulate all or a portion of the therapeutic mRNA within the delivery vehicle.

[0042] The controller may be further configured to pneumatically deflect one or more regions of a membrane of the cartridge to open and / or close a valve of the cartridge when driving the first blank, driving the second blank, and driving the third blank. In some examples, the controller is further configured to pneumatically deflect one or more regions of a membrane of the cartridge to drive the first blank solution, the second blank solution, and / or the sample solution into the UV measurement chamber. In some examples, the driving force may be applied by providing pressure to upstream sample and blank lines or through vacuum suction in the UV chamber that draws (e.g., "sucks") sample material (e.g., the sample and / or blank of interest) into the UV chamber.

[0043] In general, the systems described herein can be configured to include one or more cartridges and a microfluidic driver device. For example, the system can include a cartridge; and a microfluidic driver device including a cartridge mount, a plurality of pressure lines, each fluid line coupled or configured to couple to a fluid source, wherein each fluid line, and at least a subset of the pressure lines, are configured to be secured to an inlet port or an outlet port on a cartridge held within a mounting mount, an ultraviolet (UV) light source, a UV light detector, and a controller configured to control the application of pressure through the pressure lines to drive fluid through the cartridge, wherein the controller is further configured to direct the formation of a therapeutic polynucleotide, and further wherein the controller controls the application of pressure through the pressure lines to drive fluid through the cartridge. and estimating the concentration of the therapeutic polynucleotide by modifying operation of the microfluidic driver device based on the concentration of the therapeutic polynucleotide determined from the first absorption measurement, the second absorption measurement, and the third absorption measurement.

[0044] All of the methods and devices described herein, in any combination, are contemplated herein and can be used to achieve the benefits described herein. [Brief explanation of the drawings]

[0045] A better understanding of the features and advantages of the methods and apparatus described herein will be obtained by reference to the following detailed description that sets forth exemplary embodiments and the accompanying drawings. [Figure 1] 10A and 10B illustrate schematic diagrams of UV measurement of polynucleotide material in a UV measurement region of a cartridge by a microfluidic driver device comprising a UV light source and a UV detector. [Figure 2A] 1 illustrates an example of a device comprising a microfluidic driver device control system described herein that can be configured to determine polynucleotide concentration and modify microfluidic driver device activity based on the determined concentration. [Figure 2B] 1 illustrates a schematic diagram of an example of a microfluidic driver device control system that can be used as described herein. [Figure 3A] 1 shows an example of a cartridge for a microfluidic driver device described herein. [Figure 3B] 1 shows an example of a cartridge for a microfluidic driver device described herein. [Figure 3C] 1 shows an example of a cartridge for a microfluidic driver device described herein. [Figure 4A] 1 is a cross-sectional view through a portion of an example cartridge for use with a microfluidic driver device. [Figure 4B] FIG. 1 is an enlarged view of the UV measurement area of ​​the cartridge. [Figure 5] 10A and 10B show schematic diagrams of exemplary UV measurement areas of a cartridge. [Figure 6] 1 is a graph showing the UV transmittance of materials forming an exemplary cartridge before and after gamma irradiation. [Figure 7] 1 is a graph of the change in detection signal over time for exemplary materials of the cartridge under long-term UV exposure. [Figure 8] 1 is an example of a dilution curve (graph) described herein. [Figure 9] 1 shows a schematic diagram of an example of a portion of a cartridge including a UV measurement region. [Figure 10A] FIG. 10 is a top view of an example cartridge for a microfluidic driver device that may include a UV measurement region. [Figure 10B]1 is an example of a cross-sectional view through one area of ​​an example cartridge that includes a UV measurement area. [Figure 10C] 1 shows an example of a portion of a cartridge for a microfluidic driver device, showing a schematic of a vacuum cap for removing air bubbles. [Figure 10D] FIG. 1 is a top view of an example cartridge ("biochip") containing a UV measurement chamber. [Figure 10E] FIG. 10 is another example of a top view of a cartridge including a UV measurement chamber. [Figure 11A] FIG. 1 shows a partial view of a cartridge containing a UV measurement area. [Figure 11B] FIG. 1 shows a partial view of a cartridge containing a UV measurement area. [Figure 12B] FIG. 11B is an enlarged view of the UV measurement area of ​​FIG. 11A. [Figure 12] 1 illustrates a schematic diagram of a method for processing polynucleotides, the method including using UV absorption measurements that can be used to estimate the concentration of the polynucleotide and / or to modify the processing of the polynucleotide. [Figure 13A] 1 shows another example of a UV measurement chamber. [Figure 13B] 1 shows another example of a UV measurement chamber. [Figure 14] 1 shows a schematic diagram of an example of a microfluidic device that includes a subsystem for determining polynucleotide concentration. [Figure 15A] 15A and 15B are graphs illustrating the range of intensity adjustment for an example UV light source (FIG. 15A) and the resulting light intensity (FIG. 15B). [Figure 15B] 15A and 15B are graphs illustrating the range of intensity adjustment for an example UV light source (FIG. 15A) and the resulting light intensity (FIG. 15B). [Figure 16A] 16A and 16B show examples of UV detection of concentration using the device described herein. 16A is a graph showing the detected signal (measured as voltage output from the UV detector) for a blank, a first sample, a blank, a third sample, a fourth sample, and a blank. 16B is a graph showing a comparison of a standard concentration detection system with the system described herein. [Figure 16B] 16A and 16B show examples of UV detection of concentration using the device described herein. 16A is a graph showing the detected signal (measured as voltage output from the UV detector) for a blank, a first sample, a blank, a third sample, a fourth sample, and a blank. 16B is a graph showing a comparison of a standard concentration detection system with the system described herein. [Figure 17] FIG. 10 is a top view of another example of a cartridge including a UV measurement chamber. [Figure 18] FIG. 18 is an enlarged top view of the cartridge area of ​​FIG. 17. [Figure 19] 1 is a flowchart illustrating an example of a method that can be used to assess the limit of quantitation. [Figure 20] FIG. 1 is a top view of an example backscatter shield. [Figure 21] FIG. 21 is a schematic cross-sectional side view of an arrangement including a cartridge with the backscatter shield of FIG. 20 during a UV measurement process. [Figure 22] FIG. 10 is a schematic cross-sectional side view of an arrangement including a cartridge with another example backscatter shield during a UV measurement process. [Figure 23] 10 is a flowchart illustrating an example of a method that can be used to account for bubbles in a fluid in a UV measurement chamber. DETAILED DESCRIPTION OF THE INVENTION

[0046] The devices and methods described herein can be used to generate and / or process polynucleotides, particularly therapeutic polynucleotides such as therapeutic mRNA.

[0047] These methods and devices can measure RNA or DNA concentrations as part of an integrated, closed-circuit system and use the resulting concentrations to modify polypeptide processing. Light absorption, specifically ultraviolet (UV) transmission through a UV measurement region of the closed cartridge, can be used to estimate the concentration of polynucleotides within the closed cartridge. The cartridges can have narrow path lengths and be adapted to optimize the signal from the absorption of UV light by the polynucleotide sample. Also described herein are microfluidic driver devices for operating with these cartridges. These systems can include determining the concentration of therapeutic polynucleotides at various stages of processing within the cartridge and further control operations within the closed cartridge to form and / or process (e.g., compound) the therapeutic polynucleotides, including controlling operation of a microfluidic driver device on the cartridge using the concentration data, either directly or based on UV absorption data proportional to the concentration of the therapeutic polynucleotide.

[0048] Generally, the methods and devices described herein may use UV absorption techniques to measure polynucleotide (e.g., RNA and / or DNA) concentration in a cartridge. These techniques may compare the level (and in some instances, the ratio) of transmitted UV light at wavelengths of about 250-270 nm (e.g., centered around 260 nm) from a polynucleotide sample (e.g., a therapeutic polynucleotide sample) to one or more "blank" solutions taken immediately before, after, or both. Based on the absorption of UV light by the polynucleotide and the path length of the UV light through the fluid (e.g., sample or blank), the device or method can determine the concentration of the polynucleotide.

[0049] These devices (and related methods) are particularly compact and can use readily available and easy-to-manufacture materials, including polymeric materials (e.g., plastics) that themselves absorb UV light. These methods and devices also allow for tight control of path length, which can be relatively small (e.g., 1 mm or less, e.g., 0.5 mm, 0.4 mm, 0.2 mm, 0.1 mm, etc.), thereby enabling sampling of very small volumes while achieving high accuracy and extended dynamic range. These methods and devices can also accommodate a large dynamic range of concentrations and noise levels by adjusting the dilution of the sample and / or the intensity of the UV light source and / or the sensitivity of the UV detector. This adjustment can be performed automatically, semi-automatically, or manually. In general, these methods and devices can be used with disposable or limited-use cartridges, along with durable / reusable microfluidic driver devices that operate with the cartridges. However, any of these techniques can be adapted for use with reusable cartridges and / or cartridgeless systems in which cartridge mechanisms are integrated into the microfluidic driver device.

[0050] In any of these methods and devices, a sample (e.g., a polynucleotide sample) can be microfluidically driven into a UV measurement chamber made from a relatively UV-absorbing material while minimizing the amount of UV-absorbing material (e.g., plastic) in the optical path. The measurement chamber may set the critical path length of light through the sample, which may be fixed. A UV light source and detector may be positioned on either side of the measurement chamber when the cartridge is installed in the microfluidic driver device. The optical path may be perpendicular to the cartridge. For example, FIG. 1 schematically illustrates an example microfluidic driver device 102 operating on a cartridge 105. The microfluidic driver device includes a UV light source (e.g., UV LED 101) positioned above the cartridge 105 and a UV detector 103 positioned directly below it. Alternatively or additionally, optical fibers may be used as either or both the detector and emitter (light source) to transmit and collect light. The light source transmits a UV light beam 111, which may be configured to contact a detection region within the measurement chamber 107. In any of these examples, the light source and detector may be spaced apart so that the beam (which in some examples is conical, but may also be parallel) interrogates only a defined region of the UV measurement field, resulting in the volume of the sample within the short optical path being known by the system.

[0051] Device The methods described herein can generally be implemented using a device that may be used with and / or include one or more cartridges (e.g., biochips) and a system (e.g., a microfluidic control system) configured to control operations within the cartridges. These microfluidic control systems may also be referred to herein as microfluidic devices, microfluidic control devices, microfluidic driver device control systems, microfluidic control systems, or microfluidic systems. The cartridges may be disposed within the microfluidic control system and may operate in a closed-circuit manner that prevents exposure to the atmosphere of some, or more preferably substantially all, or all of the component parts of the system's fabrication components. In particular, portions of the device that contact fluids within the system are prevented from exposure to the atmosphere. FIG. 2A illustrates an example of a microfluidic driver device control system that includes a microfluidic driver device management system 203 (including hardware for holding cartridges, applying positive / negative pressure to operate microfluidic operations within the cartridges, heating / cooling the entire microfluidic driver device, or regions thereof, detecting one or more mechanisms from the cartridges, and / or recording operations performed on one or more cartridges), a controller (not shown), and a refrigerated container 205 (e.g., an ISO Class 5 cabinet). The system may use or may include one or more cartridges 201. These cartridges may be any of the cartridges described herein, any of which may include one or more UV measurement regions.

[0052] The microfluidic device may be a microfluidic device for forming therapeutic polynucleotides (e.g., mRNA therapeutics) that may include: a mount for removably holding a cartridge; a plurality of pressure lines; a plurality of fluid vials, each fluid vial comprising or configured to couple with a fluid line, and each fluid line and at least a subset of the pressure lines configured to be biased against the cartridge held in a mounting mount to form a closed fluid path; and a controller configured to control the application of pressure through the pressure lines to drive fluid movement within the cartridge when the cartridge is held in the mount, the controller being configured to direct synthesis of a synthetic template, direct an in vitro transcription (IVT) reaction using the template to form a therapeutic polynucleotide, and estimate the concentration of the therapeutic polynucleotide, and / or direct purification of the therapeutic polynucleotide in one or more cartridges held in the mounting mount. Any of these devices may include an optical or UV concentration detection subsystem that includes one or more UV light sources and one or more UV detectors configured to be positioned over the UV detection area of ​​the cartridge when the cartridge is held or installed in the device.

[0053] A microfluidic device (e.g., a microfluidic device for forming therapeutic polynucleotides such as therapeutic mRNA) includes a mount (e.g., a mounting mount) for removably holding a cartridge; a plurality of pressure lines; a plurality of fluid vials, each fluid vial comprising or configured to couple with a fluid line, and each fluid line and at least a subset of the pressure lines configured to be biased against the cartridge held in the mounting mount to form a closed fluid path; and a device for controlling application of pressure through the pressure lines to drive fluid movement within the cartridge when the cartridge is held in the mounting mount. and a controller configured to: identify the contents of the fluid vial; transfer sub-microliter quantities of material from the fluid vial to one or more reactors in the cartridge held in the mounting mount; direct the synthesis of a synthetic template; direct an in vitro transcription (IVT) reaction using the template to form a therapeutic polynucleotide; direct the purification of the therapeutic polynucleotide in one or more microfluidic driver devices held in the mounting mount; and detect the concentration of the therapeutic polynucleotide and / or adjust the activity of the device based on the concentration.

[0054] The controller may be configured to perform any of the methods described herein, and in particular may be configured to receive inputs (e.g., light input, pressure input, temperature / heat input, UV absorption input, etc.) and process the inputs to control fluid movement within the microfluidic driver device, temperature (including thermocycling) of various regions of the microfluidic driver device, rinsing / bonding, opening / closing valves of the microfluidic device, sensing of the microfluidic device, etc. The controller may comprise one or more microprocessors, communication circuitry, memory, etc. The controller may comprise firmware, hardware, and / or software.

[0055] Any of these devices may include one or more (e.g., multiple) optical fiber sensors positioned around the mounting mount and reagent storage frame for monitoring fluid levels in the reagent storage frame and fluid movement within the microfluidic driver device when the microfluidic driver device is mounted in the mounting mount.

[0056] Alternatively or additionally, the optical sensor may be present at the bottom of the device (eg, below the installation mount) and may face upward to detect fluid volume, movement, etc.

[0057] As shown in FIG. 1, any of these devices may include a UV detection subsystem that includes a UV light source and a UV detector.

[0058] The described methods and devices generally include one or more fluid power circuits for moving materials (liquid materials) between fluid chambers (reservoirs, wetted sides, reactors, etc.) and channels of a microfluidic driver device, or within the microfluidic driver device, and in some cases between the microfluidic driver device and fluid reservoirs (vials, bottles, containers, etc.) within the device. The fluid power circuits may be hydraulic or pneumatic circuits that may include one or more pressure channels and pressure-receiving sides of a microfluidic device, particularly a chamber within the microfluidic device. The fluid power circuits are sometimes referred to as microfluidic power circuits. A single microfluidic chip may include multiple fluid power circuits, and the fluid power circuits may also include one or more pressure lines and interfaces between pressure lines of a microfluidic control device and one or more microfluidic chips within the microfluidic driver device. One or more fluid power circuits may share components (valves, pressure lines, vacuum caps, etc.) with other overlapping fluid power circuits. Furthermore, also for convenience, where the term "pneumatic" is used, it should be understood that general fluid power circuits (e.g., hydraulic and / or pneumatic) may be used instead or in addition. The fluid material driven by the fluid power lines may be any suitable fluid (e.g., gas or liquid, such as air, water, oil, etc.).

[0059] Also described herein are cartridges (e.g., closed-path microfluidic driver devices) for processing therapeutic polynucleotides in a closed path. As noted above, these cartridges may also be referred to herein as microfluidic chips, microfluidic pathway plates, process chips, biochips, process plates, etc. Generally, cartridges may be substantially flat, plate-like structures, and these structures may be relatively thin (e.g., less than a few millimeters thick, e.g., about 0.5 to about 20 mm, about 0.5 to about 15 mm, about 0.5 to about 10 mm, etc.). The cartridges described herein generally may be at least partially transparent, particularly a transparent top portion of the cartridge, such that one or more optical sensors (e.g., cameras, CCDs, fiber optics, etc.) may be used to sense, detect, monitor, record, etc., motions, including fluid movement and / or elastic layer movement, by the microfluidic driver device when used with the microfluidic devices described herein.

[0060] 2B is a schematic diagram of one example of a microfluidic driver device control system that can be used as described herein. In this example, the device includes a housing 233 that encloses a mount 215 that can hold one or more cartridges 211, which can be single-use devices. The housing can be a chamber, enclosure, etc., which can include a lid or opening and can be sealed when closed. The housing can enclose a thermal regulator and / or be configured to be enclosed within a thermally regulated environment (e.g., a refrigeration unit, etc.). The housing can form a sterility barrier. In some examples, the housing can form a humidified or humidity-controlled environment.

[0061] The mount 215 may be configured to secure the cartridge using one or more pins or other components configured to hold the cartridge in a fixed and predetermined orientation.

[0062] In some examples, a thermal control 213 may be located adjacent to the mount 215 to regulate the temperature to one or more cartridges 211. The thermal control may include a thermoelectric component (e.g., a Peltier element) and / or one or more heat sinks to control the temperature of all or a portion of the cartridge. In some examples, one or more thermal controls may be included to separately regulate the temperature of one or more regions of the cartridge. The thermal control may include one or more thermal sensors (e.g., thermocouples, etc.) that may be used for feedback control of the microfluidic driver device and / or the thermal control.

[0063] 2B, fluid interface assembly 209 couples liquid reagents and / or pressure (e.g., gas) to cartridge 211 held in location mount 215 and may assist in the delivery of fluid materials and positive / negative gas pressure from pressure source 217 to the interior of cartridge 211. The fluid interface assembly may optionally assist in securing the cartridge, as described in more detail below. The fluid interface assembly may be removably coupled to the device (and may be detached, or portions may be removed) for sterilization between uses.

[0064] The reagent storage frame 207 may be configured to include a plurality of fluid sample holders, each of which may hold a fluid vial configured to hold a reagent (e.g., nucleotides, solvents, water, etc.) for delivery to the cartridge 211, or the fluid vial may be configured to receive a product from inside the cartridge 211. The reagent storage frame may also be referred to as a reagent rack. In some examples, the reagent rack includes a plurality of pressure lines and / or manifolds configured to divide one or more pressure sources 217 into multiple pressure lines that may be applied to the cartridge and controlled independently or collectively (in subcombinations). Alternatively, the fluid reservoirs (e.g., vials) may be configured to secure and seal directly to the cartridge.

