Apparatus and method for measuring DNA / RNA production in biochips

Abstract

Microfluidic devices (e.g., systems, apparatuses, etc.) and microfluidic methods provide detection of polynucleotide concentrations. The devices may include removable, single-use, or reusable microfluidic components, which may include one or more channels, chambers, and / or substrates for performing microfluidic manipulations and are suitable for detection of polynucleotide concentrations. For example, the microfluidic component may be a microfluidic cartridge that may be inserted, retained, and / or disposed in a microfluidic driver device that may supervise and control operation within one or more cartridges based in part on the detected concentration of the polynucleotide.

Description

[0001] Claiming priority

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 396,765, filed on August 10, 2022, entitled “Apparatuses and Methods for Measuring DNA / RNA Production in Biochips.”

[0003] Incorporation by Reference

[0004] 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 Art

[0005] UV spectrophotometry is a standard technique for nucleic acid quantification. In UV spectrophotometry, a nucleic acid sample is placed in a chamber (usually a quartz cuvette), which is then placed in a UV spectrophotometer. UV light is passed through the sample at a specified path length, and the absorbance of the sample at a specific wavelength is measured. The absorbance at 260nm (A260) is typically used to measure nucleic acids. Nucleic acids (e.g., DNA or RNA) contain conjugated double bonds in their purine and pyrimidine rings, which have a specific absorption peak at about 260nm. The maximum absorbance of nucleic acids occurs at a wavelength of 260nm. The intensity of this absorbance is proportional to the concentration of nucleic acids. Due to these physical properties, the nucleic acid concentration of a sample can be determined. Based on the absorbance reading, the concentration of a sample can be determined. The purity of a sample can also be estimated by comparing it with the absorbance at 280nm or 230nm.

[0006] However, this traditional UV spectrophotometric determination for the quantification of polynucleotides usually requires the removal of the sample and transfer to a dedicated spectrophotometer device. Typically, the minimum sample volume must be at least 1 μL to 75 μL to obtain accurate instrument readings. If the sample is highly diluted, or if the extraction technique requires the nucleic acid to be eluted into a low volume, this may be problematic. Pollution is also a problem. For example, if DNA is measured, biomolecules such as proteins, RNA, and chaotropic salts from the extraction process may mistakenly increase the nucleic acid concentration estimate. Buffer salts such as Tris, EDTA, and guanidine isothiocyanate strongly absorb at 230nm and diffuse into (bleed into) the 260nm absorbance range. In addition, free nucleotides present in the sample may affect the UV quantitative method. The change in sample pH also changes the UV reading. The alternative to the photometer is to add a fluorescent probe and measure the sample in a fluorometer, which requires further processing of RNA / DNA samples and labeling such molecules with fluorescent molecules, thereby increasing cost and complexity. All of these methods have major disadvantages and may require manual intervention and output measurements that are not associated with the microfluidic control of the system.

[0007] Described herein are devices, and in particular microfluidic devices, that may address these shortcomings. Summary of the invention

[0008] Microfluidic devices (e.g., systems, devices, etc.) and methods for microfluidic detection of polynucleotide concentrations are described herein. These devices may include removable, single-use or reusable microfluidic members, which may include one or more channels, chambers and / or substrates for performing microfluid manipulation. For example, microfluidic members may be microfluidic boxes, microfluidic chips ("biochips"), etc., and may be referred to as "boxes" in this article. Any box in these boxes may be a roughly flat planar structure, which may be inserted, maintained and / or placed in a microfluidic drive device, which may monitor and control the operation in one or more boxes. Microfluidic drive devices may be referred to as microfluidic path devices in this article and may include actuators, sensors, controllers (including control circuits, processors, etc.). As described in detail below, box may be suitable for including one or more polynucleotide quantitative regions, which may be suitable for optical sensing and quantification of polynucleotides such as DNA, RNA (e.g., mRNA), etc. The polynucleotide quantification region is configured for spectrophotometric reading by an emitter and a detector, which may be part of a microfluidic drive device. The microfluidic drive device may also be adapted to control the activity of the microfluidic drive device and the cartridge based on feedback from the spectrophotometric reading.

[0009] For example, the devices and methods described herein can be configured to quantitatively generate RNA and / or DNA in a microfluidic device (e.g., in a box driven by a microfluidic drive device) via an exponential biochemical reaction. These devices can change or control and adjust the output to correct for changes caused by reagents, biochips, or temperature defects. Specifically, the methods and devices described herein can measure the concentration of RNA and / or DNA generated when the device is operated microfluidically, and can control the operation of the device when preparing therapeutic mRNA (including but not limited to mRNA vaccines). These methods and devices can make it possible to confirm the success of various amplification and synthesis steps, and normalize multiple batches of outputs. This can also allow the reuse and optimization of the box. The repetition of the synthesis steps in the box can produce discrete outputs, which traditionally require detailed quality analysis for release. Alternatively, the methods and devices described herein can allow the output to be aggregated into a single batch, thereby requiring only a single quality analysis process and also simplifying manual sample collection and sorting. If the concentration of the RNA / DNA product measured for each replicate is within nominal parameters, the device can automatically add it to the pool; if the concentration is outside the parameter range, it can be microfluidically transferred to a "further testing" or "waste" container. This measurement-determined transfer can be operator-driven or can be automatically determined by the microfluidic system.

[0010] These methods and devices may also allow for standardization of output. The complex biochemical processes that produce RNA and DNA can be sensitive to parameters such as reagent supply, temperature, and biomolecule stability; yields can vary.

[0011] Stabilizing the output can remove the measurement and dilution / concentration steps between the production of the DNA / RNA and the next step in the production process of the therapeutic material. To stabilize the output, the methods and devices described herein can select a low target concentration and dilute the output of the system so it is in a more controlled range. In order to automatically perform this dilution, we need to know the pre-dilution concentration of the DNA / RNA and use that to determine the amount of diluent to add.

[0012] The methods and devices described herein may also allow for iterative improvement of automated microfluidic RNA / DNA production to achieve better control. For example, once a process performed by a device (e.g., in a box) is demonstrated to be in tight control, with regular and consistent output concentrations, the product can be released without performing detailed analysis. Instead, a "parametric release" may be performed, in which metrics such as temperature, reagent consumption, and intermediate RNA / DNA concentrations are used to demonstrate that the process is nominally proceeding.

[0013] In general, described herein are boxes ("biochips") that are particularly suitable for allowing an automated system or semi-automated system (e.g., a microfluidic drive device) to determine the concentration of a therapeutic polynucleotide processed (e.g., formed, mixed, synthesized) in the box, and / or modifying the operation of the microfluidic drive device on the box based on the determined concentration. For example, described herein are boxes (e.g., box devices) for processing polynucleotides, the box 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, a region of the second layer in the UV measurement region has a thickness less than the second thickness, and wherein there is no elastic material between a region of the first layer in the UV measurement region and a region of the second layer in the UV measurement region to form a UV measurement chamber having a separation thickness. The therapeutic polynucleotide may refer to a solution (e.g., an aqueous solution) of the polynucleotide.

[0014] Any box in these boxes can be formed by polymeric materials such as cyclic olefin copolymer (COC) material or cyclic olefin polymer COP. Although these materials are not very UV transmissive and can (as surprisingly found herein) change their UV transmission characteristics with UV exposure, the method and apparatus described herein can be adapted or adjusted to allow sensitive and accurate estimation of UV absorption, and therefore allow sensitive and accurate estimation of the concentration of polynucleotides (e.g., therapeutic polynucleotides). The first layer and the second layer can be formed by the same material or different materials. For example, the second layer can include COC material and / or COP material.

[0015] For example, the first thickness may be between about 0.1 mm and about 5 mm, and the second thickness may be between about 0.1 mm and about 5 mm. The first layer and the second layer may have the same thickness or different thicknesses.

[0016] The elastic material or pieces of elastic material may be sandwiched between the first and second layers (and may include additional layers above or below the first and second layers). The elastic material may form a layer. The elastic material may be held directly between the two layers and 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 thickness of the elastic layer may be uniform or non-uniform.

[0017] The path length of the UV sensing area can be relatively short and can be approximately the same as the separation thickness because the UV sensing chamber can be formed in the space between the first layer and the second layer where there is no elastic material. The elastic material can thus surround the UV sensing chamber (on the lateral sides of the UV sensing chamber). The box can have one or more UV sensing chambers. In some examples, the UV sensing chamber is in a consistent position on a variety of different box designs or configurations.