[0065] The fluid interface assembly may include multiple fluid and / or pressure lines and may include biased (e.g., spring-loaded) holders or tips that individually and independently drive each fluid and / or pressure line into the cartridge when the cartridge is held in mount 215 (alternatively, as noted, the device may be directly spring-mounted). Tubing, e.g., fluid and / or pressure lines, may be part of and / or connected to the fluid interface assembly. In some examples, each fluid line includes flexible tubing connecting between the reagent storage frame and the cartridge via a connector that couples a vial to the tubing with a locking engagement (e.g., a ferrule). The ends of the fluid pathways, in some examples, the ends of the fluid lines and pressure lines, may be configured to seal against the cartridge, as described herein, for example, at a sealing port formed in the cartridge. For example, the ends of the fluid lines may be cut or shaped to be flat (vertical inside view). The vial can be pressurized (e.g., to a pressure greater than about 1 atm, e.g., about 2 atm, about 3 atm, about 5 atm, etc.) through a connector that can also be connected to a pressure source. For example, the fluid vial can be pressurized to about 1 to about 20 psig (e.g., about 5 psig, about 10 psig, about 20 psig, etc.). Negative or positive pressure can be applied, for example, a vacuum (e.g., about -7 psig or about 7 psia) can be applied to draw the fluid back into the vial (e.g., reservoir) at the end of the process. Generally, the fluid vial can be operated at a lower pressure than the pneumatic valve, which can prevent or reduce leakage. In some examples, the pressure differential between the fluid valve and the pneumatic valve can be about 5 psi (e.g., about 7 psi, about 10 psi, about 12 psi, about 15 psi, about 20 psi, etc.).

[0066] Each vial may be coded (e.g., with an identifier that can be read by one or more sensors, as described below). The controller can monitor the fluid levels and, therefore, the amount of each material in the fluid interface assembly.

[0067] The device may also further include a magnetic field applicator 219 that may be configured to generate a magnetic field in the region of the cartridge 211. One or more sensors 205, which may be optical sensors, may be part of the device and may sense one or more of a barcode, fluid levels in fluid vials held in a reagent storage frame, and fluid movement within the cartridge 211 when the device is loaded into the mount 215.

[0068] Any of these devices may include a UV emission / detection subsystem (e.g., concentration estimation subsystem 233) capable of measuring the absorption of UV light (e.g., approximately 260 nm). Based on these measurements, the process in the device can be controlled, for example, by measuring one or more "blanks" containing no polynucleotides and comparing them to samples containing processed polynucleotides. In some examples, visual / optical markers can be used to estimate yield. For example, fluorescence can be used to detect process yield or residual material by tagging with a fluorophore. Alternatively or additionally, dynamic light scattering (DLS) can be used to measure particle size distribution within a portion of the microfluidic driver device (e.g., a mixing section). In some examples, sensor measurements can be performed using one or two optical fibers to carry light (e.g., laser light) and detect the emerging light signal. The instrument package may be mounted remotely from the device. Such non-contact sensing may be preferred.

[0069] In any of the methods and devices described herein, a sensor (e.g., a video sensor) may record all activity on a cartridge (e.g., a chip). For example, an entire run for synthesizing and / or processing a material (e.g., a therapeutic RNA) may be recorded by one or more video sensors, including a video sensor that can visualize the cartridge, e.g., from above. Processing on the cartridge may be visually tracked, and this record may be kept for later quality control and / or processing. Thus, video recordings of processing may be saved, stored, and / or transmitted for subsequent review and / or analysis.

[0070] The interior of the device, for example, the housing 233, may be further configured to be sterilizable. Specifically, portions of the device can be removed and sterilized separately. Sterilization may be performed, for example, by UV irradiation or any other sterilization method that may be required to limit contamination or to meet regulatory requirements. The device, including the housing, may be contained in a High Efficiency Particulate Air (HEPA) filtered environment. The device, including the housing, may be contained in a temperature-controlled enclosure. Additionally, the device itself may include one or more temperature-controlled areas. In any of the devices described herein, the device may include a temperature-controlled area (e.g., within the housing) for storing reagents and / or mRNA (e.g., therapeutic mRNA) at a storage temperature (e.g., about -10°C to about 20°C, e.g., about 10°C, about 4°C, about -10°C, etc.). Any of these devices may include a library of manufactured mRNA, which may be configured individually or in combination with one or more additional mRNAs and delivery vehicles.

[0071] As described above, the microfluidic driver device controller system may be controlled by controller 221 and includes at least applying pressure through cartridge 211 to drive fluid movement. The controller may be completely or partially external to the housing. The controller may be configured to include user input / output. For example, a user interface 223 for the system may allow for easy operation and command of the device and cartridge. Any of the devices described herein may include all or some of the components shown in FIG. 2B, although not all components are required. In FIG. 2B, only some of the connections between components are shown, and additional (or alternative) connections may be used.

[0072] The microfluidic driver device control system can support all production activities within the microfluidic driver device, such as reagent delivery, fluid control, temperature control, mixing, purification, and process monitoring. Manufacturing activities on the microfluidic driver device control system can be accessed and controlled through application software.

[0073] A cartridge can be configured to contain one or more reactors for manufacturing operations performed to precisely prepare a therapeutic (e.g., therapeutic mRNA) substance. The same microfluidic driver apparatus can operate on more than one cartridge, either in series and / or parallel, without interrupting the continuous path nature of the microfluidic driver apparatus control system. For example, when manufacturing a therapeutic using multiple processing operations performed in multiple reactors using multiple cartridges, a fluid product, including a partial product, from one cartridge can be transferred by the apparatus to one or more additional cartridges in a closed-loop manner, for example, by moving a fluid containing the cartridge product into a storage reservoir of the microfluidic driver apparatus control device.

[0074] Each cartridge can be configured to contain one or more reactors for processing during the manufacturing process. For example, Figures 3A-3C show three example cartridges. These examples show three different types of cartridges: a template cartridge (Figure 3A), an in vitro transcription (IVT) cartridge (Figure 3B), and a formulation cartridge (Figure 3C). Each of these example cartridges can be configured to contain mechanisms for performing a set of unit operations in a controlled and highly reproducible manner.

[0075] In some examples, the cartridge may be configured as a multi-layer structure consisting of two more rigid layers and a flexible membrane sandwiched between two raised layers. Figure 4A shows a cross-sectional view (across the plane of the cartridge) of an example cartridge having multiple layers forming a reactor for processing a therapeutic agent described herein. The reactor may include a chamber, including seals, channels, valves, and a pumping chamber, formed from multiple layers. For example, the cartridge may be formed of two or more rigid or semi-rigid plates 403, 405 and at least one elastic layer 407. The elastic layer 407 may be a sheet of elastic material that is liquid-impermeable. The elastic layer may be somewhat gas-permeable or may include various regions that are treated to be more or less gas-permeable. While a single continuous sheet of elastic material may be used, in some examples, multiple sheets of elastic material may be used, or multiple sheet pieces may form a "sheet." The layers and elastic sheets may be laminated together. Generally, chambers for holding, valving, and / or pumping fluids may be formed in the plates on either side of the elastic layer, such that the elastic layer bisects the chamber into a liquid-containing side and a pressure (e.g., gas) application side. The total volume of the chambers may be constant and formed in both the first (e.g., upper) and second (e.g., lower) plates, but this volume may be divided into a pressure side and a liquid side. Applying positive or negative pressure to the pressure side can deform the elastic sheet, decreasing the volume of the liquid-containing side (down to zero, closing the chamber) or increasing the volume of the liquid-containing side (up to a predetermined maximum). The pressure application sides of the chambers may be connected, for example, via pressure ports 443 in the upper plate (first layer) 403, which connect to pressure channels 447, to apply negative or positive pressure to the pressure-receiving sides 419 of one or more chambers. The liquid-containing side 417 opposite the pressure application side of each chamber may be connected to a fluid port 423 via a fluid channel 421.Both fluid and pressure ports may be formed by openings in the upper plate (upper layer) 403 and elastic layer 407, so that pressure lines are pressed into the elastic layer 407, which is supported on the underside of the port by an opposing rigid or semi-rigid layer 405, allowing for sealed connections isolated from the atmosphere, even when there are multiple different input lines.

[0076] In FIG. 4A , cartridge 400 includes a first (e.g., upper) layer (in this example, a plate) 403 having a first (e.g., top or upper) surface 411, a second (e.g., bottom or lower) surface 429, and a thickness therebetween. First surface 411 may form the exposed outer surface. The cartridge also includes a second layer (e.g., a plate) 405 having a first (e.g., upper or top) surface 431, a second (e.g., lower or bottom) surface 433, and a thickness therebetween. Resilient layer 407 is sandwiched between second surface 429 of first plate 403 and first surface 431 of second plate 405. The layers shown in FIG. 4A may not be to scale (e.g., resilient layer 407 may be thinner than the plates).

[0077] 4A may also include multiple chambers 415, 416, 420, each having a fixed volume. These chambers are formed by cut-out areas (e.g., rounded / curved cuts) in the second (bottom) surface 429 of the first plate 403 and the first (upper) surface 431 of the second plate 405. The elastic layer 407 bisects these chambers 415, each including a liquid-containing side 417 and a pressure (e.g., gas-containing) side 419. The cartridge 400 may also include multiple liquid (e.g., fluid) channels. In FIG. 4A , a single fluid channel 421 is shown extending from a fluid port 423 through the thickness of the first plate 403 to a fluid channel opening 425, through the elastic layer 407, through most of the thickness of the second plate 405, and down to the bottom region 433 of the second plate, where a length of the fluid channel 421 is formed that extends parallel to the bottom surface of the third plate.

[0078] With respect to fluid port 423, the diameter of the opening into the first plate forming fluid port 423 that extends through the thickness of first plate 403 may be larger than the diameter of fluid channel opening 425 that extends through elastic layer 407 into liquid (e.g., fluid) channel 421. Fluid channel opening 425 may be centered with respect to the bottom of the fluid port opening and may be offset from the wall of the fluid port opening by at least the expected wall thickness of the fluid line or fluid line connection interface that connects to the fluid port.

[0079] Fluid channel 421 connects to the liquid-containing side 417 of first chamber 415. This first chamber has a relatively small holding volume (a fixed volume), but can be configured as a valve that can be fully opened and closed by movement of elastic layer 407.

[0080] The cartridge 400 also includes multiple pressure channels that can be independently controlled to apply positive and / or negative pressure. While FIG. 4A shows a single pressure port 443 connected to the fourth chamber 420, chambers 415, 416 may each be connected to a separate pressure port and pressure channel to independently manipulate and control the movement of the portion of the elastic layer 407 that bisects the chambers, independently valving and / or pumping each chamber. In some examples, a pressure port may be shared among multiple chambers. In FIG. 4A, the pressure (e.g., gas) port 443 is similar to the fluid (e.g., liquid) port 425 and includes an opening that extends completely through the first plate 403, into the exposed elastic layer 407, and into an opening through the elastic layer to form a pressure (e.g., gas) channel opening 445. Pressure channel opening 445 connects with pressure (e.g., gas) channel 447 that extends from pressure port 443, penetrates most of the thickness of first plate 403, enters a channel along the second plate, passes through the second plate and elastic layer 407, and returns upward to the region of the pressure channel in the first plate that connects to pressure (e.g., gas) containing portion 419 of fourth chamber 420. As described for analogous fluid (e.g., liquid) ports, the diameter of pressure port 443 through the thickness of first plate 403 may be larger than the diameter of pressure channel opening 445 through elastic layer 407, and may be centered or offset greater than the wall thickness of the pressure line or pressure line connecting interface that connects to the pressure port.

[0081] In the cross section through cartridge 400 shown in FIG. 4A , there are multiple connections to other fluid (e.g., liquid) lines, fluid ports, pressure lines, and pressure ports, which are not shown, but may be outside the plane of the illustration. For example, in FIG. 4A , the liquid-containing side or portion 417 of the fourth chamber may be connected to additional valves (chambers) and / or channels, including, for example, an outlet channel extending from the liquid-containing side 417. Although not shown, additional chambers (e.g., configured as valves) may be formed as described above. In some examples, the outlet channel may deliver fluid from one or more chambers through another fluid port (not shown) to a fluid-receiving reservoir, e.g., a vial, tube, or the like. This receiving reservoir may be held within a reagent storage frame.

[0082] In general, this configuration of microfluidic driver device and cartridge is configured so that multiple complex operations can be performed by the device on the cartridge in a fully enclosed (sealed and protected from the atmosphere) manner without the need for manual intervention. Fluids are metered using fixed volume chambers and can be moved, mixed, filtered, etc. by applying air pressure to deflect regions of the elastic layer.

[0083] In some examples, a chamber in the cartridge may be configured as a mixing chamber for mixing fluids within the cartridge. In some examples, a chamber may be configured as a purification chamber that may contain a filter material. In some examples, one or more chambers may be configured as a concentrator for concentrating a therapeutic substance.

[0084] Various cartridges may have different arrangements of channels, ports, and chambers, but they may share a similar basic architecture and numerous functional elements that can be used in different configurations to perform different protocols. Functional elements include input ports, metering valves, pumps, reaction chambers, mixing and purification structures, and / or UV sensing regions, as described herein. In FIG. 4A, UV sensing region 493 includes a thin UV measurement chamber 481, and the passage of materials into and out of this UV sensing region can be regulated by a controller using one or more valves and by controlling the pressure within these channels.

[0085] Any of these cartridges may include one or more bubble removal chambers, or any of the chambers on the wetted side of the chamber may be configured as a bubble removal chamber, allowing air bubbles in the fluid on the fluid-receiving side to be removed. A bubble removal chamber, sometimes referred to as a vacuum cap, may generally be configured to apply negative pressure to the other side of a membrane while fluid is held within the wetted side of the chamber. The membrane may be at least partially gas-permeable, as described above. FIG. 10C shows an example of a bubble removal chamber. All or more preferably a portion 1988 of the membrane dividing the chamber (e.g., only the cap region) may be in contact with a vacuum through a vacuum line 1987, for example, on the top surface or upper plate of the device, as shown in FIG. 10C. During operation, the vacuum cap 1938 retains fluid within the wetted side of the chamber, and by applying negative pressure to the upper (pressure-receiving) side of the chamber, air bubbles within the line may be removed or reduced. The membrane dividing the chamber into a liquid-contacting side and a pressure-receiving side may be gas-permeable, such that gas (e.g., air, nitrogen, etc.) can be drawn through the membrane covering the flow path, thereby removing gas from the liquid (fluid) side by negative pressure. For example, the membrane (or a region of the membrane within a vacuum cap) may be, for example, a PolyDiMethylSilicone (PDMS) elastomer film that is sufficiently gas-permeable to allow gas to be removed from the liquid side of the membrane. A fluid chamber (e.g., formed between a first plate and a second plate) having a fixed volume as described herein may comprise or be coupled to one or more bubble elimination chambers (vacuum caps) and / or may be configured as a bubble elimination chamber. In some examples, the portion of the elastic layer disposed between the first and second surfaces that forms the chamber, e.g., that divides the liquid-wetted side of the second surface (and / or second plate) from the pressure-receiving side of the first surface (and / or first plate), may be minimally deflected (or may not be deflected at all). For example, the upper pressure-receiving side may be minimally spaced from and / or nearly flush with the relaxed membrane (e.g., flat), while the liquid-wetted side is concave and extends to the second surface (second plate).The controller may retain fluid within the vacuum cap region by, for example, applying positive pressure to the pressure-receiving side of the valve and negative pressure to the pressure-receiving side of the vacuum cap, for example, by closing valves on one or both sides (inlet and outlet) of the vacuum cap. The absolute amount of negative pressure applied (e.g., magnitude of negative pressure) may be less than that applied to deflect the membrane (e.g., less than the absolute value of the positive pressure applied to close the valve and / or pump). Alternatively, in some examples, the membrane may be configured to deflect (e.g., deflect upward) against the first surface and / or plate, for example, to draw fluid from the input 1989 into the enlarged, wetted side of the chamber. The membrane may be held by negative pressure applied against the first top surface, which may allow bubbles (e.g., air bubbles) to be removed. The controller may hold the fluid in the vacuum chamber for a period of time sufficient to remove all or a portion of the gas (e.g., about 1 second or more, about 5 seconds or more, about 10 seconds or more, about 20 seconds or more, about 30 seconds or more, about 1 minute or more, about 1.5 minutes or more, about 2 minutes or more, about 5 minutes or more, about 1 second to about 5 minutes, about 2 seconds to about 5 minutes, about 5 seconds to about 5 minutes, etc.). In FIG. 10C, pressure may be applied through pressure line 1987, which communicates with the pressure-receiving side of a chamber formed between the first and second surfaces (e.g., the first and second plates) of the device. Vacuum cap 1938 may be valved by one or more valves 1992. Fluid may exit the wetted side from fluid line 1989 on the other side of the vacuum cap.

[0086] The cartridge can interface with the microfluidic driver device control system through a set of spring-loaded connections for both reagents, as well as pneumatic lines used to manage fluid movement and valve control. The reagent and gas lines can be sealed by pressure against an elastomer layer embedded within the cartridge, creating a completely sealed pathway from the reagent vial into the cartridge and from the cartridge to the export vial. The sealed pathway can be maintained throughout the entire reaction within the cartridge, effectively eliminating any contact with the atmosphere and minimizing the risk of contamination.

[0087] The microfluidic driver device control systems described herein may provide a sterile controlled environment and may include interfaces for loading reagents and retrieving outputs. In any of the devices (e.g., systems) described herein, the device may include an enclosure that provides a controlled environment, and the enclosure may be located within the controlled environment. For example, the enclosed device may be a Class 5 environment that may be located within a Class 7 environment.

[0088] The microfluidic driver device may control the system of the microfluidic driver device and provide single-step connection to all actuators. These control systems may also scan all reagent and cartridge identifiers (e.g., barcodes) and monitor fluid levels. Generally, these microfluidic driver device control systems may automate all or some of the microfluidic driver device functions and generate visual records of all process operations that may be monitored (e.g., for optical quality control analysis of intermediate process outputs, etc.), stored, transmitted, or later reviewed.

[0089] As described above, the microfluidic driver device control system may include hardware such as a nest (microfluidic driver device holder) designed to precisely align cartridges so they can only be inserted in a single orientation. This can be achieved, for example, through two pins and / or notches in the nest that are adapted to the shape of the cartridge. The microfluidic driver device management system (control system) also includes vial racks for holding reagent and export vials, a downward-facing camera for recording all fluid and valve movements, and product export. A side camera on a rail for capturing barcodes and detecting fluid levels, and a robotic arm with magnets for bead manipulation, hold the cartridge in place using a vacuum chuck that ensures good contact with the Peltier element for temperature control. Once the cartridge is in place, mating with all connectors is achieved in a single motion by lowering the top of the microfluidic driver device management system via a dowel pin guide system.