[0018] Generally speaking, any of these boxes 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 upper surface of the second layer, and may be at least partially covered by an elastic material / layer. In some examples, the fluid channels (also referred to herein as jet channels) are tunneled in the second layer. The second layer may be formed by a plurality of sublayers.

[0019] The box device may also include a plurality of pneumatic valves arranged to control the flow in and out of the UV measurement chamber. The plurality of pneumatic valves may each be formed by a pneumatic chamber in the first layer and a fluid chamber in the second layer; the portion or region of the elastic material separating the pneumatic chamber from the fluid chamber may be moved up / down to open / close (or remain open in an intermediate position) the fluid chamber, which may be in fluid communication with the fluid channel and / or other fluid chambers in the box. The pneumatic chamber of each valve may be in fluid communication with the pneumatic channel in the first layer, which is configured to communicate with a pressure port on an external region of the device to actuate the pneumatic valve (e.g., to move the region of the elastic member between the pneumatic chamber and the fluid chamber). The fluid chamber may be in fluid communication with the UV measurement chamber via the fluid channel in the second layer (e.g., so that when the pneumatic valve is opened, the fluid may flow between the fluid chamber and the UV measurement chamber).

[0020] Any of these boxes 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. These boxes may also include one or more mixing chambers (mixers), reaction chambers, etc. In general, the microfluidic drive device may include one or more thermal control elements (e.g., Peltier devices, heaters / coolers, etc.) for controlling the temperature of one or more regions of the box.

[0021] Generally speaking, the cartridge device may include one or more vacuum ports on an external region of the device that are configured to be coupled to a negative pressure source to draw fluid into and / or out of the UV measurement chamber (e.g., to a waste port and / or waste chamber). For example, the device 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., a solution typically comprising the therapeutic polynucleotide), the negative pressure generated may draw the therapeutic polynucleotide into the UV measurement chamber.

[0022] Any of these box devices may include a first inlet channel within the second layer and a second inlet channel within the second layer, the first inlet channel being connected to a first chamber fluid configured to hold a polynucleotide sample fluid, and the second inlet channel being connected to a second chamber fluid configured to hold a blank sample fluid.

[0023] Any of these cartridges may also be configured for continuous mixing of solutions. For example, any of these cartridges may include more dilution mixing chambers in fluid communication with the UV measurement chamber, the more dilution mixing chambers being configured to dilute the fluid sample.

[0024] In any of these examples, the box can be configured to prevent bubbles from being present in the sensing area of ​​the UV measurement chamber (so that they will not interfere with UV detection of therapeutic nucleotide absorption signals). In some examples, the UV measurement chamber may include an undercut region, for example, an area surrounded by an elastic material 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 can be greater than the diameter of the region of the first layer in the UV measurement region (and greater than the diameter of the region of the second layer in the UV measurement region) to prevent bubbles in the central region of the UV measurement chamber.

[0025] For example, a box device for processing polynucleotides may include: a first layer, the first layer comprising a polymer material having a first thickness; a second layer, the second layer comprising a polymer material having a second thickness; an elastic material, the elastic material extending between the first layer and the second layer; an ultraviolet (UV) measurement region, the ultraviolet (UV) measurement region formed through the first layer and the second layer, wherein an area of ​​the first layer in the UV measurement region has a thickness less than or equal to the first thickness, and an area of ​​the second layer in the UV measurement region has a thickness less than or equal to the second thickness, and wherein the elastic material is removed from between an area of ​​the first layer in the UV measurement region and an area of ​​the second layer in the UV measurement region to form a UV measurement chamber; and an in vitro transcription (IVT) chamber, the in vitro transcription (IVT) chamber being at least partially formed within the second layer and being fluidically connected to the UV measurement chamber.

[0026] Also described herein are methods of using any of these boxes with any of the microfluidic drive devices described herein. Generally speaking, these methods include on-box ("closed path") detection and determination of the concentration of therapeutic polynucleotides. Any of these methods may also include techniques for optimizing or enhancing detection, including adjusting one or more parameters of the microfluidic drive device based on feedback from a detected concentration for the therapeutic polynucleotide (e.g., from a detected UV absorption signal) or from a differential signal between a UV absorption signal for a therapeutic polynucleotide signal and one or more blanks.

[0027] For example, the present invention describes a method for manufacturing polynucleotides using a microfluidic actuator device operating on a box, the method comprising: forming a therapeutic polynucleotide in the box; and determining the concentration of the therapeutic polynucleotide by the microfluidic actuator device through the following steps: driving a first blank solution into an ultraviolet (UV) measurement chamber of the box and performing a first absorption measurement through the box; driving a sample solution of the therapeutic polynucleotide into the UV measurement chamber of the box and performing a second absorption measurement through the box; driving a second blank solution into the UV measurement chamber of the box and performing a third absorption measurement through the box; and estimating the concentration of the therapeutic polynucleotide from the first absorption measurement, the second absorption measurement, and the third absorption measurement.

[0028] As mentioned, any of these methods can include adjusting the operation of the microfluidic drive device based on the estimated concentration of the therapeutic polynucleotide in the processor of the microfluidic drive device. For example, the adjustment can include directing the resulting therapeutic polynucleotide to a pool for additional therapeutic polynucleotides. Additional therapeutic polynucleotides can also be formed in the box. In some examples, adjusting the operation of the microfluidic drive device can include modifying the synthesis (formation) of the therapeutic polynucleotide, including during the amplification step, where the sample can be removed and detected.

[0029] For example, any of these methods can include comparing, by a processor of the microfluidic drive device, an estimated concentration of the therapeutic polynucleotide to a concentration range, and based on the comparison, directing the therapeutic polynucleotide for one of the following: pooling with one or more additional batches of therapeutic polynucleotides formed in the box, discarding, or further analysis.

[0030] In some examples, the method may include diluting the therapeutic polynucleotide to a standard concentration for output under the control of a processor of the microfluidic drive device. Thus, the microfluidic drive device (e.g., a controller of the microfluidic drive device) can use the determined concentration and / or volume estimate (which can be, for example, optically sensed and / or based on the known volume of the chamber and / or channel of the cartridge) to add fluid to achieve the target concentration.

[0031] In some examples, the method may include automatically (or semi-automatically, e.g., using user confirmation, input, or modification) adjusting one or more parameters of forming the therapeutic polynucleotide by the microfluidic actuator device based on the estimated concentration of the therapeutic polynucleotide. Examples of the one or more parameters may include one or more of the following: temperature, reagent volume, reagent concentration and / or amount, reaction time, mixing amount and / or time, etc.

[0032] Generally speaking, the apparatus and methods described herein can be configured to automatically adjust so that accurate concentration can be determined. For example, the methods and apparatus described herein can be configured to prevent saturation (for example, wherein the minimum UV light is absorbed by the therapeutic polynucleotide), improve the detection limit when the concentration is too high, low sensitivity, or reduce background noise, such as the noise is absorbed by the wall (first layer and / or second layer) of the UV measurement chamber. For example, adjust the light power, detector sensitivity, or in some methods and devices, a serial dilution of the therapeutic polynucleotide can be performed to generate a curve graph of the absorption by the diluent. Therefore, any of these methods may include measuring the concentration of the therapeutic polynucleotide, may include performing a serial dilution in one or more chambers of the box under the control of a microfluidic drive device, and repeating the step of driving the sample solution of the therapeutic polynucleotide for each dilution in one or more dilutions formed by the serial dilution, to generate a dilution curve. In addition, estimating the concentration of the therapeutic polynucleotide may include estimating the concentration of the therapeutic polynucleotide from the dilution curve.

[0033] In any of these methods, estimating the concentration of the therapeutic polynucleotide can include changing one or more of the sensitivity of the UV detector and the intensity of the UV emitter (e.g., UV light source) of the microfluidic drive device in response to a second absorption measurement by the box. The method or device may include automatically adjusting one or more of the sensitivity of the UV detector and the intensity of the UV emitter based on the detected absorption measurement of the sample including the therapeutic polynucleotide. Therefore, any of these devices and methods can automatically, semi-automatically (e.g., using user input) and / or manually adjust the LED intensity based on the detector reading. In some examples, the device of the method can adjust the LED intensity and / or detector sensitivity during the measurement (on the fly), which can extend the dynamic range.

[0034] Any method described herein may include pre-treating the UV measurement chamber with UV light before carrying out the first absorption measurement through the box. As described herein, it is surprisingly found that the absorption curve of the material forming the chamber changes with time as the amount of UV light applied. Surprisingly, the absorption of the box material (e.g., COC, COP and other polymers) can actually be initially reduced (e.g., in the first 60 minutes, 55 minutes, 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, etc.), thereby resulting in an increase in the detection signal. Since the reading from a specific box may be shorter (e.g., in 5 minutes or less, 4 minutes or less, 3 minutes or less, 2 minutes or less, 1 minute or less, 50 seconds or less, 45 seconds or less, 30 seconds or less, 20 seconds or less, etc.), it may be beneficial to pre-treat the material so as to provide a reading with a larger detection signal. Furthermore, the absorption of a material may increase with further gamma irradiation, and thus the methods and devices described herein may include features to improve or increase transmission through the material, for example, but thinning the material in the UV measurement region to improve transmission of UV light.

[0035] Any of these methods may further include performing an in vitro transcription (IVT) reaction to form a therapeutic polynucleotide, and / or generating a template for an IVT reaction in the cassette. In any of these methods, the therapeutic polynucleotide comprises a therapeutic mRNA. Any of these methods may further include encapsulating the therapeutic mRNA in the cassette with a delivery vehicle.

[0036] Generally, the microfluidic drive device can supervise 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 a higher pressure on the blank fluid or the sample fluid and / or by creating a vacuum in the UV measurement chamber before opening the valve. For example, the method can include causing the microfluidic drive 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.

[0037] For example, a method for manufacturing a polynucleotide using a microfluidic actuator device operating on a box may include: forming a therapeutic polynucleotide in the box, wherein the therapeutic polynucleotide includes a therapeutic mRNA; determining the concentration of the therapeutic mRNA by the microfluidic actuator device through the following steps: driving a first blank solution into an ultraviolet (UV) measurement chamber of the box and performing a first absorption measurement through the box; driving a sample solution of the therapeutic polynucleotide into the UV measurement chamber of the box and performing a second absorption measurement through the box; driving a second blank solution into the UV measurement chamber of the box and performing a first absorption measurement through the box; and estimating the concentration of the therapeutic polynucleotide from the first absorption measurement, the second absorption measurement, and the third absorption measurement; and adjusting the operation of the microfluidic actuator device based on the estimated concentration of the therapeutic polynucleotide in a processor of the microfluidic actuator device.

[0038] Also described herein is a device (e.g., system) configured to perform these methods, which may include a microfluidic drive device. Any of these systems may include software, hardware and / or firmware for performing these methods. Specifically, any of these systems may include a controller with a control circuit (including one or more processors) for coordinating actions described herein (and performing these methods), specifically including estimating the concentration of therapeutic polynucleotides. Any of these controllers may include a non-transient computer-readable storage medium storing a set of instructions that may be performed by the system.

[0039] For example, described herein are systems for forming and / or manufacturing polynucleotides, or in some examples, systems for forming therapeutic polynucleotides such as (but not limited to) therapeutic mRNAs. Any of these systems may include: a box mount; a plurality of pressure lines; a plurality of fluid lines, each fluid line connected to or configured to be connected 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 box held in the 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 the fluid through the box, wherein the controller is also configured to direct the formation of a therapeutic polynucleotide in the box, and to estimate the concentration of the therapeutic polynucleotide by: driving a first blank solution through a UV measurement chamber of the box and performing a first absorption measurement using a UV light source and a UV light receiver; driving a sample solution containing the therapeutic polynucleotide through the UV measurement chamber of the box and performing a second absorption measurement using the UV light source and the UV light receiver; and driving a second blank solution through the UV measurement chamber of the box and performing a third absorption measurement using the UV light source and the UV light receiver, further wherein the controller is configured to estimate the concentration of the therapeutic polynucleotide from the first absorption measurement, the second absorption measurement, and the third absorption measurement.

[0040] Controller can also be configured to modify the operation of system based on the estimated concentration of therapeutic polynucleotide.For example, controller can also be configured to compare the estimated concentration of therapeutic polynucleotide with concentration range, and based on this comparison, therapeutic polynucleotide is guided for one of the following: gather, abandon or further analyze with the therapeutic polynucleotide of one or more additional batches formed in box.Controller can be configured to dilute therapeutic polynucleotide to standard concentration for output.In some examples, controller is configured to automatically adjust one or more parameters of the formation of therapeutic polynucleotide based on the estimated concentration of therapeutic polynucleotide.These one or more parameters can include one or more of the following: temperature, reagent volume, reagent concentration, time and mixing.

[0041] The controller can also be configured to perform serial dilution in one or more chambers of the box, and for each dilution of the sample solution of the therapeutic polynucleotide formed by the serial dilution, the step of repeating the driving of the sample solution of the therapeutic polynucleotide is to generate a dilution curve. The controller can be configured to estimate the concentration of the therapeutic polynucleotide from the dilution curve. Alternatively or in addition thereto, the controller can be 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 by the box.

[0042] The controller may be configured to pre-treat the UV measurement chamber with UV light prior to taking a first absorption measurement through the cartridge.

[0043] The controller can also be 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 in the box. Generally speaking, the therapeutic polynucleotide can be a therapeutic mRNA. The controller can be configured to encapsulate all or a portion of the therapeutic mRNA with a delivery vehicle.

[0044] The controller may also be configured to pneumatically deflect one or more regions of the film of the box to open and / or close the valve of the box when driving the first blank, driving the second blank, and driving the third blank. In some examples, the controller is also configured to pneumatically deflect one or more regions of the film of the box 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 on the upstream sample line and the blank line or by vacuum suction of the sample material (e.g., a sample of interest and / or a blank sample) in the LTV chamber in the UV chamber.

[0045] Generally speaking, the systems described herein can be configured to include one or more boxes and a microfluidic drive device. For example, the system can include: a box; and a microfluidic drive device, the microfluidic drive device including: a box mounting; a plurality of pressure lines; a plurality of fluid lines, each fluid line coupled to or configured to be coupled 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 box held in the mounting; 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 box, wherein the controller is further configured to direct the formation of a therapeutic polynucleotide, and further, wherein The controller is configured to estimate the concentration of the therapeutic polynucleotide by the following steps: driving a first blank solution through the UV measurement chamber of the box and performing a first absorption measurement using a UV light source and a UV light receiver; driving a sample solution containing the therapeutic polynucleotide through the UV measurement chamber of the box and performing a second absorption measurement using the UV light source and the UV light receiver; and driving a second blank solution through the UV measurement chamber of the box and performing a third absorption measurement using the UV light source and the UV light receiver; and modifying the operation of the microfluidic actuator device based on the concentration of the therapeutic polynucleotide determined from the first absorption measurement, the second absorption measurement, and the third absorption measurement.

[0046] All methods and apparatus described herein, in any combination, are contemplated herein and may be used to achieve the benefits as described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] A better understanding of the features and advantages of the methods and apparatus described herein will be obtained by referring to the following detailed description and accompanying drawings which set forth exemplary embodiments, in which:

[0048] Figure 1 The UV measurement of the polynucleotide material within the UV measurement area of ​​the cartridge by a microfluidic drive device including a UV light source and a UV detector is schematically illustrated.

[0049] Figure 2A An example of a device comprising a microfluidic actuator device control system as described herein is illustrated, which microfluidic actuator device control system can be configured to determine a polynucleotide concentration and modify the activity of the microfluidic actuator device based on the determined concentration.

[0050] Figure 2B One example of a microfluidic actuator device control system that can be used as described herein is schematically illustrated.

[0051] FIG. 3A to FIG. 3C Examples of cartridges for use with a microfluidic actuator device as described herein are illustrated.

[0052] Figure 4A is a cross section through a portion of one example of a cartridge for use with a microfluidic actuator device.

[0053] Figure 4B is a magnified view of the UV measurement area of ​​the box.

[0054] Figure 5 An example of a UV measurement area of ​​a cassette is schematically illustrated.

[0055] Figure 6 is a graph showing the UV transmittance of the material forming an example cell before and after gamma irradiation.

[0056] Figure 7 is a graph of the detection signal change over time for exemplary materials of the cartridge under extended UV exposure.

[0057] Figure 8 is an example of a dilution curve (graph) as described herein.

[0058] Fig. 9 One example of a portion of a cassette including a UV measurement area is schematically illustrated.