[0090] FIG. 4B schematically illustrates an example of a UV measurement region of a cartridge described herein. In this example, similar to FIG. 4A, the cartridge includes an upper first layer 403 and a lower second layer 405. A resilient material 407 forms a layer between the first and second layers. UV measurement region 493 is formed by cutting down the thickness 495 of the first region to form first UV measurement region 496, and similarly cutting down the thickness 494 of the second layer to potentially form second UV measurement region 498. One or more fluid channels 421 may be positioned to fluidly connect UV measurement chamber 481 to a source of therapeutic polynucleotide and / or a source of “blank” solution (e.g., water, buffer, saline, etc.).

[0091] 4B, a UV measurement chamber 481 is formed between the first and second layers in the area where the elastic material 407 has been removed or is not present. Thus, the UV measurement chamber has the same thickness as the elastic layer 407. UV light may enter the first UV measurement area (cut-out area), pass through the area of ​​the first layer thickness 491, then pass through any material such as the sample material and / or bank material, be retained within the UV measurement chamber 481, and then exit through the area of ​​the second layer thickness 499 into the second UV measurement area 489, where the remaining (unabsorbed) light may be detected.

[0092] In the example shown in FIG. 4B, the UV measurement region is formed by an undercut region in first plate 496 and an undercut region in second plate 498 (in a 1.5 mm thick first layer), each having dimensions of approximately 2.5 mm width and approximately 1.3 mm depth. In this example, the second layer is a mirror image of the first layer. Thus, the polymer material forming the "windows" on each side of UV measurement chamber 481 has a thickness of approximately 0.2 mm on each side of the UV measurement chamber. The UV measurement chamber can hold a known volume and can have a thickness of, for example, approximately 0.1 mm to 5 mm (e.g., 0.1 to 0.5 mm, etc.).

[0093] Because the methods and devices described herein use techniques that measure a blank (e.g., a solution identical to the polynucleotide-containing solution except for the absence of polynucleotide), the overall transmittance of the cartridge is not explicitly required for measuring absorbance. However, the transmittance of the cartridge window material can have strong indirect consequences through signal / noise and measurable sample concentration. UV-opaque windows could not be used to measure UV transmittance. Most microfluidic materials (e.g., cyclic olefin copolymer (COC), cycloolefin polymer (COP), and polydimethylsiloxane (PDMS)) significantly absorb UV at the same wavelengths where DNA / RNA characteristically absorbs. Therefore, in any of these examples, the measurement region (UV measurement chamber) can be thinned, for example, to approximately 0.1 mm to 0.5 mm. The layered structure of the cartridge may be modified to control the critical path length. For example, one of the cartridge layers may be completely removed from the UV measurement region (e.g., an elastic layer). This can be advantageous because the layer thickness can be more tightly controlled than is typically possible by machining / molding structures within the layer. Generally, for samples with higher concentrations, a thinner path length can provide better measurements (or enable measurements) compared to samples with lower concentrations. For example, for measurements of lower concentrations (e.g., when forming a template material for synthesizing polynucleotides), a chamber with a base material with a height of about 1 mm (e.g., reflecting a 1 mm PDMS elastic layer) may be used. In some instances, for measurements of higher concentrations (e.g., when performing an IVT process), a chamber with a height of about 0.1 mm (e.g., a 1 mm PDMS elastic layer) may be preferred.

[0094] Figure 5 shows the profile of an example cartridge demonstrating that thin material and short path lengths can be achieved by using elastomer layer thicknesses within the cartridge. In Figure 5, the cartridge comprises a first layer 503, a second layer 501, and a channel 521. In this example, the UV measurement chamber 581 has a thickness of approximately 0.1 mm (path length is 0.1 mm).

[0095] For sterilization, cartridges are often treated with gamma radiation before use. While this has the advantage of destroying contaminants, it can also change the material properties of the cartridge; gamma radiation treatment has been found to change (e.g., decrease) the US transmittance of various materials used in some of the exemplary cartridges herein. For example, the UV transmittance of one component of the cartridge (COC) becomes significantly more UV-opaque after gamma treatment (see FIG. 6). Without the thin-walled design shown in FIGS. 4B and 5, it would be completely opaque to a UV detector.

[0096] UV transmittance through gamma-treated COC can depend on the degree of UV exposure, following an unusual trend of becoming more transparent with moderate exposure and eventually becoming less transparent with prolonged exposure. See, for example, Figure 7. In this example, the transmittance of the polymeric material (e.g., COC) initially and surprisingly increases with exposure, followed by a decrease in admittance over time. For example, because the transmittance of the cartridge changes during exposure, in some of the devices and methods described herein, it is beneficial to accurately measure the concentration, but first scan with a blank, and possibly (optionally) after using three novel mitigation measures. That is, for example, a "blank" measurement can be taken before and after a "sample" measurement to trend the cartridge's transmittance change and use these values ​​to estimate the material response during the sample measurement.

[0097] The methods and devices described herein may pre-treat the cartridge with UV light so that it is in a stable, high-transmission region of the curve during measurement ( FIG. 7 ). Generally, UV light may be applied (e.g., by pulsing or by using a dimming mechanism) only when needed to obtain measurements, thereby minimizing exposure, and a dimming mechanism is utilized so that the LED light is just bright enough to obtain a good signal and avoid overexposure. The LED light can be adjusted automatically or manually based on the detector reading to always ensure a good signal reading from the detector.

[0098] Any of the devices described herein may be configured to perform measurements on a series of dilutions (dilution series), as shown in FIG. 8. Generally, a microfluidic device may be configured to perform serial dilutions around a measurement chamber. The process may consist of microfluidically transferring an aliquot of a reaction to the measurement chamber, performing a measurement, and then diluting the aliquot for subsequent measurements. The diluted aliquot can be diluted multiple times and measured each time to generate a graph such as that shown in FIG. 8. At the end of the measurement, a pure buffer solution is passed through the measurement chamber to rinse, collect a "blank" reading, and correct for UV exposure effects. The graph in FIG. 8 illustrates the mechanics and utility of this process. For example, a sample may initially be too concentrated and therefore completely opaque to the UV wavelength of interest (e.g., 260 nm). This is shown at point 801 in the upper right corner of the graph in FIG. 8. Serial dilutions (illustrated at 70%) result in measurements of a lower concentration but still effectively opaque stage. After multiple dilutions, the measurements enter a linear regime, eventually reaching point 805 in the lower left corner of the graph in Figure 8, where the aliquot becomes indistinguishable from the blank. UV absorbance can be used to directly determine concentration along the linear regime until point 805 is reached, at which point UV absorbance measurements may no longer be useful. From the slope of the linear regime, the device or method can also determine the dilution ratio (i.e., 70% to 73%). Using the number of dilution steps, the dilution ratio, and the measured concentration at that point in the dilution, the device can determine the original aliquot concentration. Additionally, the linear portion of the curve indicates the validity of the measurement, as a nonlinear component may indicate that the signal is close to the detector noise level or that there is an unexpected time-varying component or dilution error.

[0099] Figure 9 shows one possible configuration of a portion of a cartridge configured for serial dilution. In this example, the device includes a UV measurement chamber 981 flanked by fluid chambers 984, 985 on either side of the UV measurement chamber, along with a series of valves 976, 977, 978. The chambers are in fluid communication with a source 972 of polynucleotide input and a source 973 of buffer used for dilution ("blank"), as well as a waste output 975. By selectively opening and closing the valves as well as pressurizing the measurement chambers 985, 984, the device (e.g., a controller) can empty one chamber to waste and refill it with fresh buffer to generate a dilution. Additionally, controlling the pressure on either side of the measurement chamber can enable bubble removal techniques such as fluid sweeping and forcing air through a gas-permeable flexible membrane, as described above.

[0100] Generally, these methods and devices can use the determined nucleotide concentration to control the operation of the device. For example, these devices and methods can be configured to pool multiple batches of polynucleotides. The measured concentration can be automatically compared by a controller against nominal and acceptable ranges. If the comparison is favorable, the batches can be pooled into a common container. If not, the controller can microfluidically direct it to a waste container or for further analysis. In some examples, the device can use the determined concentration to normalize the system output. The RNA / DNA concentration can be measured and sent to a product container. The volume of the added product can be read in the product container (e.g., by mass or imaging), and this concentration and volume, along with the target concentration, can be used to calculate and microfluidically add a volume of diluent. Alternatively, or in addition, these methods and devices can adjust or control any of the processing (parametric) elements when processing polynucleotides. For example, the measured concentration can be automatically compared by a computer against nominal and acceptable ranges. The product can be parametrically released for use, in addition to other process parameters such as temperature history and reagent usage. Abnormal products may be screened and the process investigated.

[0101] 10A-10E and 11A-11B show additional examples of cartridges that can be used as described herein. FIG. 10A shows one example of a cartridge 1900. This example also includes a permeable insert 1969 within the liquid-wetted side of chamber 1957. In FIG. 10A, cartridge 1900 can include at least one pair of chambers 1953, 1957, 1957′, each of which can include a liquid-wetted side 1917, a pressure (e.g., gas) side 1919, fluid connections, pressure connections, and fluid / pressure lines that can be formed within the thickness of the microfluidic driver device. In some examples, the chambers can be paired, with each chamber of the pair connected to one another by a fluidic connector 1955. The fluidic connector 1955 can be used in coordination with positive and / or negative pressure applied to the pressure sides of the chambers to drive liquid on the liquid side between the two chambers and mix the liquid within each chamber. The chamber may be bisected by an elastic material (e.g., an elastic layer or membrane), and by deflecting the elastic material within a fixed volume of the chamber, any liquid within may be driven into or out of the liquid-contacting side of the chamber (e.g., between the two chambers).

[0102] The cartridge 1900 may include more than one pair of chambers, any of which may include a permeable insert. Each pair of chambers may be used for a different process. For example, the first pair of chambers 1953 may be used for RNA synthesis. The second pair of chambers 1957, 1957' may be used for purification of the synthesized polynucleotides. Fluid from the first pair of chambers 1953 may be driven into the second pair of chambers by applying pressure to the pressure-receiving side 1919 of each chamber and opening a valve 1959 between the first pair of chambers 1953 and the second pair of chambers 1957. The valve chamber 1959 may be formed by an elastic layer 1907 within the connector channel between the two pairs of chambers.

[0103] 10A and 10B can have multiple pressure and fluid ports 1923, 1923′, which can be disposed adjacently around the periphery of the cartridge and configured to connect to the fluid interface assembly 109, as described above.

[0104] Valve 1961, which can control the timing of reagent delivery driven from fluidic port 1923 but can also enable metering into chambers of the device when placed in series with one or more similarly constructed valves, can have ports (e.g., sealing valves) formed from the elastic layer along the length of connecting channel 1939 (either a pressure channel or a fluidic channel), as shown, for example, in FIG. 10A. For example, in FIG. 10A, three valve chambers are shown (described in more detail below), and the first of these three valves can act as a peristaltic pump, while the intermediate valves can be metering chambers that meter small volumes (e.g., having metering volumes of about 10 nL, about 20 nL, about 25 nL, about 50 nL, about 75 nL, about 100 nL, etc.). The size of the channels, and particularly the size of the chambers connected to the channels, can meter the volume dispensed along fluidic connecting channel 1939, 1921 and delivered into chamber 1953 connected to fluidic connecting channel 1939, 1921. In some examples, the metered volume may be as little as 50 nL. Metered volumes of about 100 nL, about 1 microliter, about 5 microliters, or more may be transferred. Various valve sizes may be preselected for incorporation into the microfluidic driver device 1900, and reagents may be connected to the appropriate metered size at the user's choice.

[0105] Additionally, more than one valve body 1961 may be included in a row along the fluid connecting channel 1939. A series of valves 1961 may also act as a peristaltic pump for moving fluids, including (but not limited to) viscous fluids. The ability to function as a peristaltic pump for fluids may be particularly advantageous for moving fluids that may be viscous or that may contain suspended particles, such as purification or capture beads.

[0106] As noted above, cartridge 1900 may also include a delivery or export reservoir or reservoir 1963. In FIG. 10A, the preselected volume may be formed similarly to the chamber structure described above, or may include only a metering side, as desired. In either case, a valve can be used to meter the desired volume into reservoir 1963. Valve 1965 can control the delivery of fluid from reservoir 1963. If a larger volume is desired, delivery may be repeated. Alternatively, if reservoir 1963 is preselected to be the export reservoir, valve 1965 can be opened to deliver fluid from chamber 1957, while valve 1967 remains closed, allowing only a measured volume of fluid to be exported to reservoir 1963. This fluid can then be exported to a fluid vial on the reagent storage frame for further processing or testing. In some examples, the chamber, reservoir, or storage (e.g., 1963) may be configured as a metering section of a 1 μL pump formed, for example, by three valve structures (1967, 1965, 1967). The chamber may be configured to transport waste from, for example, mixing chamber 1957.

[0107] The cartridge 1900 can be a closed pathway structure. While the fluid vials, fluid lines, and microfluidic driver devices are connected, device operations can be performed without any exchange of materials in or out of the system, particularly in or out of the cartridge flow channels, for processing, including synthesizing polynucleotides (e.g., RNA) and preparing them for biological delivery (as therapeutics, such as drugs, vaccines, etc.). Thus, the entire system can operate as a closed pathway and / or individual microfluidic driver devices can operate as closed pathways (protected from the atmosphere) within the system.

[0108] Some examples of cartridge 1900 may further include a concentrator within the chamber, which may be disposed within the thickness of the second plate and in fluid communication with an outlet channel such as 1949. The polynucleotides may be concentrated by expelling excess fluid medium, and the concentrated polynucleotide mixture may be exported from cartridge 1900 for further handling or use. In some examples, the concentrator may be a dialysis chamber. For example, a dialysis membrane may be present within or between the plates of the microfluidic driver device.

[0109] The cartridge 1900 may be formed from a material that is at least substantially translucent to visible light, and in some examples, the material may be substantially transparent to ultraviolet light (or both visible and UV light). In some examples, the microfluidic driver device 1900 may be formed from a material that is substantially transparent to visible and / or ultraviolet light.

[0110] As described above, the cartridge may be formed of two or more plates stacked on top of each other, forming chambers and / or channels between the plates, and a resilient material may be sandwiched between the first and second plates. The first and / or second plates may be formed from a rigid material. The plates may be formed from the same material or different materials. For example, the rigid material may be a polymer or glass. The polymer or glass may be biocompatible, e.g., it does not leach any monomers or soluble small molecules that are toxic to living cells. Any suitable biocompatible polymer may be used, including medical-grade polycarbonate urethane, silicone polycarbonate urethane, and polyether urethane, among others. In some examples, the polymer may be a cycloolefin copolymer.

[0111] FIG. 10B shows a cross section of a portion of the cartridge, showing the UV measurement chamber 1981 of the UV measurement region 1993. Thus, the microfluidic driver device may be configured as a multi-layer structure composed of two more rigid layers 1903, 1905 and a flexible membrane 1907 sandwiched between two ridged layers. FIG. 10B shows a portion of a cross section (across the plane of the cartridge) of an example cartridge having multiple layers forming a reactor for processing therapeutic agents described herein. The reactor may include seals, channels, valves, and chambers, including a pumping chamber, formed from the multiple layers. For example, the cartridge may be formed of two or more rigid or semi-rigid plates 1903, 1905 and at least one elastic layer 1907. The elastic layer 1907 may be a sheet of elastic material that is liquid impermeable. The elastic layer may be somewhat gas permeable or may be treated to include various regions to be more or less gas permeable. While a single continuous sheet of elastic material may be used, in some instances, multiple sheets of elastic material may be used, or multiple pieces of sheet may form a "sheet." Layers and elastic sheets may be laminated together. Generally, chambers for holding, valving, and / or pumping fluids may be formed in the plates on either side of the elastic layer, such that the elastic layer bisects the chamber into a liquid-containing side and a pressure (e.g., gas) application side. The total volume of the chambers may be constant and formed in both the first (e.g., upper) and second (e.g., lower) plates, but this volume may be divided into a pressure side and a liquid side. Applying positive or negative pressure to the pressure side can deform the elastic sheet, either decreasing the volume of the liquid-containing side (down to zero, closing the chamber) or increasing the volume of the liquid-containing side (up to a predetermined maximum). The pressure-application sides of the chambers may be connected, for example, via pressure ports in the upper plate 1903 that connect to pressure channels 1947, to apply negative or positive pressure to one or more pressure-receiving sides 1919 of the chambers. The liquid-containing side 1917 of each chamber opposite the pressure-applied side may be connected to a fluid port 1923 via a fluid channel 1921 .Both the fluid and pressure ports may be formed by openings in the upper plate 1903 and the elastic layer 1907, so that the pressure lines are pressed into the elastic layer 1907 which is supported on the underside of the port by an opposing rigid or semi-rigid layer 1905, allowing for sealed connections isolated from the atmosphere even when there are multiple different input lines.

[0112] 10B, cartridge 1900 includes a first (e.g., upper) plate 1903 having a first (e.g., top or upper) surface 1911, a second (e.g., bottom or lower) surface 1929, and a thickness therebetween. First surface 1911 may form the exposed outer surface. Microfluidic driver device also includes a second plate 1905 having a first (e.g., upper or top) surface 1931, a second (e.g., lower or bottom) surface 1933, and a thickness therebetween. Elastic layer 1907 is sandwiched between second surface 1929 of first plate 1903 and first surface 1931 of second plate 1905.

[0113] 10B may also include multiple chambers 1915, 1916, 1918, 1920, each having a fixed volume. These chambers are formed by cut-out areas (e.g., rounded / curved cuts) into the second (bottom) surface 1929 of the first plate 1903 and the first (top) surface 1931 of the second plate 1905. The resilient layer 1907 bisects these chambers 1915, each including a liquid-containing side 1917 and a pressure-receiving (e.g., gas-containing) side 1919.

[0114] The cartridge 1900 may also include a plurality of liquid (e.g., fluid) channels. In Figure 10B, a fluid channel 1921 is shown extending from a fluid port 1923 through the thickness of the first plate 1903 to a fluid channel opening 1925, through the elastic layer 1907, and through most of the thickness of the second plate 1905, where it forms a length of the liquid channel 1921 that extends parallel to the bottom surface of the third plate.

[0115] With respect to fluid port 1923, the diameter of the opening into first plate 1903 forming fluid port 1923 that extends through the thickness of the first plate may be larger than the diameter of fluid channel opening 1925 that extends through elastic layer 1907 into liquid (e.g., fluid) channel 1921. Fluid channel opening 1925 may be centered with respect to the bottom of the fluid port opening and may be offset from the wall of the fluid port opening by at least the expected wall thickness of the fluid line or fluid line connection interface that connects to the fluid port.

[0116] Fluid channel 1921 connects to the liquid-containing side 1917 of first chamber 1915. This first chamber has a relatively small holding volume (a fixed volume), but can be configured as a valve that can be fully opened and closed by movement of elastic layer 1907.