[0059] Fig. 10A is a top view of an example of a cartridge for a microfluidic actuator device that may include a UV measurement region.

[0060] Fig. 10B is an example of a cross section through an area of ​​an example of a box including a UV measurement area.

[0061] Fig. 10C An example of a portion of a cartridge for a microfluidic drive device is illustrated, schematically showing a vacuum cover for removing air bubbles.

[0062] Fig. 10D is a top view of an example of a cartridge ("biochip") including a UV measurement chamber.

[0063] Fig. 10E is another example of a top view of a box including a UV measurement chamber.

[0064] FIG. 11A to FIG. 11B A partial view of the cassette including the UV measurement area is shown.

[0065] Fig.12 B is Fig.11A Magnified view of the UV measurement area.

[0066] Fig.12 Methods of treating polynucleotides are schematically illustrated and include the use of UV absorbance measurements, which can be used to estimate the concentration of the polynucleotide and / or can be used to modify the treatment of the polynucleotide.

[0067] Fig.13A and Fig. 13B Another example of a UV measurement chamber is illustrated.

[0068] Fig.14 One example of a microfluidic device including a subsystem for determining polynucleotide concentration is schematically illustrated.

[0069] Fig.15A and Fig. 15B is an example showing the intensity adjustment range of the UV light source ( Fig.15A ) and the resulting light intensity ( Fig. 15B ) curve graph.

[0070] Fig.16A and Fig. 16B An example of UV detection of concentration using a device as described herein is illustrated. Fig.16A is a graph showing the detection signal (measured as the voltage output from the UV detector) for the blank, the first sample, the blank, the third sample, the fourth sample, and the blank. Fig. 16B is a graph showing a comparison between a standard concentration detection system and a system as described herein.

[0071] Fig.17 A top plan view of another example of a cassette including a UV measurement chamber is depicted.

[0072] Fig.18 Depicted Fig.17 An enlarged top plan view of an area of ​​the box.

[0073] Fig.19 Depicted is a flow chart illustrating an example of a method that can be used to assess the limit of quantitation.

[0074] Fig. 20 Depicted is a top plan view of an example of a backscatter shield.

[0075] Fig.21 Depicts the UV measurement process including Fig. 20 A cross-sectional side schematic diagram of an arrangement of a box of a backscatter shield.

[0076] Fig. 22 Depicted is a cross-sectional side schematic view of an arrangement including a cassette with another example of a backscatter shield during a UV measurement process.

[0077] Fig.23 A flow chart illustrating an example of a method that may be used to account for bubbles in a fluid in a UV measurement chamber is depicted. DETAILED DESCRIPTION

[0078] The devices and methods described herein can be used to prepare and / or process polynucleotides, particularly therapeutic polynucleotides, such as therapeutic mRNA.

[0079] These methods and devices can measure the concentration of RNA or DNA as part of an integrated closed path system, and the resulting concentration can be used to modify the processing of the polypeptide. Light absorption and, in particular, ultraviolet (UV) transmission through the UV measurement area of ​​the closed box can be used to estimate the concentration of the polynucleotides within the closed box. The box can be adapted to have a narrow path length and optimize the signal from the absorption of LTV light by the polynucleotide sample. Microfluidic drive devices for operating on these boxes are also described herein. These systems can control operations within the closed box for forming and / or processing (e.g., synthesizing) therapeutic polynucleotides, including determining the concentration of therapeutic polynucleotides at various stages of processing within the box, and also include using concentration data to control the operation of the microfluidic drive device on the box directly or based on LTV absorption data that is proportional to the concentration of the therapeutic polynucleotide.

[0080] In general, the methods and devices described herein can use UV absorbance techniques to measure the concentration of polynucleotides (e.g., RNA and / or DNA) on the box. These techniques can compare the level (in some examples, ratio) of transmitted UV light at a wavelength between about 250nm and 270nm (e.g., centered at about 260nm) from a polynucleotide sample (e.g., a sample of therapeutic polynucleotides) to the level of one or more "blank" solutions collected immediately before, immediately after, or both. Based on the absorption of UV light by the polynucleotides, and based on 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 polynucleotides.

[0081] These devices (and related methods) are particularly compact and can use readily available and easy to manufacture materials, including polymer materials (e.g., plastics) that absorb UV light themselves. These methods and devices can also tightly control the path length, and the path length ("critical path length") can be relatively small (e.g., 1 mm or less, such as 0.5 mm, 0.4 mm, 0.2 mm, 0.1 mm, etc.), thereby allowing very small volumes to be sampled while achieving high accuracy and an extended dynamic range. These methods and devices can also adapt to a larger dynamic range of concentrations and noise levels by adjusting the dilution of the sample and / or by adjusting the intensity of the UV light source and / or the sensitivity of the UV detector. This adjustment can be done automatically, semi-automatically, or manually. Generally speaking, these methods and devices can use disposable or limited-use boxes, as well as durable / reusable microfluidic drive devices that operate on the boxes. However, any of these techniques can be suitable for use with reusable boxes and / or boxes-free systems that integrate the features of the boxes into the microfluidic drive devices.

[0082] In any of these methods and devices, a sample (e.g., a polynucleotide sample) can be microfluidically driven to a UV measurement chamber that minimizes the amount of UV absorbing material (e.g., plastic) in the light path while still being made of relatively UV absorbing material. The measurement chamber can set a critical path length for light to pass through the sample, which can be fixed. When the box is placed in the microfluidic drive device, the UV light source and detector can be positioned on either side of the measurement chamber. The light path can be perpendicular to the box. For example, Figure 1 An example of a microfluidic drive device 102 operating on a box 105 is schematically illustrated. The microfluidic drive device includes a UV light source (e.g., UV LED 101) positioned above the box 105 and a UV detector 103 positioned directly below the box. Alternatively or in addition, optical fibers can be used as one or both of the detector and emitter (light source) to emit and collect light. The light source emits a UV beam 111, which can be configured to contact a detection area within a measurement chamber 107. In any of these examples, the light source and detector can be spaced apart so that the beam (which is tapered in some examples, but can be collimated) only interrogates a limited area of ​​the UV measurement area, so that the sample volume in the short light path is known to the system.

[0083] Device

[0084] The methods described herein can generally be used to perform with a device that can be used with one or more boxes (e.g., biochip) and / or can include one or more boxes and a system (e.g., microfluidic control system) configured to control the operation in the box. These microfluidic control systems may be referred to as microfluidic devices, microfluidic control devices, microfluidic drive device control systems, microfluidic control systems, or microfluidic systems in this article. The box can be placed in a microfluidic control system and can be operated in a closed path manner, which prevents some or more preferably almost all or all of the components of the manufacturing parts in the system from being exposed to the atmosphere. Specifically, the part of the fluid in the contact system of the device is prevented from being exposed to the atmosphere. Figure 2A An example of a microfluidic drive device control system is shown, which includes: a microfluidic drive device management system 203 (including hardware for holding a cartridge, applying positive / negative pressure to operate microfluidic operations in the cartridge, heating / cooling all or regions of the microfluidic drive device, detecting one or more features from the cartridge, and / or recording operations performed on one or more cartridges), a controller (not shown), and a refrigerated container 205 (e.g., an ISO 5 class cabinet). The system may be used or may include one or more cartridges 201. These cartridges may be any of the cartridges described herein, wherein any cartridge may include one or more UV measurement regions.

[0085] The microfluidic device can be a microfluidic device for forming a therapeutic polynucleotide (e.g., a therapeutic mRNA). The device may include: a mounting for removably holding a cartridge; a plurality of pressure lines; a plurality of fluid vials, wherein each fluid vial includes a fluid line or is configured to be coupled to a fluid line, wherein each fluid line and at least a subset of the pressure lines are configured to bias against a cartridge held in the mounting to form a closed fluid path; and a controller configured to control the application of pressure through the pressure lines to drive fluid movement in the cartridge when the cartridge is held in the mounting, wherein the controller is configured to direct the 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 the purification of the therapeutic polynucleotide in one or more cartridges held in the mounting. Any of these devices may include an optical or UV concentration detection subsystem, which includes one or more UV light sources and one or more UV detectors, which are configured to be positioned above the UV detection area of ​​the box when the box is held by or placed in the device.

[0086] A microfluidic device (e.g., a microfluidic device for forming therapeutic polynucleotides such as therapeutic mRNA) may include: a mount (e.g., a placement mount) for removably retaining a box; a plurality of pressure lines; a plurality of fluid vials, wherein each fluid vial includes a fluid line or is configured to be connected to a fluid line, wherein each fluid line and at least a subset of the pressure lines are configured to be biased against a box retained in the placement 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 in the box while the box is retained in the placement mount, wherein the controller is configured to measure the contents of the fluid vials, transfer sub-microliter amounts of material from the fluid vials to one or more reactors in the box retained in the placement mount, direct the synthesis of a synthetic template, use the template to direct an in vitro transcription (IVT) reaction to form a therapeutic polynucleotide, direct the purification of the therapeutic polynucleotide in one or more microfluidic drive devices retained in the placement mount, and detect the concentration of the therapeutic polynucleotide and / or regulate the activity of the device based on the concentration.

[0087] The controller may be configured to perform any of the methods described herein, and specifically may be configured to receive inputs (e.g., optical inputs, pressure inputs, temperature / heat inputs, UV absorption inputs, etc.) and process the inputs to control the movement of fluids in the microfluidic drive device, the temperature of various regions of the microfluidic drive device (including thermal cycling), flushing / combination, opening / closing of valves of the microfluidic device, detection of the microfluidic device, etc. The controller may include one or more microprocessors, communication circuits, memory, etc. The controller may include firmware, hardware, and / or software.

[0088] Any of these devices may include one or more (e.g., multiple) optical sensors arranged about the mounting mount and the reagent storage rack to monitor fluid levels within the reagent storage rack and fluid movement in the microfluidic drive device when the microfluidic drive device is positioned in the mounting mount.

[0089] Alternatively or in addition, an optical sensor may be present on the bottom of the device (e.g., below the mounting mount) and may be pointed upward to detect fluid volume, movement, etc.

[0090] like Figure 1 As shown, any of these devices may include a UV detection subsystem including a UV light source and an anti-UV detector.

[0091] The described methods and devices generally include one or more fluid power circuits to move materials (liquid materials) between fluid chambers (reservoirs, liquid contacting sides, reactors, etc.) and channels of microfluidic drive devices or within microfluidic drive devices, and in some cases between microfluidic drive devices and fluid reservoirs (vials, bottles, containers, etc.) within the device. The fluid power circuit can be a hydraulic circuit or a pneumatic circuit, which can include a microfluidic device, and specifically one or more pressure channels and a pressure receiving side of a chamber in a microfluidic device. The fluid power circuit may also be referred to as a microfluidic power circuit. A single microfluidic chip may include multiple fluid power circuits; the fluid power circuit may also include one or more pressure lines and an interface between the pressure lines of a microfluidic control device and one or more microfluidic chips within a microfluidic drive device. One or more fluid power circuits may share components (valves, pressure lines, vacuum covers, etc.) with other overlapping fluid power circuits. In addition, for convenience, it should be understood that where the term "pneumatic" is used, a general fluid power circuit (e.g., hydraulic and / or pneumatic) may be used alternatively or additionally. The fluid material driven by the fluid power lines may be any suitable fluid (eg, a gas or liquid such as air, water, oil, etc.).

[0092] Also described herein are boxes (e.g., closed path microfluidic drive devices) for processing therapeutic polynucleotides in a closed path. As mentioned, these boxes may be referred to herein as microfluidic chips, microfluidic path plates, processing chips, biochips, processing plates, etc. In general, boxes may be generally flat, plate-like structures; these structures may be relatively thin (e.g., less than a few mm thick, e.g., between about 0.5 mm and about 20 mm thick, between about 0.5 mm and about 15 mm thick, between about 0.5 mm and about 10 mm thick, etc.). The boxes described herein may generally be at least partially transparent, and in particular, may be transparent at the top of the box, so that one or more optical sensors (cameras, CCDs, optical fibers, etc.) may be used to sense, detect, monitor, record (etc.) actions, including fluid movement and / or movement of an elastic layer, using the microfluidic drive device when the microfluidic drive device is used by the microfluidic device described herein.

[0093] Figure 2B is a schematic diagram of one example of a microfluidic actuator 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 cover or opening; when closed, it can be sealed. The housing can enclose a thermal regulator and / or can be configured to be enclosed in a thermally regulated environment (such as a refrigeration unit, etc.). The housing can form a sterile barrier, and in some examples, the housing can form a humid or humidity-controlled environment.

[0094] Mount 215 may be configured to secure the cartridge using one or more pins or other features configured to hold the cartridge in a fixed and predefined orientation.

[0095] In some examples, a thermal control device 213 may be located near the mount 215 to regulate the temperature of one or more cartridges 211. The thermal control device may include a thermoelectric component (e.g., a Peltier device) and / or one or more heat sinks for controlling the temperature of all or a portion of the cartridge. In some examples, more than one thermal control device may be included for individually regulating the temperature of one or more regions of the cartridge. The thermal control device may include one or more thermal sensors (e.g., thermocouples, etc.) that may be used for feedback control and / or thermal control of the microfluidic actuator device.

[0096] exist Figure 2BIn the embodiment of the present invention, the fluid interface assembly 209 couples liquid reagents and / or pressure (e.g., gas) to the box 211 held in the mounting mount 215 and can help deliver fluid materials and positive / negative gas pressure from the pressure source 217 to the interior of the box 211. The fluid interface assembly can optionally help secure the box, as described in more detail below. The fluid interface assembly can be removably coupled to the device (and can be removed or a portion can be removed) for sterilization between uses.

[0097] The reagent storage rack 207 can be configured to include a plurality of fluid sample holders, each fluid sample holder can keep a fluid bottle configured to keep a reagent (e.g., nucleotides, solvents, water, etc.) for delivery to the box 211, or alternatively, the fluid bottle can be configured to receive products from the inside of the box 211. The reagent storage rack can be referred to as a reagent rack. In some examples, the reagent rack includes a plurality of pressure lines and / or a manifold, which are configured to divide one or more pressure sources 217 into a plurality of pressure lines, which can be applied to the box and can be independently or jointly (with a sub-combination) controlled. Alternatively, the fluid reservoir (bottle, etc.) can be configured to directly fix and seal against the box.

[0098] The fluid interface assembly can include multiple fluid lines and / or pressure lines, and can include a biased (e.g., spring-loaded) retainer or end that individually and independently drives each fluid line and / or pressure line to the box when the box is held in the mounting member 215 (or, as mentioned, alternatively, the device can be directly spring-mounted). The pipeline (e.g., fluid line and / or pressure line) can be a part of the fluid interface assembly and / or can be connected to the fluid interface assembly. In some examples, the fluid line includes a flexible pipe that is connected between the reagent storage rack and the box via a connector that is coupled to the pipe in a locking engagement (e.g., ferrule) with a vial. The end of the fluid path (in some examples, the end of the fluid line, the pressure line) can be configured to, for example, seal against the box at a sealing port formed in the box, as described herein. For example, the end of the fluid line can be cut or formed to be flat (vertical internal view). The vial can be pressurized (e.g., > about 1 atm pressure, such as about 2 atm, about 3 atm, about 5 atm, etc.) via a connector that can also be connected to a pressure source. For example, a fluid vial can be pressurized to between about 1 psig and 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 at the end of the process to draw the fluid back into the vial (e.g., a reservoir). Generally speaking, the fluid vial can be driven at a lower pressure than the pneumatic valve, which can prevent or reduce leakage. In some examples, the pressure difference between the fluid valve and the pneumatic valve can be between about 5 psi (e.g., about 7 psi, about 10 psi, about 12 psi, about 15 psi, about 20 psi, etc.).

[0099] Each vial may be coded (eg, by an identifier that can be read by one or more sensors, as described below). The controller may monitor the fluid level, and therefore the amount of each material in the fluid interface assembly.

[0100] The device may also include a magnetic field applicator 219, which may be configured to generate a magnetic field at the area 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 within fluid vials held within a reagent storage rack, and fluid movement within the cartridge 211 when the device is mounted within a mount 215.

[0101] Any of these devices can include a UV emission / detection subsystem (e.g., concentration estimation subsystem 233) that can make measurements of the absorption of UV light (e.g., at about 260 nm) and can control processes on the device based on these measurements, for example, by measuring one or more "blanks" without polynucleotides and comparing with samples with processed polynucleotides. In some examples, visual markers / optical markers can be used to estimate yield. For example, fluorescence can be used to detect process yield or residual materials by labeling with fluorophores. Alternatively or in addition, dynamic light scattering can be used to measure particle size distribution within a portion of the microfluidic drive device (e.g., a mixing portion). In some examples, sensor measurements can be accomplished using one or two optical fibers to transmit light (e.g., laser) in and detect an optical signal out. The instrument package can be mounted away from the device. Such non-contact sensing can be preferred.