[0117] The cartridge 1900 also includes multiple pressure channels that can be independently controlled to apply positive and / or negative pressure. Chambers 1915, 1916, 1918 may each be connected to a separate pressure port and pressure channel to independently manipulate and control the movement of the portion of the elastic layer 1907 that bisects the chambers, thereby independently valving and / or pumping each chamber. In some examples, pressure ports may be shared between multiple chambers.

[0118] In the cross section through cartridge 1900 shown in FIG. 10B , there are multiple connections to other fluid (e.g., liquid) lines, fluid ports, pressure lines, and pressure ports, which are not shown, but may be outside the plane of the illustration. For example, in FIG. 10B , the liquid-containing side or portion 1917 of the fourth chamber may be connected to additional valves (chambers) and / or channels, including, for example, an outlet channel extending from the liquid-containing side 1917. Although not shown, additional chambers (e.g., configured as valves) may be formed as described above. In some examples, the outlet channel may deliver fluid from one or more chambers through another fluid port (not shown) to a fluid-receiving reservoir, e.g., a vial, tube, or the like. This receiving reservoir may be held within a reagent storage frame.

[0119] In FIG. 10E, cartridge 1980 includes four PCR chambers 1983, 1983′, 1983″, and 1983′′, each of which may include a liquid-contacting side, a pressure (e.g., gas) side, and is fluidly connected to an adjacent PCR chamber. Each PCR chamber has a fixed volume and is formed between a first surface of a first plate and a second surface of a second plate, as described above for a general chamber, and the first and second plates may be joined to each other with an elastically deformable membrane (e.g., an elastic layer) between them to divide the chamber. The elastic layer divides each chamber into a liquid-contacting side at the second surface and a pressure-receiving side at the first surface.

[0120] In the cartridge shown in FIG. 10D, the pressure-receiving side 1919 of each chamber is further partitioned by one or more fluidly connected serpentine paths 1985. These serpentine paths in the pressure-receiving surface are configured to distribute the positive and negative pressures (e.g., negative pressures) applied through the channels more evenly across the relatively large chamber surface. The subdivision of the pressure-receiving surface in each PCR chamber (e.g., by one or more serpentine paths in some examples) can support the deflectable membrane when negative pressure is applied to pull it away from the fluid-containing chamber. This can also prevent the formation of air bubbles and maintain a fixed, predictable volume.

[0121] The cartridge shown in Figure 10D also includes multiple fluidic channels, each extending from fluidic ports 1923, 1923' through the first plate region to the second plate region, to fluidically connect with the wetted side of one or more of the multiple chambers (similar to the configuration shown in Figures 10A and 10B). In Figure 10D, a subset of the fluidic ports are labeled, including those providing fluidic connection to (off-device) plasmid 1923, PCR buffer, a source of primers (e.g., T7 primers), oligo-dT, enzymes (e.g., polymerases), purification substrates (e.g., Ampure™ beads), RNASE-free air, dNTPs, product output ("OUT") 1923, output from the UV yield detection channel ("OUT UV"), buffer for UV detection ("UV buffer"), water, ethanol (e.g., 70% ethanol rinse), and waste ("Waste"). Additional fluidic ports are also included and may be redundant or unused. As explained, the pressure ports can provide communication for applying positive and / or negative pressure to the pressure-receiving side of each chamber, channel, vacuum cap, valve, etc. Thus, by applying positive or negative pressure to a particular pressure port 1943 or combination of pressure ports, the controller can control fluid movement within the device, including mixing, pumping, valving, etc. The pressure and fluid ports can be located on the top side of the first plate, typically around the periphery of the plate, as shown in Figures 10A and 19D.

[0122] 10D, the device also includes a plurality of pressure channels 1947 (similar to those shown in FIG. 10B), each extending from one or more pressure ports through the first plate region and elastic layer, into the second plate region, and back through the elastic layer into the first plate region, with each pressure channel of the plurality of pressure channels extending into the first plate region and fluidly connecting with one or more pressure-receiving sides of one or more of the plurality of chambers. As shown in FIG. 10B, application of positive or negative pressure (from the pressure ports) through the pressure channels by a system controller including the fluid reservoir and pneumatic drive can open or close valves 1915, 1918 and pump fluid through chambers 1916, 1920, 1983.

[0123] As mentioned above, any of the cartridges described herein may include one or more UV measurement chambers (e.g., UV yield detection chambers) 1990 in fluid communication with one or more of the PCR chambers. The UV yield detection chamber may include a UV yield detection window configured to allow UV light to pass through for quantification of polynucleotides in the UV yield detection chamber. The UV measurement chamber 1990 may also be connected to a buffer source to perform UV detection. The UV detection may measure the absorbance of the buffer containing the DNA. A system including a controller coordinating operations in the microfluidic driver device may be configured to control the operation of the UV yield detection chamber, as described in detail below. For example, the controller of a system configured to use the microfluidic driver device may first test the absorbance of a UV buffer without product, and then add a predetermined amount of (e.g., purified) product for comparison. The system (e.g., a controller) can then automatically or semi-automatically use the determined concentration to alert the user, and / or make a decision to discard or mark the sample for further analysis, and / or dilute the product before it is exported from the cartridge or transferred to another cartridge.

[0124] Figure 10E shows another example of a cartridge (e.g., "biochip") 1980' similar to that shown in Figure 10D, including a UV yield detection chamber 1990' in fluid communication with a sample and / or blank (e.g., buffer) chamber. In Figure 10E, the cartridge device also includes a waste port 1923' (e.g., a negative pressure port that can be connected to a waste line).

[0125] Generally, the devices shown in Figures 10D and 10E are similar and may include any of the features of the devices shown in Figures 10A-10C. For example, ports may be formed from the elastic layer along the length of the connecting channel 1939 (either a pressure channel or a fluid channel), as shown in Figure 10A. One or more valve bodies 1961 may be included in a row along the fluid connecting channel 1939.

[0126] The PCR chambers can be configured to optimize the PCR process described herein. For example, a microfluidic driver device can include PCR chambers with an area much greater than the height of the wetted side. For example, the wetted side of each PCR chamber can have a thickness that is 1.5 cm or less, e.g., 1.3 cm or less, 1.2 cm or less, 1.1 cm or less, 1.0 cm or less, 0.9 cm or less, 0.8 cm or less, 0.7 cm or less, 0.6 cm or less, or 0.5 cm or less, among others. Typically, a lower height (e.g., the "thickness" of the chamber) can result in more efficient heat transfer during thermal cycling, but a smaller overall volume. The cartridges described herein can be used for PCR in the PCR chambers without the need for adding oil / hydrophobic materials, as evaporation can be limited by the closed (or closable) configuration of the chambers.

[0127] Generally, any of these cartridges can include a purification chamber in fluid communication with a purification substrate (eg, Ampure beads).

[0128] The cartridges described herein may also be configured to provide mixing (e.g., bubble mixing) by applying air (e.g., RNase-free air) from the cartridge through the wetted side of the device, from a fluidic port, and into a reservoir connected to the fluidic port. In FIG. 10D , for example, the cartridge can be controlled by a controller (of the system to which the cartridge is connected) to mix and resuspend substrate beads (e.g., Ampure™ beads) in a reservoir connected to the fluidic port by driving RNase-free air through a fluidic channel communicating with the fluidic port. This can result in bubbling and mixing of the substrate beads in the reservoir. The controller can control mixing by application of RNase-free air (using positive and / or negative pressure applied through a pressure port of the microfluidic driver device), and after mixing, can direct the resuspended substrate from the reservoir to a purification chamber for purifying the template product.

[0129] As described above, any of these devices may be configured as a removable cartridge configured to engage a fluid reservoir and a pneumatic drive, and may be coupled to a system, for example, a microfluidic driver device control system including a controller for coordinating the operation of the microfluidic driver device to generate the template.

[0130] Generally, the cartridge may be of any suitable size / volume. For example, the cartridge may be configured to have a total PCR reactor size of about 3 mL to about 10 mL (e.g., about 4 mL to about 8 mL, about 5 mL to 7 mL, etc.). In the example shown in Figure 10D, the total PCR reactor volume (all four PCR chambers combined) is about 6.03 mL. Thus, in Figure 10D, the PCR reaction volume is about 3 mL, which has been found to produce about 130 ng / µL of template product (in 3 mL).

[0131] 11A and 11B show examples of cartridges that include one or more valves 1176, 1177 and a UV measurement channel 1181 connected to an input (e.g., a source of polynucleotide material such as polynucleotide formation chamber 1179 and / or a source of buffer or other blank solution). In FIG. 11A, the UV measurement chamber is also connected to a suction (e.g., waste) port 1172. By controlling the valves, suction may be used to load the UV measurement chamber 1181. FIG. 11B shows a slightly enlarged view of the UV measurement chamber of FIG. 11A.

[0132] Another example of a UV measurement chamber is shown in Figures 13A and 13B. In this example, UV measurement chamber 1381 is formed by thinned regions of upper and lower plates in space (between which UV chamber 1381 is formed), and may alternatively include an elastic layer. The UV measurement chamber in this example is formed of COC polymer (top and bottom layers) and has a diameter of approximately 2.5 mm. The elastic layer (e.g., PDMS) is cut out to form a chamber with dimensions of 3 mm x 4.5 mm. In this example, the UV chamber includes a fluid inlet 1383 connected to valve 1376 and a fluid outlet 1385 connected to a second valve 1377. Fluid inlet valve 1376 is in fluid communication with polynucleotide sample line 1339 and water (or buffer / blank) fluid line 1338, with flow through each of these lines also controlled by valves 1378, 1378'. In operation, the valve may be open and fluid from either the polynucleotide sample line 1339 or the water / buffer / blank line 1338 may be pumped into the UV measurement chamber 1381, or the fluid may be drawn into the UV measurement chamber by applying negative pressure (suction) from the suction / waste port 1388.

[0133] FIG. 12 illustrates the operation of the above-described apparatus for processing polynucleotides using a microfluidic driver device operating on a cartridge. In this example, the method may be for therapeutic polynucleotides (e.g., in a cartridge) 1201, as described above. The polynucleotides may undergo further processing (e.g., purification, concentration, etc.). Additionally, the UV measurement subsystem may be prepared for acquiring measurements 1203. For example, the apparatus may pre-irradiate the chamber as described above and / or form one or more dilutions of the polynucleotide solution. The apparatus may then determine the concentration of the therapeutic polynucleotide 1205. This may include sequentially driving a first blank solution into the UV measurement chamber and acquiring a first absorption measurement 1207, then driving the sample solution (or a diluted version thereof) and acquiring a second absorption measurement 1209. Optionally, a second blank measurement may be acquired 1210, in which case the first and second blank measurements may be compared and / or combined. For example, the two blank measurements may be averaged. The device can then estimate 1211 the concentration of polynucleotide in the sample from the first and second (and optionally third) absorption measurements.

[0134] The method (or device implementing it) may then adjust 1213 the operation of the device (eg, a microfluidic driver device) based on the absorption measurements and / or the concentration of the therapeutic polynucleotide.

[0135] example FIG. 14 shows an example of a portion of an apparatus described herein, including a UV detection subsystem. For example, the apparatus may include a controller (shown here as a separate processor 1401 for data processing), or in some examples, the controller may be integrated into the apparatus. In this example, the UV light source (shown as LED 1411) and UV detector 1415 both include power supplies 1407, 1407′, which may allow them to be adjusted separately. For example, by adjusting the power supplied by power supply 1407 to LED driver 1409, which controls the output of LED 1411 and therefore the illumination through the sample or blank in UV detection chamber 1413, the LED intensity can be increased or decreased (e.g., brighter / dimmer 1405). Similarly, the power supplied by power supply 1407′ to UV detector 1415 can be adjusted to increase or decrease the LED detector's sensitivity to absorption. The resulting UV absorption signal may be stored and / or analyzed by the device, as described above and shown in Figure 14, which includes a signal grabber circuit 1403 for capturing, storing, and / or transmitting the resulting absorbance. The controller may also provide feedback based on the detected absorbance to adjust either or both the intensity of the UV light source 1409 and / or the detector sensitivity of the UV detector 1415.

[0136] As described above, any device configured to determine polynucleotide concentration using UV light absorption can be configured to adjust the UV light intensity over a range of values ​​to achieve a linear output range for absorption by the polynucleotide sample, thereby avoiding saturation of the absorption signal output. Figure 15A shows the dimming voltage versus current for an example of a UV light source (LED) that can be used. The dimming voltage over this range is linear and can be linearly related to the resulting light intensity, as shown in Figure 15B. Figure 15B shows voltage readings for different applied dimming voltages. Referring back to Figure 14, the LED can therefore be adjusted (e.g., by adjusting the dimming voltage) to increase or decrease its intensity based on the detected absorption signal from the sample and blank. For example, if the absorption signal received for a particular polynucleotide signal is too high (e.g., at or near saturation), the voltage applied to the UV LED can be reduced by adjusting the dimming voltage. Alternatively, or in addition, the sensitivity of the UV detector can be decreased or increased. The same adjustment (e.g., dimming voltage) can be used for both the sample and the blank.

[0137] Generally, the devices and methods described herein can determine polynucleotide concentrations with high accuracy and repeatability. For example, FIG. 16A shows an example of detecting the concentrations of various samples (and corresponding blanks). In FIG. 16A, the device described herein can be used to first apply a blank 1602 and then measure UV absorption, as shown in FIG. 16A. In this example, the absorption of each polynucleotide sample or blank is performed for 10 seconds, with a stable reading obtained throughout this period; in some instances, the absorption may be measured for a shorter time (e.g., 5 seconds, 1 second, 0.5 seconds, 0.1 seconds, etc.) or longer. After measuring the first blank, a first RNA sample (e.g., undiluted) is moved to the UV measurement chamber to replace the blank, and UV absorption is measured 6104. The sample fluid can then be removed from the UV measurement chamber, and a second blank can be moved and measured 1602', followed by a second sample (e.g., a half-diluted RNA sample) 1606. A second sample is measured 1608 (e.g., a 10-fold diluted RNA sample), followed by a third blank measurement 1602''. The sample measurements may be different polynucleotides and / or different dilutions of the same polynucleotide (as shown in Figure 16A).

[0138] 16B shows a comparison between measurements obtained as described above and a commercially available concentration detection system that requires a different method and technique to determine analyte concentration, showing very close agreement. In this example, the commercially available analyte concentration detector may be, for example, a Nanodrop™ system (Thermo Fisher). Thus, the methods and apparatus described herein, which are particularly well-suited for on-cartridge (e.g., "on-chip") UV and therefore concentration measurements, show comparable or even better results compared to other larger and more expensive systems.

[0139] 17-18 show another example of a cartridge (e.g., "biochip") 2000. The cartridge 2000 of this example may be configured and operable similarly to any of the other cartridges described herein, with example differences described below. The cartridge 2000 of this example includes a plurality of pressure ports 2043 and a plurality of fluid ports 2023. A plurality of pressure channels 2047 extend from the pressure ports 2043. The pressure ports 2043 and pressure channels 2047 may provide for the application of positive and / or negative pressure to the pressure-receiving side of each chamber, channel, vacuum cap, valve, etc. within the cartridge 2000. Thus, a controller can control the movement of fluids within the cartridge 2000, including mixing, pumping, valving, etc., by applying positive or negative pressure to a particular pressure port 2043 or combination of pressure ports 2043. The pressure channels 2047 may therefore be understood as pneumatic channels.

[0140] The pressure port 2043 and the fluid port 2023 may be located on the top side of the cartridge 2000 (e.g., around the periphery of the cartridge 2000), as shown in Figure 17. Alternatively, the pressure port 2043 and / or the fluid port 2023 may be located on the bottom side of the cartridge 2000.

[0141] Each fluid port 2023 is fluidly coupled to a corresponding fluid channel in cartridge 2000, such that each fluid channel extends from a respective fluid port 2023. By way of example only, one or more fluid ports 2023 may be fluidly connected to a source of plasmid, PCR buffer, primer (e.g., T7 primer), oligo dT, enzyme (e.g., polymerase), purification substrate (e.g., Ampure™ beads), RNASE-free air, dNTPs, product output collection vessel, output from a UV yield detection channel, buffer for UV detection, water, ethanol (e.g., 70% ethanol rinse), or waste. Fluid ports 2023 (and corresponding channels) may be understood as providing a path for fluid communication. In some cases, one or more fluid ports 2023 are overlapping and therefore not used during a particular process.

[0142] Cartridge 2000 in this example further includes PCR chambers 2083, 2083', 2083", 2083'", each of which may include a liquid-contacting side, a pressure (e.g., gas / air) side, and is fluidly connected to an adjacent PCR chamber. Each PCR chamber has a fixed volume and is formed between a first surface of a first plate and a second surface of a second plate, as described above for a general chamber, and the first and second plates may be joined to each other with an elastically deformable membrane (e.g., an elastic layer) between them to divide the chamber. The elastic layer divides each chamber into a liquid-contacting side at the second surface and a pressure-receiving side at the first surface.

[0143] The pressure-receiving side of each PCR chamber 2083, 2083', 2083", 2083'" is bounded by one or more fluidly connected serpentine paths 2085. These serpentine paths 2085 in the pressure-receiving surface are configured to distribute the positive and negative pressures (especially negative pressures) applied through the corresponding pressure channels 2047 more evenly across the relatively large chamber surface. The subdivision of the pressure-receiving surface in each PCR chamber 2083, 2083', 2083", 2083'" by the serpentine paths 2085 can support the deflectable membrane when negative pressure is applied to pull it away from the fluid-containing chamber. This can also prevent the formation of air bubbles and maintain a fixed, predictable volume.

[0144] Cartridge 2000 in this example further comprises a UV measurement chamber 2020 in fluid communication with one or more of PCR chambers 2083, 2083′, 2083″, 2083′″ and / or blank (e.g., buffer) chambers such that UV measurement chamber 2020 can receive an aliquot of liquid from one or more of PCR chambers 2083, 2083′, 2083″, 2083′″ and / or blank (e.g., buffer) chambers. UV measurement chamber 2020 may comprise a UV yield detection window configured to pass UV light for quantification of polynucleotides in UV measurement chamber 2020 as described herein. As best seen in FIG. 18 , inlet liquid channel 2026 leads into UV measurement chamber 2020, while outlet liquid channel 2050 leads out of UV measurement chamber 2020.

[0145] A vacuum cap 2024 is positioned upstream of the inlet liquid channel 2026. Another vacuum cap 2052 is positioned downstream of the outlet liquid channel 2050. In some versions, the vacuum caps 2024, 2052 are configured and operative similarly to the vacuum cap 1938 described above with reference to FIG.