[0102] In any of the methods and devices described herein, a sensor (e.g., a video sensor) can record all activities on a box (e.g., a chip). For example, the entire run for synthesizing and / or processing a material (such as a therapeutic RNA) can be recorded by one or more video sensors, including a video sensor that can, for example, visualize the box from above. The processing on the box can be visually tracked, and the record can be retained for later quality control and / or processing. Therefore, the video record of the processing can be saved, stored, and / or transmitted for subsequent viewing and / or analysis.

[0103] The internal part of the device (e.g., in the housing 233) can also be configured to be sterilizable. Specifically, parts of the device can be removed and sterilized separately. Sterilization can be performed, for example, by UV irradiation or any other sterilization method required to limit contamination or meet regulatory requirements. The device including the housing can be contained in a high-efficiency particulate air (HEPA) filtered environment. The device including the housing can be contained in a temperature-controlled package. In addition, the device itself can include one or more temperature-controlled areas. In any of the devices described herein, the device can (e.g., in the housing) include a temperature-controlled area for storing reagents and / or for storing mRNA (e.g., therapeutic mRNA) at a storage temperature (e.g., a temperature between about -10°C and about 20°C, such as about 10°C, about 4°C, about -10°C, etc.). Any of these devices can include a library of manufactured mRNAs, which can be synthesized individually or in combination with one or more additional mRNAs and delivery vectors.

[0104] As described above, the microfluidic actuator device controller system can be controlled by a controller 221, including applying pressure through the box 211 to at least drive the fluid to move. The controller can be completely or partially outside the housing. The controller can be configured to include user input / output. For example, the user interface 223 of the system can allow simple operation and guidance of the device and box.

[0105] Any of the devices described herein may include Figure 2B All or some of the components shown in the figure may be included; not all components are required. Figure 2B In the drawings, only some of the connections between components are shown; additional (or alternative) connections may be used.

[0106] The microfluidic drive device control system can support all production activities inside the microfluidic drive device, such as reagent supply, fluid control, temperature control, mixing, purification and process monitoring. The manufacturing activities on the microfluidic drive device control system can be accessed and controlled by the application software.

[0107] The box can be configured to include one or more reactors for manufacturing operations that are performed to accurately prepare therapeutic (e.g., therapeutic mRNA) materials. The same microfluidic drive device can be operated on one or more boxes in series and / or parallel without interrupting the continuous path nature of the microfluidic drive device control system. For example, when a therapeutic agent is manufactured using multiple processing operations performed in multiple reactors using multiple boxes, the fluid product (including a portion of the product from one box) can be transferred to one or more additional boxes in a closed path manner by the device, including by moving the fluid containing the box product into a storage reservoir portion of the microfluidic drive device control device.

[0108] Each cartridge may be configured to include one or more reactors for processing during the manufacturing process. FIG. 3A to FIG. 3C Three examples of boxes are illustrated. These examples illustrate three different types of boxes: template boxes ( Figure 3A )、In vitro transcription (IVT) cassette ( Figure 3B ) and preparation kit ( Figure 3C ). Each of these cartridge examples can be configured to include features for performing a set of unit operations in a controlled and highly reproducible manner.

[0109] In some examples, the cartridge may be constructed as a multi-layer structure consisting of two more rigid layers and a flexible membrane sandwiched between the two ridged layers. Figure 4AIllustrated is a cross-sectional view (transverse to the plane of the box) through an example of a box with multiple layers, which form a reactor for processing therapeutic agents as described herein. The reactor may include seals, channels, valves and chambers including pumping chambers formed by multiple layers. For example, the box may be formed by two or more rigid or semi-rigid plates 403, 405 and at least one elastic layer 407. The elastic layer 407 can be a sheet of elastic material that is impermeable to liquid. The elastic layer can be slightly gas permeable, or can be treated to be more or less (including in various regions) gas permeable. Although a single continuous sheet of elastic material can be used, in some examples, multiple sheets of elastic material can be used, or a "sheet" can be formed by sections of multiple sheets. These layers and elastic sheets can be laminated together. Generally speaking, a chamber for holding, valving and / or pumping fluids can be formed in a plate on either side of the elastic layer so that the elastic layer divides the chamber into two equal parts: a liquid containing side and a pressure (e.g., gas) application side. The total volume of the chamber can be constant and can be formed in both the first (e.g., upper) plate and the second (e.g., lower) plate, but the volume can be divided into a pressure side and a liquid side. By applying positive or negative pressure to the pressure side, the elastic sheet can be deformed to reduce the volume of the liquid containing side (down to zero, closing the chamber) or increase the volume of the liquid containing side (to a predetermined maximum value). The pressure applying side of the chamber can be connected, for example, via a pressure port 443 in the upper plate (first layer) 403 connected to a pressure channel 447 so as to apply negative or positive pressure to the pressure receiving side 419 of one or more chambers. The liquid containing side 417 opposite to the pressure applying side of each chamber can be connected to a fluid port 423 via a fluid channel 421. Both fluid ports and pressure ports can be formed by openings to the upper plate (upper layer) 403 and the elastic layer 407, thereby allowing a sealed connection isolated from the atmosphere even when multiple different input pipelines are present, because the pressure line is pushed into the elastic layer 407 supported on the underside of the port by the relatively rigid or semi-rigid layer 405.

[0110] exist Figure 4A , the box 400 includes a first (e.g., upper) layer (a plate in this example) 403 having a first (e.g., top or upper) surface 411 and a second (bottom or lower) surface 429 and a thickness therebetween. The first surface 411 may form an exposed outer surface. The box also includes a second layer (e.g., plate) 405 having a first (e.g., upper or top) surface 431 and a second (e.g., lower or bottom) surface 433 and a thickness therebetween. An elastic layer 407 is sandwiched between the second surface 429 of the first plate 403 and the first surface 431 of the second plate 405. Figure 4A The layers shown in FIG. 4 may not be to scale (eg, elastic layer 407 may be thinner relative to the plates).

[0111] Figure 4A The illustrated cartridge 400 may also include a plurality of chambers 415, 416, 420, each having a fixed volume. The chambers are formed by cutout regions (e.g., circular / curved cutouts) 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 the chambers 415 so that each chamber includes a liquid containing side 417 and a pressure (e.g., gas containing) side 419. The cartridge 400 may also include a plurality of liquid (e.g., fluid) channels. In Figure 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, and through most of the thickness of the second plate 405, down to the bottom area 433 of the second plate, where it forms the length of the liquid channel 421 extending parallel to the bottom surface of the third plate.

[0112] With respect to fluid port 423, the diameter of the opening into first plate 403 forming fluid port 423 extending through the thickness of the first plate may be greater than the diameter of fluid channel opening 425 extending through elastic layer 407 and into liquid (e.g., fluid) channel 421. Fluid channel opening 425 may be centered relative 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 a fluid line or fluid line coupling interface to be connected to the fluid port.

[0113] The fluid channel 421 is connected to the liquid receiving side 417 of the first chamber 415. The first chamber may be configured as a valve having a relatively low holding volume (fixed volume) but can be fully opened or closed by movement of the elastic layer 407.

[0114] The cartridge 400 also includes a plurality of pressure channels that can be independently controlled to apply positive and / or negative pressure. Figure 4A , a single pressure port 443 is shown connected to the fourth chamber 420, but each of the chambers 415, 416 can be connected to a separate pressure port and pressure channel for independently operating and controlling the movement of the portion of the elastic layer 407 that bisects these chambers to independently valve and / or pump each chamber. In some examples, the pressure port can be shared between multiple chambers. Figure 4A4, the pressure (e.g., gas) port 443 is similar to the fluid (e.g., liquid) port 425 and includes an opening that passes completely through the first plate 403, down to the exposed elastic layer 407, to form a pressure (e.g., gas) passage opening 445 through the elastic layer. The pressure passage opening 445 is connected to a pressure (e.g., gas) passage 447 that extends from the pressure port 443 through the majority of the thickness of the first plate 403, and in a channel along the second plate and back up through the second plate and elastic layer 407 to an area of ​​the pressure passage within the first plate that is connected to the pressure (e.g., gas) containing portion 419 of the fourth chamber 420. As described for the similar fluid (e.g., liquid) port, the diameter of the pressure port 443 through the thickness of the first plate 403 can be larger than the diameter of the pressure passage opening 445 through the elastic layer 407, and the pressure port and pressure passage opening can be concentric or offset by more than the wall thickness of the pressure line or pressure line coupling interface to be connected to the pressure port.

[0115] Passing through Figure 4A In the cross-section of the cassette 400 shown in FIG. 4 , there are multiple connections to other fluid (e.g., liquid) lines, fluid ports, pressure lines, and pressure ports that are not shown because they may be out of the plane shown. For example, in FIG. Figure 4A In the embodiment of the present invention, the liquid-receiving side or portion 417 of the fourth chamber can be connected to additional valves (chambers) and / or channels, including, for example, an outlet channel extending from the liquid-receiving side 417. Additional chambers (e.g., configured as valves) not shown can be formed as described above. In some examples, the outlet channel can deliver fluid from one or more chambers to a fluid receiving reservoir, such as a vial, tube, etc., through another fluid port (not shown). The receiving reservoir can be maintained in a reagent storage rack.

[0116] Generally speaking, this configuration of the microfluidic drive device and cartridge is constructed so that a number of complex operations can be performed by the device on the cartridge in a fully encapsulated (sealed and protected from the atmosphere) manner without the need for manual intervention. Fluids can be metered using fixed volume chambers and moved, mixed, filtered, etc. by applying pneumatic pressure to deflect regions of an elastic layer.

[0117] In some examples, the chambers within the cartridge can be configured as mixing chambers for mixing fluids within the cartridge. In some examples, the chambers can be configured as purification chambers, which can include filter materials. In some examples, one or more chambers can be configured as concentrators for concentrating therapeutic materials.

[0118] Although the various boxes can have different arrangements of channels, ports, and chambers, they can also share similar basic architectures and multiple functional elements that can be used in different configurations to perform different protocols. As described herein, the functional elements include input ports, metering valves, pumps, reaction chambers, mixing structures and purification structures, and / or UV sensing areas. Figure 4A In FIG. 4 , the UV sensing region 493 includes a thin UV measurement chamber 481 and the passage of material into and out of the UV sensing region can be regulated by a controller using one or more valves and by controlling the pressure within these passages.

[0119] Any of these boxes may include one or more bubble removal chambers, or any of the chambers on the liquid contact side of the chamber may be configured as a bubble removal chamber, wherein bubbles within the fluid on the fluid containing side may be removed. The bubble removal chamber may be referred to as a vacuum cover; and may generally be configured to apply a negative pressure on the opposite side of the membrane while the fluid is held within the liquid contact side of the chamber. As described above, the membrane may be at least partially gas permeable. Fig. 10C An example of a bubble removal chamber is shown. All or more preferably a portion 1988 of the membrane dividing the chamber (e.g., only the cover area) can be exposed to vacuum via a vacuum line 1987, for example, in the upper surface or upper plate of the device, such as Fig. 10CAs shown. In operation, the vacuum cover 1938 can remove or reduce bubbles in the pipeline by keeping the fluid in the liquid contact side of the chamber and applying negative pressure on the upper (pressure receiving) side of the chamber. The membrane that divides the chamber into the liquid contact side and the pressure receiving side can be gas permeable so that the negative pressure can remove gas from the liquid (fluid) side by sucking gas (e.g., air, nitrogen, etc.) through the membrane covering the fluid path. For example, the membrane (or the area of ​​the membrane in the vacuum cover) can be, for example, a polydimethylsiloxane (PDMS) elastomer membrane that is sufficiently gas permeable to allow gas to be removed from the liquid side of the membrane. A fluid chamber with a fixed volume (e.g., formed between a first plate and a second plate) as described herein may include or be coupled to one or more bubble removal chambers (vacuum covers) and / or may be configured as a bubble removal chamber. In some examples, the portion of the elastic layer disposed between the first surface and the second surface forming the chamber may be deflected only minimally (or not at all), the portion dividing, for example, the liquid contact side in the second surface (and / or the second plate) and the pressure receiving side in the first surface (and / or the first plate). For example, the upper pressure receiving side can be minimally spaced and / or nearly flush (e.g., flat) with the relaxed membrane, while the liquid contacting side is concave and extends into the second surface (second plate). The controller can, for example, maintain the fluid within the vacuum cover region by blocking the valve on either or both sides (inlet and outlet) of the vacuum cover, for example, by applying a positive pressure to the pressure receiving side of the valve, and can apply a negative pressure to the pressure receiving side of the vacuum cover. The absolute amount of the negative pressure applied (e.g., the magnitude of the negative pressure) can be less than the amount 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 can 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 liquid contacting side of the chamber. The membrane can be maintained on the first upper surface by the applied negative pressure, thereby allowing the removal of gas bubbles (e.g., air bubbles). The controller may maintain the fluid in the vacuum chamber for a period of time sufficient to remove all or some 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, between about 1 second and about 5 minutes, between about 2 seconds and about 5 minutes, between about 5 seconds and about 5 minutes, etc.). Fig. 10C In the embodiment of the present invention, pressure can be applied through a pressure line 1987 that communicates with a pressure receiving side of a chamber formed between a first surface and a second surface of the device (e.g., a first plate and a second plate). The vacuum cover 1938 can be valved by one or more valves 1992. Fluid can exit the liquid contacting side from a fluid line 1989 on the opposite side of the vacuum cover.

[0120] The box can be connected to the microfluidic drive device control system through spring-loaded connectors for two reagents and pneumatic lines for managing fluid movement and valve control. The reagent and gas lines can be sealed by pressure on the elastomer layer embedded in the box, which creates a completely sealed path from the reagent vial into the box and from the box to the output vial. The sealed path can be maintained during all reactions inside the box, effectively preventing any contact with the atmosphere and minimizing the risk of contamination.

[0121] The microfluidic drive device control system described herein can provide a sterile controlled environment and can include an interface for loading reagents and retrieving outputs. In any of the devices described herein (e.g., systems), the device can include a package that provides a controlled environment; the package can also be placed in a controlled environment. For example, the packaged device can be a Class 5 environment, which can be placed in a Class 7 environment.

[0122] The microfluidic drive device controls the system of the microfluidic drive device and can provide a single-step connection to all actuators. These control systems can also scan all reagents and box identifiers (e.g., bar codes) and can monitor fluid levels. Generally speaking, these microfluidic drive device control systems can automate all or some of the microfluidic drive device functions and can generate a visual record of all process operations that can be monitored (such as for optical quality control analysis of, for example, intermediate process outputs), stored, transmitted, or reviewed later.

[0123] As described above, the microfluidic drive device control system may include a microfluidic drive device management system, which includes hardware such as a nest (microfluidic drive device holder), which can be designed so that the box is correctly aligned and can be inserted only in a single orientation. This can be managed, for example, by two pins and / or a notch in the nest, which matches the shape of the box. The microfluidic drive device management system (control system) also includes a vial rack for holding reagents and output vials, a downward camera that records all liquids and valve movements, and product output. The side camera on the guide rail will capture the barcode and detect the fluid level, and the robot arm has a magnet for bead manipulation. The box is fixed in place with a vacuum suction cup, which ensures good contact with the Peltier device for temperature management. Once the box is in place, the top portion of the microfluidic drive device management system is lowered through the dowel pin guide system to achieve cooperation with all connectors in a single operation.

[0124] Figure 4B An example of a UV measurement area of ​​a box as described herein is schematically illustrated. In this example, Figure 4AAs shown, the box includes an upper first layer 403 and a lower second layer 405. An elastic material 407 forms a layer between the first layer and the second layer. The UV measurement region 493 is formed by cutting the thickness of the first region 495 to form a first UV measurement region 496 and also potentially by cutting the thickness of the second layer 494 to form a second UV measurement region 498. One or more fluid channels 421 can connect the UV measurement chamber 481 to a source of therapeutic polynucleotides and / or a source of "blank" solution (e.g., water, buffer, saline, etc.) fluid.