[0146] In this example, the vacuum caps 2024, 2052 are operable to remove air bubbles from the liquid within the UV measurement chamber 2020. The vacuum caps 2024, 2052 are pneumatically coupled to one another via a pneumatic channel 2040, thereby facilitating simultaneous operation of the vacuum caps 2024, 2052. In some versions, one or both of the vacuum caps 2024, 2052 may also function as a valve to selectively allow or block communication of a liquid aliquot / sample to / from the UV measurement chamber 2020. In such a scenario, the vacuum caps 2024, 2052 may be pneumatically actuated similar to other valves described herein.

[0147] A pair of liquid channels 2028, 2030 lead to vacuum cap 2024. A valve 2076 selectively allows or prevents the flow of liquid into vacuum cap 2024 via channel 2028. Similarly, valve 2032 selectively allows or prevents the flow of liquid into vacuum cap 2024 via channel 2030. Another vacuum cap 2070 is in fluid communication with valve 2076 and is pneumatically coupled to vacuum cap 2024 via channel 2022. Yet another vacuum cap 2072 is also pneumatically coupled to vacuum cap 2070 and in fluid communication with another valve 2074. Thus, vacuum caps 2024, 2052, 2070, 2072 can be pneumatically coupled to one another via channels 2022, 2040 to facilitate simultaneous operation of vacuum caps 2024, 2052, 2070, 2072.

[0148] In this example, valve 2076 fluidly intervenes in the pathway between PCR chambers 2083, 2083′, 2083″, 2083′′ and channel 2028, such that valve 2076 is operable to selectively block or allow liquid communication from PCR chambers 2083, 2083′, 2083″, 2083′ to channel 2028 and, in turn, to UV measurement chamber 2020 via vacuum cap 2024 and channel 2026. In some versions, PCR chambers 2083, 2083′, 2083″, 2083′′ can be pressurized to force a liquid aliquot / sample into UV measurement chamber 2020, while valves in the pathway can be open. Alternatively, liquid can be driven into UV measurement chamber 2020 by applying a vacuum through output port 2060, while valves in the pathway can be open. In yet other versions, the main reservoir may be pressurized, and valves 2076, 2052 (and possibly vacuum caps 2024, 2052) may be actuated in turn to peristaltically pump liquid from PCR chambers 2083, 2083', 2083'', 2083''' into UV measurement chamber 2020. Alternatively, any other suitable structural mechanism and / or technique may be used.

[0149] Channel 2030 provides a path for fluid communication of a blank or buffer solution (e.g., water, etc.) to UV measurement chamber 2020 via vacuum cap 2024 and inlet liquid channel 2026. As described above, valve 2032 selectively allows or prevents the flow of liquid through channel 2030 into vacuum cap 2024 (and thus into inlet liquid channel 2026 and UV measurement chamber 2020). Channel 2034 extends from valve 2030 to pump chamber 2036. Pump chamber 2036 may further be fluidly coupled to a source of blank or buffer solution and may be pneumatically operated to drive blank or buffer fluid through channels 2034, 2030, 2026 towards UV measurement chamber 2020 when valve 2032 is in an open state. Alternatively, blank or buffer solution may be driven into UV measurement chamber 2020 by applying a vacuum through output port 2060 while valves in the pathway may be in an open state.

[0150] Another channel 2054 and valve 2056 are further coupled to the vacuum cap 2052. A pneumatic channel 2062 further extends from the valve 2056 and provides a path for pressurizing the valve 2056, thereby transitioning it between an open and a closed state. Another channel 2058 also extends from the valve 2056 and provides a path for fluid communication from the valve 2056 to an output port 2060. The output port 2060 may operate in a similar manner to the aspiration / waste port 1388 described above.

[0151] Cartridge 2000 in this example further includes a pooling output port 2002. As best seen in FIG. 18 , pooling output port 2002 is coupled to a liquid communication channel 2004 that leads to a valve 2006. Valve 2006 is in fluid communication with pneumatic channel 2010, through which valve 2006 can be selectively pressurized, thereby transitioning valve 2006 between an open state and a closed state. Liquid communication channel 2008 is also coupled to valve 2006. Channel 2008 is further fluidly coupled to PCR chambers 2083, 2083′, 2083″, 2083′″ such that when valve 2006 is in an open state, fluid can be transported from PCR chambers 2083, 2083′, 2083″, 2083′″ to pooling output port 2002 via channels 2004, 2008. Thus, the pooling output port 2002 can be used to pool the outputs of the PCR chambers 2083, 2083', 2083'', 2083''' into a single batch in a container external to the cartridge 2000, as described above.

[0152] Some versions of cartridge 2000 may further include an additional output port (not shown) fluidically coupled to PCR chambers 2083, 2083', 2083", 2083'" so that fluid can be delivered from PCR chambers 2083, 2083', 2083", 2083'" to the additional output port (not shown) instead of being delivered to pooling output port 2002. In other words, output fluid from PCR chambers 2083, 2083', 2083", 2083'" can be selectively delivered to either pooling output port 2002 or the additional output port, such that PCR chambers 2083, 2083', 2083", 2083'" effectively have two separate, individually controlled outputs. In some scenarios, fluid communicated from PCR chambers 2083, 2083′, 2083″, 2083′′ to an additional output port (not shown) may be retained for further analysis or otherwise processed. In some such scenarios, fluid from PCR chambers 2083, 2083′, 2083″, 2083′′ may be communicated to an additional output port (not shown) instead of to pooling output port 2002, depending on concentration measurement data obtained through UV measurement chamber 2020. It should also be understood that at least a portion of the fluid output from PCR chambers 2083, 2083′, 2083″, 2083′′ may be communicated out through output port 2060. In some such scenarios, fluid communicated out through output port 2060 is treated as waste.

[0153] In some examples, the intensity of the UV light source (e.g., UV LED 101) may change over time. Additionally or alternatively, UV transmittance may vary between different cartridges. Such differences in UV light intensity and / or UV transmittance may affect the Limit of Quantitation (LoQ) of the UV measurement process. In other words, variations in UV light intensity, UV transmittance, and / or other noise sources may affect the maximum and minimum sample concentrations that can be reliably detected with some given confidence through the UV measurements described herein. Therefore, it may be desirable to provide a process for establishing upper and lower LoQs for a cartridge prior to using the cartridge to obtain polynucleotide concentration measurements during a process for forming a therapeutic polynucleotide.

[0154] 19 shows an example of a process 2100 that can be used to estimate the upper and lower LoQ of a cartridge prior to using the cartridge to obtain polynucleotide concentration measurements during a process for forming a therapeutic polynucleotide. This process 2100 may be used in conjunction with any of the various cartridges described herein. Process 2100 may be performed by controller 221, processor 1401, and / or any other suitable hardware component. Process 2100 begins with measuring the maximum voltage of a UV detector (e.g., UV detector 103, 1415), as shown in block 2102. Next, the standard deviation of the UV detection signal is measured, as shown in block 2104. Using these measurements, process 2100 then includes determining an upper LoQ, as shown in block 2106, and determining a lower LoQ, as shown in block 2108.

[0155] By way of example only, the upper LoQ may be determined by the following equation (I) (block 2106):

[0156]

number

[0157] By way of further example only, the lower LoQ may be determined by the following equation (II) (block 2108):

[0158]

number

[0159] The determined upper LoQ may be compared to a threshold value to determine whether the upper LoQ is acceptable, as shown in block 2110. Similarly, the determined lower LoQ may be compared to a threshold value to determine whether the lower LoQ is acceptable, as shown in block 2112. If either the upper LoQ or the lower LoQ is unacceptable, a warning may be provided to the operator, as shown in block 2112. By way of example only, the warning may include a message indicating that the UV light source should be replaced, a message indicating that the measured concentration is too close to one of the upper or lower LoQs and, as a result, the measured concentration is unreliable, and / or any other suitable message. In some versions, the controller 221, processor 1401, and / or any other suitable hardware component may prevent further use of the cartridge and / or UV light source if the upper LoQ or lower LoQ is found to be unacceptable unless corrective action is taken by the operator and then the upper and lower LoQs are subsequently found to be acceptable. In either case, if both the upper LoQ and the lower LoQ are found to be acceptable, the controller 221, processor 1401, and / or any other suitable hardware components may enable the cartridge to be used to form therapeutic polynucleotides (and / or perform any other suitable process), as shown in block 2118.

[0160] In some cases, particularly when the sample is at a substantially high concentration level (e.g., resulting in a substantially low UV transmittance of the sample), the sample may tend to induce substantial backscattering when illuminated with UV light. Such backscattering of UV light can adversely affect the accuracy of readings by a UV detector (e.g., UV detector 103, 1415) by adversely affecting the dynamic range. In some cases, such backscattering can be corrected through data processing techniques, but such data processing techniques may require certain geometric assumptions. Another option may include using a UV light-blocking material to suppress scattered light and thereby improve the dynamic range of the UV detector.

[0161] 20-21 illustrate one example of how a UV light-blocking material can be used to suppress scattered light and thereby improve the dynamic range of a UV detector. In particular, FIGS. 20-21 illustrate a UV-blocking member 2200 that includes a disk-shaped body 2202 having a central opening 2204 formed therethrough. Body 2202 includes a material configured to prevent the transmission of UV light therethrough, while central opening 2204 allows the transmission of UV light therethrough.

[0162] 21 illustrates an arrangement 2300 in which a UV blocking member 2200 is applied to a cartridge 2310. The cartridge 2310 may be configured and operable similarly to any other cartridge described herein. In this example, the cartridge 2310 includes an upper plate 2320, a lower plate 2330, and a resilient layer 2340 sandwiched between the plates 2320, 2330. A recess 2322 formed in the upper plate 2320 provides a first UV measurement area, while a recess 2332 formed in the lower plate 2330 provides a second UV measurement area below the recess 2322. A UV measurement chamber 2350 is interposed between the recesses 2322, 2332 and may be configured and operable similarly to the other UV measurement chambers described herein.

[0163] The UV blocking member 2200 is positioned on the upper plate 2320, directly above the recess 2322. In some versions, the UV blocking member 2200 is adhered to the top surface of the upper plate 2320. Alternatively, the UV blocking member 2200 may be secured to the upper plate 2320 in any other suitable manner. As shown in FIG. 21 , the diameter of the opening 2204 is smaller than the diameter of the recess 2322. The opening 2204 allows UV light 2304 from the UV light source 2302 to pass through the UV blocking member 2200 and enter the recess 2322, thereby irradiating the sample in the UV measurement chamber 2350 with the UV light 2304. To the extent that the sample in the UV measurement chamber 2350 provides backscattering of the UV light 2304, the body 2202 substantially limits such backscattering. UV light 2360 exits UV measurement chamber 2350 through recess 2332 and reaches UV detector 2362, which generates a signal indicative of the concentration of the sample in UV measurement chamber 2350, as described herein.

[0164] FIG. 22 illustrates another example of an arrangement 2400 that can be used to mitigate backscattering of UV light from a UV measurement chamber. Similar to arrangement 2300 described above, arrangement 2400 of this example includes a UV light source 2402, a cartridge 2410, and a UV detector 2462. Similar to cartridge 2310 described above (and similar to other cartridges described herein), cartridge 2410 of this example includes an upper plate 2420, a lower plate 2430, and an elastic layer 2440 sandwiched between plates 2420, 2430. A recess 2422 formed in upper plate 2420 provides a first UV measurement area, while a recess 2432 formed in lower plate 2430 provides a second UV measurement area below recess 2422. A UV measurement chamber 2450 is interposed between recesses 2422, 2432 and is configured and operable similarly to the other UV measurement chambers described herein.

[0165] Similar to arrangement 2300, arrangement 2400 of the present example also includes a UV blocking member 2470, however, UV blocking member 2470 of this example is configured differently than UV blocking member 2200. In particular, UV blocking member 2470 of this example comprises a cylindrical body 2472 that defines a central passageway 2472 through UV blocking member 2470.

[0166] The body 2472 comprises a material configured to prevent the transmission of UV light therethrough, while the central passage 2472 allows the transmission of UV light therethrough.

[0167] The body 2472 is sized to fit within the recess 2422 such that an outer sidewall of the body 2472 contacts an inner sidewall of the recess 2422. In some versions, the body 2472 is secured within the recess 2422 by a friction fit, using an adhesive, or using any other suitable technique. In this example, the inner sidewall of the central passage 2472 is configured to provide an inner diameter that is substantially constant along the height of the body 2472. In some other versions, the central passage 2472 is tapered. For example, some variations may provide an opening at the top of the body 2472 that is smaller than the opening at the bottom of the body 2472. Some other variations may provide an opening at the top of the body 2472 that is larger than the opening at the bottom of the body 2472. Alternatively, the central passage 2472 may have any other suitable configuration.

[0168] As shown in FIG. 22 , the diameter of the central passage 2472 is smaller than the diameter of the recess 2422. It should be understood that the diameter of the central passage 2472 shown in FIG. 22 is merely an example, and that the diameter of the central passage 2472 may be increased or decreased relative to that shown in FIG. 22 . In either case, the central passage 2472 allows UV light 2404 from the UV light source 2402 to pass through the UV blocking member 2470 and enter the recess 2422, thereby irradiating the sample in the UV measurement chamber 2450 with UV light 2404. To the extent that the sample in the UV measurement chamber 2450 provides backscattering of the UV light 2404, the body 2472 substantially limits such backscattering. UV light 2460 exiting the UV measurement chamber 2450 through the recess 2432 reaches the UV detector 2462, which generates a signal indicative of the concentration of the sample in the UV measurement chamber 2450, as described herein.

[0169] 21 and 22 show UV blocking members 2200, 2470 used in separate arrangements 2300, 2400, some other arrangements may include a combination of UV blocking members 2200, 2470. For example, in a variation of arrangement 2400 shown in FIG. 22, UV blocking member 2200 may be positioned above UV blocking member 2470. In some such arrangements, the diameter of opening 2204 is smaller than the diameter of central passageway 2472. In some such arrangements, UV blocking member 2200 substantially prevents backscattering that would otherwise occur through the upper opening of recess 2422, while UV blocking member 2470 prevents backscattering that would otherwise occur through the sidewalls of recess 2422. As yet another example of a variation, the sidewalls of recess 2422 may be laser carbonized, coated with a UV blocking material, or otherwise treated to prevent transmission of UV light through the sidewalls of recess 2422.

[0170] In some cases, air bubbles may reach the UV measurement chamber, and the presence of such bubbles in the UV measurement chamber may cause the concentration of the sample in the UV measurement chamber to be underestimated. Therefore, it may be desirable to provide a measurement algorithm that accounts for the possibility of inaccurate measurements that may be caused by the presence of air bubbles in the UV measurement chamber. An example of such a process 2500 is shown in FIG. 23. This process 2500 may be used in conjunction with any of the various cartridges described herein. Process 2500 may be performed by controller 221, processor 1401, and / or any other suitable hardware component. As shown in block 2502 of FIG. 23, process 2500 begins by obtaining four concentration measurements based on readings from a UV detector described herein (e.g., UV detectors 103, 1415, 2362, 2462). Next, as shown in block 2504, the average of these four measurements is calculated. Next, as shown in block 2506, the four measurements are compared to the calculated average, and outlier measurements are discarded. In some versions, the outlier measurement is identified as the measurement whose value is furthest from the average. In some other versions, outliers are identified based on a T-test. As yet another variation, the measurement providing the lowest concentration value may be automatically identified as an outlier. In some such versions, calculating the mean (block 2504) may be omitted.

[0171] After discarding the outlier measurements (block 2506), the average of the remaining three measurements is calculated, as shown in block 2508. This updated average is then reported (e.g., as a measured concentration), as shown in block 2510. Optionally, the standard deviation is reported along with the average. The updated average (and possibly the standard deviation) may be reported to an operator and / or a control algorithm, which may automatically modify one or more subsequent actions based on the reported average (and possibly the standard deviation). By way of further example only, the updated average may be used for pooling, as described herein.

[0172] In some examples, calculating the average of the first four measurements as shown in block 2504 and then comparing the first four measurements to that average may reveal that all four measurements were substantially close to the average. In other words, the standard deviation from the first average calculation may be substantially small (e.g., less than a predetermined threshold). In some such scenarios, process 2500 may be stopped and the average reported (as shown in block 2510 and described above) without discarding the outliers (block 2506) and recalculating the average (block 2508). While this example process 2500 initially utilizes four measurements, other versions may provide more or fewer than four measurements as an initial step. Similarly, this example process 2500 discards only one outlier, while other versions may discard two or more outliers.

[0173] As an alternative to using process 2500 shown in FIG. 23 and described above, a quad detector may be used to account for the presence of bubbles in the sample. For example, differences in voltage across the quadrant axes may indicate a non-uniform sample. Such a version may warrant a calibration measurement after each cartridge is loaded to account for optical variations between cartridges. Such calibration may be automated.

[0174] As yet another alternative to using process 2500 shown in FIG. 23 and described above, an imaging detector may be used to account for the presence of air bubbles in a sample. For example, image analysis and / or machine learning techniques may be used to identify air bubbles. If an air bubble is detected, a vacuum cap (e.g., such as vacuum cap 1938 described above) may be used to automatically remove the bubble in response to optical detection of the bubble. Alternatively, any concentration measurements captured during bubble detection may be ignored. Additionally or alternatively, optically captured bubble data may be utilized to apply a correction to the concentration measurements, effectively offsetting the presence of air bubbles in the fluid.

[0175] The following examples relate to various non-exhaustive ways in which the teachings herein can be combined or applied.

[0176] It should be understood that the following examples are not intended to limit the scope of any claims that may be presented at any time in this application or any subsequent application thereto. No disclaimer is intended. The following examples are provided solely for illustrative purposes. It is contemplated that the various teachings herein may be configured and applied in numerous other ways. It is also contemplated that some variations may omit certain features referred to in the following examples. Accordingly, no aspect or feature referred to below should be deemed critical unless later expressly indicated as such by the inventors or their successor in interest. If any claim containing additional features other than those referred to below is presented in this application or any subsequent application related to this application, those additional features shall not be presumed to have been added for any reasons related to patentability. [Example]

[0177] 1. A cartridge device for processing polynucleotides, comprising: a first layer having a first thickness; a second layer having a second thickness; an elastic material extending between the first layer and the second layer and separating the first layer from the second layer by a separation thickness; and an ultraviolet (UV) measurement region formed through the first layer and the second layer, wherein a region of the first layer within the UV measurement region has a thickness less than or equal to the first thickness; and a region of the second layer; a cartridge device wherein the UV measurement area has a thickness less than or equal to the second thickness, and the elastic material is not present between the area of ​​the first layer in the UV measurement area and the area of ​​the second layer in the UV measurement area, forming a UV measurement chamber having the separation thickness. [Example]

[0178] 2. The cartridge device of example 1, wherein the first layer comprises a polymeric material. [Example]

[0179] The cartridge device of example 2, wherein the first layer comprises a cyclic olefin copolymer (COC) material. [Example]

[0180] The cartridge device according to any one of Examples 1 to 3, wherein the first thickness is 1 to 5 mm. [Example]

[0181] The cartridge device of any one of Examples 1 to 4, wherein the second layer comprises a polymeric material. [Example]

[0182] The cartridge device of example 5, wherein the second layer comprises a cyclic olefin copolymer (COC) material. [Example]

[0183] The cartridge device according to any one of Examples 1 to 6, wherein the second thickness is 1 to 5 mm. [Example]

[0184] The cartridge device according to any one of Examples 1 to 7, wherein the separation thickness is 1 mm to 0.1 mm. [Example]

[0185] The cartridge device of any of Examples 1 to 8, further comprising a fluid channel in the second layer in fluid communication with the UV measurement chamber. [Example]

[0186] A cartridge device as described in any of Examples 1 to 9, further comprising a plurality of pneumatic valves arranged to control flow into and out of the UV measurement chamber, each of the plurality of pneumatic valves being formed by an air pressure chamber in the first layer and a fluid chamber in the second layer, a portion of the elastic material separating the air pressure chamber from the fluid chamber, the air pressure chamber being in fluid communication with an air pressure channel in the first layer that is configured to communicate with a pressure port in an outer region of the cartridge device to actuate the air pressure valve, and the fluid chamber being in fluid communication with the UV measurement chamber through the fluid channel in the second layer when the air pressure valve is open. [Example]

[0187] The cartridge device of any of Examples 1 to 10, further comprising an in vitro transcription (IVT) chamber formed at least partially within the second layer and in fluid communication with the UV measurement chamber. [Example]

[0188] A cartridge device described in any of Examples 1 to 11, further comprising one or more vacuum ports in an outer region of the cartridge device configured to connect to a negative pressure source to draw fluid into the UV measurement chamber. [Example]

[0189] A cartridge device described in any of Examples 1 to 12, further comprising: a first inlet channel in the second layer in fluid communication with a first chamber configured to hold a polynucleotide sample fluid; and a second inlet channel in the second layer in fluid communication with a second chamber configured to hold a blank sample fluid. [Example]

[0190] 14. The cartridge device of any of Examples 1 to 13, further comprising one or more dilution and mixing chambers in fluid communication with the UV measurement chamber configured to dilute the fluid sample. [Example]

[0191] A cartridge device described in any of Examples 1 to 14, wherein the diameter of the UV measurement chamber is larger than the diameter of the region of the first layer within the UV measurement region to limit air bubbles in the central region of the UV measurement chamber. [Example]

[0192] a second layer comprising a polymeric material having a second thickness; an elastic material extending between the first layer and the second layer; an ultraviolet (UV) measurement region formed through the first layer and the second layer, wherein a region of the first layer in the UV measurement region has a thickness equal to or less than the first thickness, and a region of the second layer in the UV measurement region has a thickness equal to or less than the second thickness, and the elastic material is removed from between the region of the first layer in the UV measurement region and the region of the second layer in the UV measurement region to form a UV measurement chamber; and an in vitro transfer (IVT) chamber formed at least partially in the second layer and in fluid communication with the UV measurement chamber; A cartridge device comprising: [Example]

[0193] 1. A method for manufacturing a polynucleotide using a microfluidic driver device operated with a cartridge, the method comprising: forming a therapeutic polynucleotide in a cartridge; and determining a concentration of the therapeutic polynucleotide by using the microfluidic driver device to drive a first blank solution into an ultraviolet (UV) measurement chamber of the cartridge and obtain a first absorption measurement through the cartridge; driving a sample solution of the therapeutic polynucleotide into the UV measurement chamber of the cartridge and obtain a second absorption measurement through the cartridge; and determining a concentration of the therapeutic polynucleotide by extrapolating the concentration of the therapeutic polynucleotide from the first absorption measurement and the second absorption measurement. [Example]

[0194] 18. The method of example 17, further comprising, in a processor of the microfluidic driver device, adjusting operation of the microfluidic driver device based on the estimated concentration of the therapeutic polynucleotide. [Example]

[0195] The method of any of Examples 17-18, further comprising comparing the estimated concentration of the therapeutic polynucleotide to a concentration range by a processor of the microfluidic driver device, and, based on the comparison, directing the therapeutic polynucleotide to one of pooling with one or more additional batches of therapeutic polynucleotide formed in the cartridge, discarding, or further analysis. [Example]

[0196] 20. The method of any of Examples 17-19, further comprising diluting the therapeutic polynucleotide to a standard concentration for output under control of a processor of the microfluidic driver device. [Example]

[0197] 21. The method of any of Examples 17-20, further comprising automatically adjusting one or more parameters of the formation of the therapeutic polynucleotide by the microfluidic driver device based on the estimated concentration of the therapeutic polynucleotide. [Example]

[0198] 22. The method of example 21, wherein the one or more parameters include one or more of temperature, reagent volume, reagent concentration, time, and mixing. [Example]

[0199] 23. The method of any of Examples 17-22, wherein determining the concentration of the therapeutic polynucleotide further comprises performing a serial dilution in one or more chambers of the cartridge under the control of the microfluidic driver device, and repeating the step of driving the sample solution of the therapeutic polynucleotide for each of one or more dilutions formed by the serial dilution to create a dilution curve. [Example]

[0200] 24. The method of Example 23, wherein estimating the concentration of the therapeutic polynucleotide comprises estimating the concentration of the therapeutic polynucleotide from the dilution curve. [Example]

[0201] The method of any of Examples 17-24, wherein estimating the concentration of the therapeutic polynucleotide further comprises altering one or more of the sensitivity of a UV detector and the intensity of a UV emitter of the microfluidic driver device in response to the second absorption measurement through the cartridge. [Example]

[0202] 26. The method of any of Examples 17-25, further comprising pre-treating the UV measurement chamber with UV light before obtaining the first absorption measurement through the cartridge. [Example]

[0203] The method of any of Examples 17-26, wherein forming the therapeutic polynucleotide comprises performing an in vitro transcription (IVT) reaction to form the therapeutic polynucleotide. [Example]

[0204] The method of Example 27, further comprising creating a template for the IVT reaction in the cartridge. [Example]

[0205] The method of any of Examples 17 to 28, wherein the therapeutic polynucleotide comprises a therapeutic mRNA. [Example]

[0206] The method of Example 29, further comprising encapsulating the therapeutic mRNA in a delivery vehicle. [Example]

[0207] 31. The method of any of Examples 17-30, wherein the microfluidic driver device monitors the driving of the first blank solution and the driving of the sample solution by pneumatically deflecting one or more regions of a membrane of the cartridge to open and / or close valves of the cartridge. [Example]

[0208] 32. The method of example 31, wherein the microfluidic driver device pneumatically deflects one or more regions of the membrane of the cartridge to drive the first blank solution and / or the sample solution into the UV measurement chamber. [Example]

[0209] A method of manufacturing polynucleotides using a microfluidic driver device operating with the cartridge, comprising: forming a therapeutic polynucleotide in the cartridge, the therapeutic polynucleotide comprising a therapeutic mRNA; driving, with the microfluidic driver device, a first blank solution into an ultraviolet (UV) measurement chamber of the cartridge and obtaining a first absorption measurement through the cartridge; driving, with the microfluidic driver device, a sample solution of the therapeutic polynucleotide into the UV measurement chamber of the cartridge and obtaining a second absorption measurement through the cartridge; driving a second blank solution into the UV measurement chamber of the cartridge and obtaining a third absorption measurement through the cartridge; determining a concentration of the therapeutic mRNA by estimating a concentration of the therapeutic polynucleotide from the first absorption measurement, the second absorption measurement, and the third absorption measurement; and adjusting, in a processor of the microfluidic driver device, operation of the microfluidic driver device based on the estimated concentration of the therapeutic polynucleotide; A method comprising: [Example]

[0210] 1. A system for producing and / or processing polynucleotides, comprising: a cartridge mount; a plurality of pressure lines; a plurality of fluid lines each connected to or configured to connect to a fluid source, wherein each fluid line, and at least a subset of the pressure lines, is configured to be secured to an inlet or outlet port on a cartridge held within the cartridge mount; an ultraviolet (UV) light source; a UV light detector; and a controller configured to control the application of pressure through the pressure lines to drive fluid through the cartridge, the controller being further configured to: direct the formation of a therapeutic polynucleotide in the cartridge; and estimate the concentration of the therapeutic polynucleotide by driving a first blank solution through a UV measurement chamber of the cartridge and obtaining a first absorption measurement using the UV light source and UV light receiver; driving a sample solution containing the therapeutic polynucleotide through the UV measurement chamber of the cartridge and obtaining a second absorption measurement using the UV light source and UV light receiver; and further configured to estimate the concentration of the therapeutic polynucleotide from the first absorption measurement and the second absorption measurement. [Example]

[0211] 35. The system of Example 34, wherein the controller is further configured to modify operation of the system based on the estimated concentration of the therapeutic polynucleotide. [Example]

[0212] The system of any of Examples 34-35, wherein the controller is further configured to compare the estimated concentration of the therapeutic polynucleotide with a concentration range and, based on the comparison, direct the therapeutic polynucleotide to one of pooling with one or more additional batches of therapeutic polynucleotide formed in the cartridge, discarding, or further analyzing. [Example]

[0213] 37. The system of any of Examples 34-36, wherein the controller is further configured to dilute the therapeutic polynucleotide to a standard concentration for output. [Example]

[0214] The system of any of Examples 34-37, wherein the controller is further configured to automatically adjust one or more parameters of the formation of the therapeutic polynucleotide based on the estimated concentration of the therapeutic polynucleotide. [Example]

[0215] 39. The system of example 38, wherein the one or more parameters include one or more of temperature, reagent volume, reagent concentration, time, and mixing. [Example]

[0216] The system of any of Examples 34-39, wherein the controller is further configured to perform a serial dilution in one or more chambers of the cartridge and repeat the step of driving the sample solution of the therapeutic polynucleotide for each of one or more dilutions of the sample solution of the therapeutic polynucleotide formed by the serial dilution to create a dilution curve. [Example]

[0217] 41. The system of Example 40, wherein the controller is further configured to estimate the concentration of the therapeutic polynucleotide from the dilution curve. [Example]

[0218] A system described in any of Examples 34 to 41, wherein the controller is further configured to change one or more of the sensitivity of the UV detector and the intensity of the UV light source in response to the second absorption measurement value through the cartridge. [Example]

[0219] 43. The system of any of Examples 34 to 42, wherein the controller is further configured to pretreat the UV measurement chamber with UV light before obtaining the first absorption measurement value through the cartridge. [Example]

[0220] The system of any of Examples 34-43, wherein the controller is further configured to form the therapeutic polynucleotide, and includes performing an in vitro transcription (IVT) reaction to form the therapeutic polynucleotide. [Example]

[0221] 45. The system of Example 44, wherein the controller is further configured to generate a template for the IVT reaction in the cartridge. [Example]

[0222] The system of any of Examples 34 to 45, wherein the therapeutic polynucleotide comprises a therapeutic mRNA. [Example]

[0223] 47. The system of Example 46, wherein the controller is further configured to encapsulate the therapeutic mRNA in a delivery vehicle. [Example]

[0224] A system described in any of Examples 34 to 47, wherein the controller is further configured to pneumatically deflect one or more regions of the membrane of the cartridge to open and / or close a valve of the cartridge when driving the first blank solution, driving the second blank solution, and driving the sample solution. [Example]

[0225] The system of Example 48, wherein the controller is further configured to pneumatically deflect one or more regions of the membrane of the cartridge to drive the first blank solution, the second blank solution, and / or the sample solution into the UV measurement chamber. [Example]

[0226] 1. A system for making and / or processing polynucleotides, comprising: a cartridge; and a microfluidic driver device including a cartridge mount; a plurality of pressure lines, each fluid line coupled or configured to couple to a fluid source, wherein each fluid line, and at least a subset of the pressure lines, is configured to be secured to an inlet port or an outlet port on a cartridge held within the cartridge mount; an ultraviolet (UV) light source; a UV light detector; and a controller configured to control the application of pressure through the pressure lines to drive fluid through the cartridge; wherein the controller is further configured to direct formation of a therapeutic polynucleotide; and the controller is further configured to drive a first blank solution through the UV measurement chamber of the cartridge and obtain a first absorption measurement using the UV light source and UV light receiver, drive a sample solution containing the therapeutic polynucleotide through the UV measurement chamber of the cartridge and obtain a second absorption measurement using the UV light source and UV light receiver, drive a second blank solution through the UV measurement chamber of the cartridge and obtain a third absorption measurement using the UV light source and UV light receiver, and estimate the concentration of the therapeutic polynucleotide by modifying operation of the microfluidic driver device based on the concentration of the therapeutic polynucleotide determined from the first absorption measurement, the second absorption measurement, and the third absorption measurement. [Example]

[0227] 1. A method of manufacturing polynucleotides using a microfluidic driver device operating on a cartridge, the method comprising: forming a therapeutic polynucleotide in a cartridge; determining a concentration of the therapeutic polynucleotide by the microfluidic driver device driving a first blank solution into a concentration measurement chamber of the cartridge and obtaining a first measurement through the cartridge; driving a sample solution of the therapeutic polynucleotide into the concentration measurement chamber of the cartridge and obtaining a second measurement through the cartridge; and estimating the concentration of the therapeutic polynucleotide from the first measurement and the second measurement; and comparing, by a processor of the microfluidic driver device, the estimated concentration of the therapeutic polynucleotide to a concentration range; and based on the comparison, directing the therapeutic polynucleotide to one of pooling with one or more additional batches of therapeutic polynucleotide formed in the cartridge, discarding, or further analyzing. [Example]

[0228] 52. The method of claim 51, wherein the concentration measuring chamber comprises an ultraviolet (UV) measuring chamber, the first measurement value comprises a first absorption measurement value, and the second measurement value comprises a second absorption measurement value. [Example]

[0229] The method of Example 52, wherein estimating the concentration of the therapeutic polynucleotide further comprises altering one or more of the sensitivity of a UV detector and the intensity of a UV emitter of the microfluidic driver device in response to the second absorption measurement through the cartridge. [Example]

[0230] 54. The method of any of Examples 52-53, further comprising pretreating the UV measurement chamber with UV light before obtaining the first absorption measurement through the cartridge. [Example]

[0231] 55. The method of any of Examples 51-54, further comprising, in a processor of the microfluidic driver device, adjusting operation of the microfluidic driver device based on the estimated concentration of the therapeutic polynucleotide. [Example]

[0232] 56. The method of any of Examples 51-55, further comprising diluting the therapeutic polynucleotide to a standard concentration for output under control of a processor of the microfluidic driver device. [Example]

[0233] 56. The method of any of Examples 51-55, further comprising automatically adjusting one or more parameters of the formation of the therapeutic polynucleotide by the microfluidic driver device based on the estimated concentration of the therapeutic polynucleotide. [Example]

[0234] 58. The method of example 57, wherein the one or more parameters include one or more of temperature, reagent volume, reagent concentration, time, and mixing. [Example]

[0235] The method of any of Examples 51 to 58, wherein determining the concentration of the therapeutic polynucleotide further comprises performing a serial dilution in one or more chambers of the cartridge under the control of the microfluidic driver device, and repeating the step of driving the sample solution of the therapeutic polynucleotide for each of one or more dilutions formed by the serial dilution to create a dilution curve. [Example]

[0236] 60. The method of Example 59, wherein estimating the concentration of the therapeutic polynucleotide comprises estimating the concentration of the therapeutic polynucleotide from the dilution curve. [Example]

[0237] The method of any of Examples 51-60, wherein forming the therapeutic polynucleotide comprises performing an in vitro transcription (IVT) reaction to form the therapeutic polynucleotide. [Example]

[0238] The method of Example 61, further comprising creating a template for the IVT reaction in the cartridge. [Example]

[0239] The method of any of Examples 51 to 62, wherein the therapeutic polynucleotide comprises a therapeutic mRNA. [Example]

[0240] The method of Example 63, further comprising encapsulating the therapeutic mRNA in a delivery vehicle. [Example]

[0241] 65. The method of any of Examples 51-64, wherein the microfluidic driver device monitors the driving of the first blank solution and the driving of the sample solution by pneumatically deflecting one or more regions of a membrane of the cartridge to open and / or close valves of the cartridge. [Example]

[0242] 66. The method of Example 65, wherein the microfluidic driver device pneumatically deflects one or more regions of the membrane of the cartridge to drive the first blank solution and / or the sample solution into the measurement chamber. [Example]

[0243] 1. A system for producing and / or processing polynucleotides, comprising: a cartridge mount; a plurality of pressure lines; a plurality of fluid lines each connected to or configured to connect to a fluid source, wherein each fluid line, and at least a subset of the pressure lines, is configured to be secured to an inlet or outlet port on a cartridge held in the cartridge mount; and a controller configured to control the application of pressure through the pressure lines to drive fluid through the cartridge, the controller being further configured to: direct the formation of a therapeutic polynucleotide in the cartridge; and estimate the concentration of the therapeutic polynucleotide by driving a first blank solution through a measurement chamber of the cartridge and obtaining a first measurement value of the first blank solution in the measurement chamber; driving a sample solution containing the therapeutic polynucleotide through the measurement chamber of the cartridge and obtaining a second measurement value of the sample solution in the measurement chamber; and further configured to estimate the concentration of the therapeutic polynucleotide from the first and second measurements. [Example]

[0244] The system of Example 67 further comprises an ultraviolet (UV) light source and a UV light detector, wherein the controller is configured to obtain the first measurement in the form of a first absorption measurement using the UV light source and the UV light receiver, the controller is configured to obtain the second measurement in the form of a second absorption measurement using the UV light source and the UV light receiver, and the controller is configured to estimate the concentration of the therapeutic polynucleotide from the first absorption measurement and the second absorption measurement. [Example]

[0245] The system described in Example 68, wherein the controller is further configured to change one or more of the sensitivity of the UV detector and the intensity of the UV light source in response to the second absorption measurement through the cartridge. [Example]

[0246] A system described in any of Examples 68 to 69, wherein the controller is further configured to pre-treat the UV measurement chamber with UV light before obtaining the first absorption measurement value through the cartridge. [Example]

[0247] The system of any of Examples 67-70, wherein the controller is further configured to modify operation of the system based on the estimated concentration of the therapeutic polynucleotide. [Example]

[0248] 72. The system of any of Examples 67-71, wherein the controller is further configured to dilute the therapeutic polynucleotide to a standard concentration for output. [Example]

[0249] The system of any of Examples 67-73, wherein the controller is further configured to automatically adjust one or more parameters of the formation of the therapeutic polynucleotide based on the estimated concentration of the therapeutic polynucleotide. [Example]

[0250] The system of Example 73, wherein the one or more parameters include one or more of temperature, reagent volume, reagent concentration, time, and mixing. [Example]

[0251] The system of any of Examples 67-74, wherein the controller is further configured to perform serial dilutions in one or more chambers of the cartridge and repeat the step of driving the sample solution of the therapeutic polynucleotide for each of one or more dilutions of the sample solution of the therapeutic polynucleotide formed by the serial dilutions to create a dilution curve. [Example]

[0252] 76. The system of Example 75, wherein the controller is further configured to estimate the concentration of the therapeutic polynucleotide from the dilution curve. [Example]

[0253] [Example]

[0254] The system of any of Examples 67-76, wherein the controller is further configured to form the therapeutic polynucleotide, and includes performing an in vitro transcription (IVT) reaction to form the therapeutic polynucleotide. [Example]

[0255] 79. The system of Example 78, wherein the controller is further configured to create a template for the IVT reaction in the cartridge. [Example]

[0256] The system of any of Examples 67 to 79, wherein the therapeutic polynucleotide comprises a therapeutic mRNA. [Example]

[0257] 81. The system of Example 80, wherein the controller is further configured to encapsulate the therapeutic mRNA in a delivery vehicle. [Example]

[0258] A system described in any of Examples 67 to 81, wherein the controller is further configured to pneumatically deflect one or more regions of the membrane of the cartridge to open and / or close a valve of the cartridge when driving the first blank solution, driving the second blank solution, and driving the sample solution. [Example]

[0259] The system of Example 82, wherein the controller is further configured to pneumatically deflect one or more regions of the membrane of the cartridge to drive the first blank solution, the second blank solution, and / or the sample solution into the measurement chamber. [Example]

[0260] emitting ultraviolet (UV) light through a UV measurement chamber of a cartridge, the UV measurement chamber being configured to receive a fluid; receiving the UV light that has passed through the UV measurement chamber of the cartridge with a UV photodetector; measuring a maximum voltage of the UV photodetector; measuring a standard deviation of a signal from the UV photodetector; determining an upper limit of quantitation (LoQ) based on the measured maximum voltage and based on the measured standard deviation; determining a lower LoQ based on the measured maximum voltage and based on the measured standard deviation; determining whether the upper LoQ and the lower LoQ are acceptable; and if the upper LoQ and the lower LoQ are acceptable, allowing the cartridge to be used to make a therapeutic polynucleotide, or providing a warning if either or both of the upper LoQ and the lower LoQ are not acceptable; A method comprising: [Example]

[0261] The method of Example 84, further comprising forming or processing a therapeutic polynucleotide in the cartridge. [Example]

[0262] 1. A cartridge device for processing polynucleotides, comprising: a first layer defining a first recess providing a first ultraviolet (UV) measurement area; a second layer defining a second recess providing a second UV measurement area; an elastic material extending between the first layer and the second layer and separating the first layer from the second layer; a UV measurement chamber formed between the first UV measurement area and the second UV measurement area, the UV measurement chamber configured to receive a fluid, the first layer and the second layer configured to allow UV light to pass through the UV measurement chamber; and a UV blocking mechanism disposed on or in the first layer, the UV blocking mechanism configured to limit backscattering of UV light through or from the first layer. [Example]

[0263] 87. The cartridge device of Example 86, wherein the elastic material is not present along the UV measurement chamber. [Example]

[0264] A cartridge device described in any of Examples 86 to 87, wherein the first recess has a first diameter and the UV blocking mechanism has an opening having a diameter smaller than the first diameter of the first recess. [Example]

[0265] A cartridge device described in any of Examples 86 to 88, wherein the UV blocking mechanism has a main body arranged on the upper surface of the first layer, and the first recess is formed through the upper surface of the first layer. [Example]

[0266] 89. The cartridge device of claim 89, wherein the body has a disk shape. [Example]

[0267] A cartridge device described in any of Examples 86 to 90, wherein the UV blocking mechanism has a cylindrical body disposed within the first recess. [Example]

[0268] A cartridge device described in any of Examples 86 to 91, wherein the UV blocking mechanism is configured to block transmission of UV light through the sidewall of the first recess. [Example]

[0269] emitting ultraviolet (UV) light through a UV measurement chamber of a cartridge containing a fluid; receiving the UV light at a UV light detector, the received UV light passing through the UV measurement chamber of the cartridge; obtaining a first plurality of concentration measurements, each concentration measurement of the first plurality indicating a respective concentration of fluid in the UV measurement chamber; calculating an average of the first plurality of concentration measurements and comparing each concentration measurement of the first plurality to the average of the first plurality of concentration measurements; identifying an outlier from the first plurality of concentration measurements relative to the average of the first plurality of concentration measurements; establishing a second plurality of concentration measurements, the second plurality of concentration measurements including the first plurality of concentration measurements excluding the identified outlier; calculating an average of the second plurality of concentration measurements and reporting the average of the second plurality of concentration measurements; A method comprising: [Example]

[0270] 94. The method of example 93, wherein the first plurality of concentration measurements includes four concentration measurements. [Example]

[0271] 95. The method of any of Examples 93-94, wherein the second plurality of concentration measurements comprises three concentration measurements. [Example]

[0272] 96. The method of any of Examples 93-95, wherein the identified outlier provides a concentration measurement that substantially deviates from the average of the first plurality of measurements due to the presence of air bubbles in the fluid in the UV measurement chamber. [Example]

[0273] The method of example 96, further comprising executing a bubble removal algorithm in response to the identified outlier deviating from the average of the first plurality of measurements by a predetermined degree.

[0274] It should be understood that all combinations of the foregoing concepts, and the additional concepts discussed in more detail below (provided that such concepts are not mutually inconsistent), are contemplated to be part of the inventive subject matter disclosed herein and may be used to achieve the benefits described herein.

[0275] The process parameters and order of steps described and / or illustrated herein are provided by way of example only and can be varied as desired. For example, although the steps illustrated and / or described herein may be illustrated or discussed in a particular order, these steps do not necessarily have to be performed in the order illustrated or discussed. The various exemplary methods described and / or illustrated herein may also omit one or more of the steps described or illustrated herein or may include additional steps in addition to those disclosed.

[0276] Any of the methods described herein (including user interfaces) may be implemented as software, hardware, or firmware, and may also be described as a non-transitory computer-readable storage medium storing a set of instructions executable by a processor (e.g., a computer, tablet, smartphone, etc.) that, when executed by the processor, causes the processor to control the performance of any of the steps, including, but not limited to, displaying, communicating with a user, analyzing, modifying parameters (including timing, frequency, intensity, etc.), determining, alerting, etc. For example, any of the methods described herein may be performed at least in part by an apparatus comprising one or more processors having a memory storing a non-transitory computer-readable storage medium that stores a set of instructions for the process of the method.

[0277] While various embodiments have been described and / or illustrated herein in the context of a fully functional computing system, one or more of these exemplary embodiments may be distributed as a program product in various forms, regardless of the particular type of computer-readable medium actually used for distribution. The embodiments disclosed herein may also be implemented using software modules that perform particular tasks. These software modules may include scripts, batch files, or other executable files that may be stored on a computer-readable storage medium or on a computing system. In some embodiments, these software modules may configure a computing system to implement one or more of the exemplary embodiments disclosed herein.

[0278] Although illustrated as separate elements, the method steps described and / or illustrated herein may represent parts of a single application. Additionally, in some embodiments, one or more of these steps may represent or correspond to one or more software applications or programs that, when executed by a computing device, cause the computing device to perform one or more tasks, such as method steps.

[0279] Additionally, one or more of the devices described herein may transform data, physical devices, and / or representations of physical devices from one form to another. Additionally or alternatively, one or more of the modules described herein, by executing on, storing data on, and / or otherwise interacting with a computing device, may transform a processor, volatile memory, non-volatile memory, and / or any other portion of a physical computing device from one form of computing device to another form of computing device.

[0280] As used herein, the term "computer-readable medium" generally refers to any form of device, carrier, or medium capable of storing or carrying computer-readable instructions. Examples of computer-readable media include, but are not limited to, transmission-type media such as carrier waves, and non-transitory-type media such as magnetic storage media (e.g., hard disk drives, tape drives, and floppy disks), optical storage media (e.g., compact discs (CDs), digital video discs (DVDs), and Blu-ray discs), electronic storage media (e.g., solid-state drives and flash media), and other distribution systems.

[0281] As used herein, when a feature or element is referred to as being "on" another feature or element, it may be directly on the other feature or element, or intervening features and / or elements may be present. In contrast, when a feature or element is referred to as being "directly on" another feature or element, there are no intervening features or elements present. Also, when a feature or element is referred to as being "connected," "coupled," or "coupled" to another feature or element, it will be understood that it may be directly connected, coupled, or coupled to the other feature or element, or that intervening features or elements may be present. In contrast, when a feature or element is referred to as being "directly connected," "directly coupled," or "directly coupled" to another feature or element, there are no intervening features or elements present. Although described or illustrated with respect to one embodiment, features and elements so described or illustrated may apply to other embodiments. Those skilled in the art will also understand that a reference to a structure or feature disposed "adjacent" to another feature may have portions that overlap or underlie the adjacent feature.

[0282] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. For example, as used herein, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly dictates otherwise. It will be further understood that the terms "comprises" and / or "comprising," as used herein, specify the presence of stated features, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items and may be abbreviated as " / ."

[0283] Spatially relative terms, such as "under," "below," "lower," "over," and "upper," may be used herein for ease of description to describe the relationship of one element or feature to another, as illustrated in the figures. It will be understood that spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if a device in the figures were inverted, an element described as "under" or "beneath" another element or feature would then be oriented "over" that other element or feature. Thus, the exemplary term "under" can encompass both an orientation of above and below. A device may be otherwise oriented (rotated 90 degrees or at other orientations), and the spatially relative descriptors used herein will be interpreted accordingly. Similarly, terms such as "upward," "downward," "vertically," "horizontally," and the like are used herein for descriptive purposes only, unless specifically indicated otherwise.

[0284] The terms "first" and "second" may be used herein to describe various features / elements (including steps), but these features / elements should not be limited by these terms unless the context indicates otherwise. These terms may be used to distinguish one feature / element from another. Thus, a first feature / element discussed below may be referred to as a second feature / element, and similarly, a second feature / element discussed below may be referred to as a first feature / element, without departing from the teachings of the present invention.

[0285] Generally, any of the apparatus and methods described herein should be understood to be inclusive, although all or a subset of the components and / or steps may alternatively be exclusive, and may be expressed as "consisting of" or "consisting essentially of" various components, steps, subcomponents, or substeps.

[0286] As used in this specification and claims, including in the examples, unless expressly specified otherwise, all numbers may be read as if preceded by the words "about" or "approximately," even if these words do not explicitly appear. The phrase "about" or "approximately," when describing a size and / or location, may be used to indicate that the described value and / or location is within a reasonable expected range of value and / or location. For example, a numerical value may have a value that is + / - 0.1% of the stated value (or range of values), + / - 1% of the stated value (or range of values), + / - 2% of the stated value (or range of values), + / - 5% of the stated value (or range of values), + / - 10% of the stated value (or range of values), etc. Any numerical value given herein should be understood to include about or approximately that value unless the context dictates otherwise. For example, if the value "10" is disclosed, then "about 10" is also disclosed. Any numerical range recited herein is intended to include all subranges subsumed therein. It is also understood that when a value is disclosed as being "less than or equal to" that value, "greater than or equal to" that value and possible ranges between those values ​​are also disclosed, as would be understood by one of ordinary skill in the art. For example, if a value "X" is disclosed, "less than or equal to X" as well as "greater than or equal to X" (e.g., where X is a numeric value) are also disclosed. It is also understood that throughout this application, data is provided in a number of different formats, and this data represents endpoints and starting points, as well as ranges for any combination of the data points. For example, if a specific data point "10" and a specific data point "15" are disclosed, it is understood that greater than 10 and 15, greater than or equal to 10 and 15, less than 10 and 15, less than or equal to 10 and 15, and equal to 10 and 15 are considered to be disclosed, as are values ​​between 10 and 15. It is understood that each unit between two specified units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0287] While various exemplary embodiments have been described above, any of numerous modifications can be made to the various embodiments without departing from the scope of the invention, as set forth in the claims. For example, the order in which various described method steps are performed is often changed in alternative embodiments, and in other alternative embodiments, one or more method steps may be skipped entirely. Optional features of various device and system embodiments may be included in some embodiments but not in others. Accordingly, the foregoing description has been provided primarily for illustrative purposes and should not be construed as limiting the scope of the invention, as set forth in the claims.

[0288] The examples and illustrations contained herein illustrate, by way of illustration and not limitation, specific embodiments in which the subject matter may be practiced. As noted above, other embodiments may be utilized and derived therefrom, such that structural and logical substitutions and changes may be made without departing from the scope of the present disclosure. Such embodiments of the inventive subject matter may be referred to herein, individually or collectively, by the term "invention," merely for convenience, and are not intended to spontaneously limit the scope of this application to any single invention or inventive concept when more than one is actually disclosed. Thus, while specific embodiments have been illustrated and described herein, any arrangement calculated to achieve the same purpose may be substituted for the specific embodiment shown. The present disclosure is intended to cover any and all adaptations or variations of the various embodiments. Combinations of the above embodiments, as well as other embodiments not specifically described herein, will be apparent to those skilled in the art upon reviewing the above description.

Claims

1. 1. A cartridge device for processing polynucleotides, comprising: a first layer having a first thickness; a second layer having a second thickness; an elastic material extending between the first layer and the second layer and separating the first layer from the second layer by a separation thickness; an ultraviolet (UV) measurement region formed through the first layer and the second layer, wherein a region of the first layer in the UV measurement region has a thickness less than or equal to the first thickness, a region of the second layer in the UV measurement region has a thickness less than or equal to the second thickness, and the elastic material is not present between the region of the first layer in the UV measurement region and the region of the second layer in the UV measurement region, forming a UV measurement chamber having the separation thickness.

2. The cartridge device of claim 1 , wherein the first layer comprises a polymeric material.

3. The cartridge device of claim 2 , wherein the first layer comprises a cyclic olefin copolymer (COC) material.

4. The cartridge device according to any one of claims 1 to 3, wherein the first thickness is 1 to 5 mm.

5. The cartridge device according to any one of claims 1 to 4, wherein the second layer comprises a polymer material.

6. The cartridge device of claim 5 , wherein the second layer comprises a cyclic olefin copolymer (COC) material.

7. The cartridge device according to any one of claims 1 to 6, wherein the second thickness is 1 to 5 mm.

8. The cartridge device according to any one of claims 1 to 7, wherein the separation thickness is between 1 mm and 0.1 mm.

9. The cartridge device of any preceding claim, further comprising a fluid channel in the second layer in fluid communication with the UV measurement chamber.

10. 10. A cartridge device as described in any one of claims 1 to 9, further comprising a plurality of pneumatic valves arranged to control flow into and out of the UV measurement chamber, each of the plurality of pneumatic valves being formed by an air pressure chamber in the first layer and a fluid chamber in the second layer, a portion of the elastic material separating the air pressure chamber from the fluid chamber, the air pressure chamber being in fluid communication with an air pressure channel in the first layer that is configured to communicate with a pressure port in an outer region of the cartridge device to actuate the pneumatic valve, and the fluid chamber being in fluid communication with the UV measurement chamber through the fluid channel in the second layer when the pneumatic valve is open.

11. The cartridge device of any preceding claim, further comprising an in vitro transcription (IVT) chamber formed at least partially within the second layer and in fluid communication with the UV measurement chamber.

12. A cartridge device as described in any preceding claim, further comprising one or more vacuum ports in an outer region of the cartridge device configured to connect to a negative pressure source to draw fluid into the UV measurement chamber.

13. A cartridge device described in any one of claims 1 to 12, further comprising a first inlet channel in the second layer that is fluidly connected to a first chamber configured to hold a polynucleotide sample fluid, and a second inlet channel in the second layer that is fluidly connected to a second chamber configured to hold a blank sample fluid.

14. The cartridge device of any preceding claim, further comprising one or more dilution and mixing chambers in fluid communication with the UV measurement chamber configured to dilute the fluid sample.

15. A cartridge device according to any preceding claim, wherein the diameter of the UV measurement chamber is greater than the diameter of the region of the first layer within the UV measurement region to limit air bubbles within a central region of the UV measurement chamber.

16. 1. A cartridge device for processing polynucleotides, comprising: a first layer comprising a polymeric material having a first thickness; a second layer comprising a polymeric material having a second thickness; an elastic material extending between the first layer and the second layer; an ultraviolet (UV) measurement area formed through the first layer and the second layer, wherein an area of ​​the first layer in the UV measurement area has a thickness less than or equal to the first thickness, an area of ​​the second layer in the UV measurement area has a thickness less than or equal to the second thickness, and the elastic material is removed from between the area of ​​the first layer in the UV measurement area and the area of ​​the second layer in the UV measurement area to form a UV measurement chamber; an in vitro transfer (IVT) chamber formed at least partially within the second layer and in fluid communication with the UV measurement chamber; A cartridge device comprising:

17. 1. A method for producing polynucleotides using a cartridge-operated microfluidic driver device, comprising: forming a therapeutic polynucleotide in the cartridge; The microfluidic driver device driving a first blank solution into an ultraviolet (UV) measurement chamber of the cartridge and obtaining a first absorption measurement through the cartridge; driving a sample solution of the therapeutic polynucleotide into the UV measurement chamber of the cartridge and obtaining a second absorption measurement through the cartridge; determining the concentration of the therapeutic polynucleotide by estimating the concentration of the therapeutic polynucleotide from the first absorption measurement and the second absorption measurement; A method comprising:

18. 20. The method of claim 17, further comprising, in a processor of the microfluidic driver device, adjusting operation of the microfluidic driver device based on the estimated concentration of the therapeutic polynucleotide.

19. 19. The method of any of claims 17-18, further comprising: comparing, by a processor of the microfluidic driver device, the estimated concentration of the therapeutic polynucleotide to a concentration range; and, based on the comparison, directing the therapeutic polynucleotide to one of pooling with one or more additional batches of therapeutic polynucleotide formed in the cartridge, discarding, or further analysis.

20. 20. The method of any of claims 17 to 19, further comprising diluting the therapeutic polynucleotide to a standard concentration for output under control of a processor of the microfluidic driver device.

21. 21. The method of any of claims 17-20, further comprising automatically adjusting one or more parameters of the formation of the therapeutic polynucleotide by the microfluidic driver device based on the estimated concentration of the therapeutic polynucleotide.

22. 22. The method of claim 21, wherein the one or more parameters include one or more of temperature, reagent volume, reagent concentration, time, and mixing.

23. 23. The method of any of claims 17 to 22, wherein determining the concentration of the therapeutic polynucleotide further comprises: performing a serial dilution in one or more chambers of the cartridge under the control of the microfluidic driver device; and repeating the step of driving the sample solution of the therapeutic polynucleotide for each of one or more dilutions formed by the serial dilution to create a dilution curve.

24. 24. The method of claim 23, wherein estimating the concentration of the therapeutic polynucleotide comprises estimating the concentration of the therapeutic polynucleotide from the dilution curve.

25. 25. The method of any of claims 17-24, wherein estimating the concentration of the therapeutic polynucleotide further comprises altering one or more of a sensitivity of a UV detector and an intensity of a UV emitter of the microfluidic driver device in response to the second absorption measurement through the cartridge.

26. The method of any of claims 17 to 25, further comprising pre-treating the UV measurement chamber with UV light before taking the first absorption measurement through the cartridge.

27. 27. The method of any of claims 17-26, wherein forming the therapeutic polynucleotide comprises performing an in vitro transcription (IVT) reaction to form the therapeutic polynucleotide.

28. 28. The method of claim 27, further comprising creating a template for the IVT reaction in the cartridge.

29. The method of any one of claims 17 to 28, wherein the therapeutic polynucleotide comprises a therapeutic mRNA.

30. 30. The method of claim 29, further comprising encapsulating the therapeutic mRNA in a delivery vehicle.

31. 31. The method of any of claims 17 to 30, wherein the microfluidic driver device monitors the driving of the first blank solution and the driving of the sample solution by pneumatically deflecting one or more regions of a membrane of the cartridge to open and / or close valves of the cartridge.

32. 32. The method of claim 31 , wherein the microfluidic driver device pneumatically deflects one or more regions of the membrane of the cartridge to drive the first blank solution and / or the sample solution into the UV measurement chamber.

33. 1. A method for producing polynucleotides using a cartridge-operated microfluidic driver device, comprising: forming a therapeutic polynucleotide in a cartridge, the therapeutic polynucleotide comprising a therapeutic mRNA; The microfluidic driver device driving a first blank solution into an ultraviolet (UV) measurement chamber of the cartridge and obtaining a first absorption measurement through the cartridge; driving a sample solution of the therapeutic polynucleotide into the UV measurement chamber of the cartridge and obtaining a second absorption measurement through the cartridge; driving a second blank solution into the UV measurement chamber of the cartridge and taking a third absorption measurement through the cartridge; determining the concentration of the therapeutic mRNA by estimating the concentration of the therapeutic polynucleotide from the first absorption measurement, the second absorption measurement, and the third absorption measurement; adjusting, in a processor of the microfluidic driver device, operation of the microfluidic driver device based on the estimated concentration of the therapeutic polynucleotide; A method comprising:

34. 1. A system for producing and / or processing polynucleotides, comprising: Cartridge mount and a plurality of pressure lines; a plurality of fluid lines, each fluid line coupled or configured to couple to a fluid source, wherein each fluid line and at least a subset of the pressure lines are configured to be secured to an inlet or outlet port on a cartridge held within the cartridge mount; an ultraviolet (UV) light source; a UV photodetector; a controller configured to control the application of pressure through the pressure line to drive fluid through the cartridge, the controller being further configured to direct the formation of a therapeutic polynucleotide in the cartridge and to estimate a concentration of the therapeutic polynucleotide by driving a first blank solution through a UV measurement chamber of the cartridge and taking a first absorption measurement using the UV light source and UV light receiver, and driving a sample solution containing the therapeutic polynucleotide through the UV measurement chamber of the cartridge and taking a second absorption measurement using the UV light source and UV light receiver; and further configured to estimate the concentration of the therapeutic polynucleotide from the first absorption measurement and the second absorption measurement; A system comprising:

35. 35. The system of claim 34, wherein the controller is further configured to modify operation of the system based on the estimated concentration of the therapeutic polynucleotide.

36. The system of any of claims 34-35, wherein the controller is further configured to compare the estimated concentration of the therapeutic polynucleotide with a concentration range and, based on the comparison, direct the therapeutic polynucleotide to one of pooling with one or more additional batches of therapeutic polynucleotide formed in the cartridge, discarding, or further analysis.

37. The system of any of claims 34 to 36, wherein the controller is further configured to dilute the therapeutic polynucleotide to a standard concentration for output.

38. 38. The system of any of claims 34-37, wherein the controller is further configured to automatically adjust one or more parameters of the formation of the therapeutic polynucleotide based on the estimated concentration of the therapeutic polynucleotide.

39. 39. The system of claim 38, wherein the one or more parameters include one or more of temperature, reagent volume, reagent concentration, time, and mixing.

40. The system of any one of claims 34 to 39, wherein the controller is further configured to perform a serial dilution in one or more chambers of the cartridge and repeat the step of driving the sample solution of the therapeutic polynucleotide for each of one or more dilutions of the sample solution of the therapeutic polynucleotide formed by the serial dilution to create a dilution curve.

41. 41. The system of claim 40, wherein the controller is further configured to estimate a concentration of the therapeutic polynucleotide from the dilution curve.

42. 42. The system of any one of claims 34 to 41, wherein the controller is further configured to modify one or more of the sensitivity of the UV detector and the intensity of the UV light source in response to the second absorption measurement through the cartridge.

43. 43. The system of any of claims 34-42, wherein the controller is further configured to pre-treat the UV measurement chamber with UV light before obtaining the first absorption measurement through the cartridge.

44. 44. The system of any of claims 34-43, wherein the controller is further configured to form the therapeutic polynucleotide, comprising performing an in vitro transcription (IVT) reaction to form the therapeutic polynucleotide.

45. 45. The system of claim 44, wherein the controller is further configured to create a template for the IVT reaction within the cartridge.

46. The system of any one of claims 34 to 45, wherein the therapeutic polynucleotide comprises a therapeutic mRNA.

47. 47. The system of claim 46, wherein the controller is further configured to encapsulate the therapeutic mRNA in a delivery vehicle.

48. 48. The system of any one of claims 34 to 47, wherein the controller is further configured to pneumatically deflect one or more regions of a membrane of the cartridge to open and / or close a valve of the cartridge when driving the first blank solution, driving the second blank solution, and driving the sample solution.

49. 49. The system of claim 48, wherein the controller is further configured to pneumatically deflect one or more regions of the membrane of the cartridge to drive the first blank solution, the second blank solution, and / or the sample solution into the UV measurement chamber.

50. 1. A system for producing and / or processing polynucleotides, comprising: A cartridge and 1. A microfluidic driver device, comprising: Cartridge mount, Multiple pressure lines, a plurality of fluid lines, each fluid line coupled or configured to couple to a fluid source, wherein each fluid line and at least a subset of the pressure lines are configured to be secured to an inlet or outlet port on a cartridge held within the cartridge mount; ultraviolet (UV) light source, a UV photodetector, and and a microfluidic driver device including a controller configured to control the application of pressure through the pressure line to drive fluid through the cartridge, wherein the controller is further configured to direct the formation of a therapeutic polynucleotide, and the controller is further configured to: drive a first blank solution through a UV measurement chamber of the cartridge, obtain a first absorption measurement using the UV light source and UV light receiver, drive a sample solution comprising the therapeutic polynucleotide through the UV measurement chamber of the cartridge, obtain a second absorption measurement using the UV light source and UV light receiver, drive a second blank solution through the UV measurement chamber of the cartridge, obtain a third absorption measurement using the UV light source and UV light receiver, and estimate the concentration of the therapeutic polynucleotide by modifying operation of the microfluidic driver device based on the concentration of the therapeutic polynucleotide determined from the first absorption measurement, the second absorption measurement, and the third absorption measurement.

51. 1. A method for producing polynucleotides using a cartridge-operated microfluidic driver device, comprising: forming a therapeutic polynucleotide in the cartridge; The microfluidic driver device driving a first blank solution into a concentration measurement chamber of the cartridge and taking a first measurement through the cartridge; driving a sample solution of the therapeutic polynucleotide into the concentration measurement chamber of the cartridge and obtaining a second measurement through the cartridge; determining the concentration of the therapeutic polynucleotide by estimating the concentration of the therapeutic polynucleotide from the first measurement and the second measurement; comparing, by a processor of the microfluidic driver device, the estimated concentration of the therapeutic polynucleotide to a concentration range, and based on the comparison, directing the therapeutic polynucleotide to one of pooling with one or more additional batches of therapeutic polynucleotide formed in the cartridge, discarding, or further analyzing; A method comprising:

52. 52. The method of claim 51, wherein the concentration measuring chamber comprises an ultraviolet (UV) measuring chamber, the first measurement comprises a first absorption measurement, and the second measurement comprises a second absorption measurement.

53. 53. The method of claim 52, wherein estimating the concentration of the therapeutic polynucleotide further comprises altering one or more of a sensitivity of a UV detector and an intensity of a UV emitter of the microfluidic driver device in response to the second absorption measurement through the cartridge.

54. 54. The method of any of claims 52-53, further comprising pre-treating the UV measurement chamber with UV light before taking the first absorption measurement through the cartridge.

55. 55. The method of any of claims 51-54, further comprising, in a processor of the microfluidic driver device, adjusting operation of the microfluidic driver device based on the estimated concentration of the therapeutic polynucleotide.

56. 56. The method of any of claims 51-55, further comprising diluting the therapeutic polynucleotide to a standard concentration for output under control of a processor of the microfluidic driver device.

57. 56. The method of any of claims 51-55, further comprising automatically adjusting one or more parameters of the formation of the therapeutic polynucleotide by the microfluidic driver device based on the estimated concentration of the therapeutic polynucleotide.

58. 58. The method of claim 57, wherein the one or more parameters include one or more of temperature, reagent volume, reagent concentration, time, and mixing.

59. 59. The method of any of claims 51-58, wherein determining the concentration of the therapeutic polynucleotide further comprises performing a serial dilution in one or more chambers of the cartridge under the control of the microfluidic driver device, and repeating the step of driving the sample solution of the therapeutic polynucleotide for each of one or more dilutions formed by the serial dilution to create a dilution curve.

60. 60. The method of claim 59, wherein estimating the concentration of the therapeutic polynucleotide comprises estimating the concentration of the therapeutic polynucleotide from the dilution curve.

61. 61. The method of any of claims 51-60, wherein forming the therapeutic polynucleotide comprises performing an in vitro transcription (IVT) reaction to form the therapeutic polynucleotide.

62. 62. The method of claim 61, further comprising creating a template for the IVT reaction in the cartridge.

63. 63. The method of any of claims 51-62, wherein the therapeutic polynucleotide comprises a therapeutic mRNA.

64. 64. The method of claim 63, further comprising encapsulating the therapeutic mRNA in a delivery vehicle.

65. 65. The method of any of claims 51 to 64, wherein the microfluidic driver device monitors the driving of the first blank solution and the driving of the sample solution by pneumatically deflecting one or more regions of a membrane of the cartridge to open and / or close valves of the cartridge.

66. 66. The method of claim 65, wherein the microfluidic driver device pneumatically deflects one or more regions of the membrane of the cartridge to drive the first blank solution and / or the sample solution into the measurement chamber.

67. 1. A system for producing and / or processing polynucleotides, comprising: Cartridge mount and a plurality of pressure lines; a plurality of fluid lines, each fluid line coupled or configured to couple to a fluid source, wherein each fluid line and at least a subset of the pressure lines are configured to be secured to an inlet or outlet port on a cartridge held within the cartridge mount; a controller configured to control the application of pressure through the pressure line to drive fluid through the cartridge, the controller being further configured to direct the formation of a therapeutic polynucleotide in the cartridge; and to estimate the concentration of the therapeutic polynucleotide by driving a first blank solution through a measurement chamber of the cartridge, obtaining a first measurement value of the first blank solution in the measurement chamber, driving a sample solution containing the therapeutic polynucleotide through the measurement chamber of the cartridge, and obtaining a second measurement value of the sample solution in the measurement chamber; and further configured to estimate the concentration of the therapeutic polynucleotide from the first measurement value and the second measurement value.

68. an ultraviolet (UV) light source; a UV photodetector; Further provided with the controller is configured to obtain the first measurement in the form of a first absorption measurement using the UV light source and the UV light receiver; the controller is configured to obtain the second measurement in the form of a second absorption measurement using the UV light source and the UV light receiver; 68. The system of claim 67, wherein the controller is configured to estimate a concentration of the therapeutic polynucleotide from the first absorption measurement and the second absorption measurement.

69. 69. The system of claim 68, wherein the controller is further configured to modify one or more of the sensitivity of the UV detector and the intensity of the UV light source in response to the second absorption measurement through the cartridge.

70. 70. The system of any one of claims 68-69, wherein the controller is further configured to pre-treat the UV measurement chamber with UV light before obtaining the first absorption measurement through the cartridge.

71. 71. The system of any of claims 67-70, wherein the controller is further configured to modify operation of the system based on the estimated concentration of the therapeutic polynucleotide.

72. 72. The system of any of claims 67-71, wherein the controller is further configured to dilute the therapeutic polynucleotide to a standard concentration for output.

73. 74. The system of any of claims 67-73, wherein the controller is further configured to automatically adjust one or more parameters of the formation of the therapeutic polynucleotide based on the estimated concentration of the therapeutic polynucleotide.

74. 74. The system of claim 73, wherein the one or more parameters include one or more of temperature, reagent volume, reagent concentration, time, and mixing.

75. 75. The system of any one of claims 67 to 74, wherein the controller is further configured to perform a serial dilution in one or more chambers of the cartridge and repeat the step of driving the sample solution of the therapeutic polynucleotide for each of one or more dilutions of the sample solution of the therapeutic polynucleotide formed by the serial dilution to create a dilution curve.

76. 76. The system of claim 75, wherein the controller is further configured to estimate a concentration of the therapeutic polynucleotide from the dilution curve.

78. 77. The system of any of claims 67-76, wherein the controller is further configured to form the therapeutic polynucleotide, comprising performing an in vitro transcription (IVT) reaction to form the therapeutic polynucleotide.

79. 79. The system of claim 78, wherein the controller is further configured to create a template for the IVT reaction within the cartridge.

80. 80. The system of any one of claims 67 to 79, wherein the therapeutic polynucleotide comprises a therapeutic mRNA.

81. 81. The system of claim 80, wherein the controller is further configured to encapsulate the therapeutic mRNA in a delivery vehicle.

82. 82. The system of any one of claims 67 to 81, wherein the controller is further configured to pneumatically deflect one or more regions of a membrane of the cartridge to open and / or close a valve of the cartridge when driving the first blank solution, driving the second blank solution, and driving the sample solution.

83. 83. The system of claim 82, wherein the controller is further configured to pneumatically deflect one or more regions of the membrane of the cartridge to drive the first blank solution, the second blank solution, and / or the sample solution into the measurement chamber.

84. 1. A method comprising: emitting ultraviolet (UV) light through a UV measurement chamber of the cartridge, the UV measurement chamber configured to receive a fluid; receiving UV light that has passed through the UV measurement chamber of the cartridge with a UV light detector; measuring the maximum voltage of the UV photodetector; measuring the standard deviation of the signal from the UV photodetector; determining an upper limit of quantitation (upper LoQ) based on the measured maximum voltage and based on the measured standard deviation; determining a lower limit of quantitation (LoQ) based on the measured maximum voltage and based on the measured standard deviation; determining whether the upper and lower LoQs are acceptable; If the upper LoQ and the lower LoQ are acceptable, allowing the cartridge to be used to make a therapeutic polynucleotide; or providing a warning if either the upper LoQ or the lower LoQ or both are not acceptable; A method comprising:

85. 85. The method of claim 84, further comprising forming or processing a therapeutic polynucleotide in the cartridge.

86. 1. A cartridge device for processing polynucleotides, comprising: a first layer defining a first recess providing a first ultraviolet (UV) measurement area; a second layer defining a second recess providing a second UV measurement area; an elastic material extending between the first layer and the second layer and separating the first layer from the second layer; a UV measurement chamber formed between the first UV measurement area and the second UV measurement area, the UV measurement chamber configured to receive a fluid, the first layer and the second layer configured to allow UV light to pass through the UV measurement chamber; a UV blocking feature disposed on or in the first layer, the UV blocking feature configured to limit backscattering of UV light through or from the first layer; A cartridge device comprising:

87. 87. The cartridge device of claim 86, wherein the elastic material is absent along the UV measurement chamber.

88. A cartridge device as described in any of claims 86 to 87, wherein the first recess has a first diameter and the UV blocking mechanism has an opening having a diameter smaller than the first diameter of the first recess.

89. A cartridge device as described in any of claims 86 to 88, wherein the UV blocking mechanism has a body disposed on the upper surface of the first layer, and the first recess is formed through the upper surface of the first layer.

90. 90. The cartridge device of claim 89, wherein the body has a disk shape.

91. A cartridge device according to any one of claims 86 to 90, wherein the UV blocking mechanism comprises a cylindrical body disposed within the first recess.

92. A cartridge device according to any one of claims 86 to 91, wherein the UV blocking mechanism is configured to block transmission of UV light through a side wall of the first recess.

93. 1. A method comprising: emitting ultraviolet (UV) light through a UV measurement chamber of the cartridge, the UV measurement chamber contacting the fluid; receiving UV light that has passed through the UV measurement chamber of the cartridge with a UV light detector; obtaining a first plurality of concentration measurements, each concentration measurement of the first plurality of concentration measurements indicating a respective concentration of fluid in the UV measurement chamber; calculating an average of the first plurality of concentration measurements; comparing each concentration measurement of the first plurality of concentration measurements to the average of the first plurality of concentration measurements; identifying outliers from the first plurality of concentration measurements relative to the average of the first plurality of concentration measurements; establishing a second plurality of concentration measurements, the second plurality of concentration measurements including the first plurality of concentration measurements excluding the identified outlier; and calculating an average of the second plurality of concentration measurements; reporting the average of the second plurality of concentration measurements; and A method comprising:

94. 94. The method of claim 93, wherein the first plurality of concentration measurements includes four concentration measurements.

95. 95. The method of any of claims 93-94, wherein the second plurality of concentration measurements comprises three concentration measurements.

96. 96. A method according to any one of claims 93 to 95, wherein the identified outlier provides a concentration measurement that deviates substantially from the average of the first plurality of measurements due to the presence of air bubbles in the fluid in the UV measurement chamber.

97. 97. The method of claim 96, further comprising: executing a bubble removal algorithm in response to the identified outlier deviating from the average of the first plurality of measurements by a predetermined amount.

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