[0125] like Figure 4B As shown, the UV measurement chamber 481 is formed in the region between the first layer and the second layer where the elastic material 407 has been removed or does not exist. Therefore, the UV measurement chamber has the same thickness as the elastic layer 407. UV light can enter the first UV measurement region (cutout region) and pass through the region of the first layer thickness 491, then pass through any material (such as sample material and / or blank material) held in the UV measurement chamber 481, and then pass out of the region of the second layer thickness 499 and enter the second UV measurement region 489, where the remaining (unabsorbed) light can be detected.

[0126] exist Figure 4B In the example shown, the UV measurement area is formed by a cutout area in the first plate 496 and a cutout area in the second plate 498, each having a dimension of approximately 2.5 mm in width and approximately 1.3 mm in depth (in a 1.5 mm thick first layer). In this example, the second layer is a mirror image of the first layer. Thus, the polymer material forming the "window" on each side of the 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 maintain a known volume and can have a thickness of, for example, between approximately 0.1 mm and 5 mm (e.g., between 0.1 and 0.5 mm, etc.).

[0127] Because the methods and devices described herein use the technology of measuring blank (for example, the same solution as containing polynucleotide, only the solution without polynucleotide), it is not clear that the overall transmission of the measuring box is required to measure absorbance. However, the transmission of the box window material may have strong indirect results via signal / noise and measurable sample concentration. UV opaque windows cannot be used to measure UV transmission. Most microfluidic materials (for example, cyclic olefin copolymer COC, cyclic olefin polymer COP and polydimethylsiloxane PDMS) significantly absorb UV at the same wavelength as the wavelength of DNA / RNA characteristic absorption. Therefore, in any example in these examples, the measurement area (UV measurement chamber) can be thinned, for example, thinned to about 0.1mm and 0.5mm, etc. In order to control the critical path length, the layered structure of the box can be modified. For example, a box layer (for example, elastic layer) in the box layer can be completely removed from the UV measurement area. This may be advantageous because the thickness of the layer is usually more tightly controlled than that possible by machining / molding structure in the layer. Generally speaking, a thinner path length can produce better measurements (or enable measurements) for samples of higher concentrations than for samples of interest of lower concentrations. For example, for measurements of lower concentrations (e.g., when forming a template material for synthesizing polynucleotides), a chamber having a substrate with a height of about 1 mm (e.g., reflecting a 1 mm PDMS elastic layer) can be used. In some examples, for measurements of higher concentrations (e.g., when performing an IVT process), a chamber having a height of about 0.1 mm (e.g., a 1 mm PDMS elastic layer) can be preferred.

[0128] Figure 5 An outline of an example of a cartridge is shown, demonstrating the material thinning and short path length achieved by playing with the thickness of the elastomer layer in the cartridge. Figure 5 , the box includes a first layer 503, a second layer 501 and a channel 521. In this example, the UV measurement chamber 581 has a thickness of about 0.1 mm (path length is 0.1 mm).

[0129] For sterility, the cassettes are typically treated by gamma irradiation prior to use. This has the benefit of destroying contaminants, but can also change the material properties of the cassettes, and it was found herein that gamma treatment altered (e.g., reduced) the US transmittance of various materials used in some of the example cassettes. For example, the UV transmittance of one component of the cassette (COC) became significantly more UV opaque after gamma treatment (see Figure 6 ). If it is not Figure 4B and Figure 5 If the thinned design shown in is used, it will be completely opaque to the UV detector.

[0130] The UV transmission of gamma treated COC can depend on the degree of UV exposure, following an unusual trend of becoming more transparent at moderate exposure and eventually becoming more opaque at long exposure. See e.g. Figure 7 . In this example, the transmittance of a polymeric material (e.g., COC) initially and surprisingly increases with exposure, followed by a decrease in admittance over the course of many hours. For example, when the transmittance of a box changes during exposure, in some of the devices and methods described herein, it is beneficial to accurately measure the concentration, but first with a blank scan, and in some cases (optionally) after employing three novel mitigation measures: For example, "blank" measurements can be taken before and after the "sample" measurement to obtain a trend in the transmittance change of the box, and these values ​​are used to estimate the material response during the sample measurement.

[0131] The methods and apparatus described herein can be used to pre-treat the cassette with UV light so that it is in the stable and higher transmission region of the curve during measurement ( Figure 7 ). Generally speaking, UV light can be applied only when needed to make a measurement (e.g., by pulsing or using a dimming feature) to minimize exposure and the dimming feature can be used to make the LED light just bright enough to get a good signal and avoid overexposure. LED light adjustments can be done automatically or manually based on the detector reading to always ensure a good signal reading from the detector.

[0132] like Figure 8 As shown, any of the devices described herein can be configured to perform measurements on a series of dilutions (serial dilutions). Generally speaking, a microfluidic device can be configured to perform serial dilutions around a measurement chamber. The process can include microfluidically transferring an aliquot of a reaction to a measurement chamber, performing a measurement, and then diluting the aliquot for subsequent measurements. The diluted aliquot can be diluted again and again, and measured each time, to produce a series of dilutions such as Figure 8 At the end of the measurement, pure buffer was rinsed through the measurement chamber to collect a "blank" reading and correct for UV exposure effects. Figure 8 The graph in illustrates the mechanics and utility of this process. For example, the sample may initially be too concentrated so that it is completely opaque to the UV wavelength of interest (e.g., 260 nm). This Figure 8 Serial dilutions (depicted as being at 70%) cause the measurement to drop further in concentration, but remain effectively opaque. After multiple dilutions, the measurement enters a linear regime, eventually reaching Figure 8point 805 at the lower left corner of the graph of , at which point the aliquot becomes indistinguishable from the blank. UV absorbance can be used to determine the concentration directly along the linear range until point 805 is reached, at which point the UV absorbance measurement may no longer be useful. From the slope of the linear range, the device or method can also determine the dilution rate (i.e., 70% to 73%). The device can use the number of dilution steps, the dilution rate, and the concentration measured at the current dilution to solve for the concentration of the original aliquot. In addition, the linear portion of the curve validates the measurement, as nonlinear components may indicate that the signal is close to the noise level of the detector, or that there is an unexpected time-varying component or dilution error.

[0133] Fig. 9 A possible configuration of a portion of a box configured for serial dilution is illustrated. In this example, the device includes: a UV measurement chamber 981, which is adjacent to a fluid chamber 984, 985 on either side of the UV measurement chamber; and a series of valves 976, 977, 978. These chambers are in fluid communication with a polynucleotide input source 972 and a buffer ("blank") source 973 for dilution and a waste output 975. By selectively opening and closing the valve and pressurizing the measurement chambers 985, 984, the device (e.g., a controller) can empty a chamber to discard and refill with fresh buffer to produce dilution. In addition, control of the pressure on either side of the measurement chamber can realize bubble removal techniques, such as fluid sweeping and forcing air to pass through a gas-permeable flexible membrane as described above.

[0134] Generally speaking, these methods and devices can use the measured nucleotide concentration to control the operation of the device. For example, these devices and methods can be configured to collect multiple batches of polynucleotides. The measured concentration can be automatically compared with the nominal and acceptable range by the controller. If the comparison is favorable, the batch can be collected in a common container. If it is unfavorable, the controller can microfluidically guide the batch to a waste container or for further analysis. In some examples, the device can use the measured concentration to standardize the system output. The RNA / DNA concentration can be measured and guided to the product container. The volume of the added product can be read on the product container (for example, by quality or imaging), and the volume of the diluent can be calculated and added microfluidically using the concentration and volume together with the target concentration. Alternatively or in addition, these methods and devices can adjust or control any processing (parameter) element when processing polynucleotides. For example, the measured concentration can be automatically compared with the nominal and acceptable range by a computer. In addition to other process parameters (such as temperature history and reagent dosage), the product can be released for use in relation to the parameters. Abnormal products can be classified, and the process is studied.

[0135] FIG. 10A to FIG. 10E and FIG. 11A to FIG. 11B Additional examples of cartridges that may be used as described herein are illustrated. Fig. 10A An example of a cartridge 1900 is shown. This example also includes a permeable insert 1969 within the liquid contacting side of chamber 1957. Fig. 10A In the embodiment of the present invention, the box 1900 can include at least one pair of chambers 1953, 1957, 1957', each of which can include a liquid contact side 1917, a pressure (e.g., gas) side 1919, a fluid connector, a pressure connector, and a fluid / pressure line that can be formed in the thickness of the microfluidic actuator device. In some examples, the chambers are paired, and each chamber in the pair of chambers can be connected to each other by a fluid connector 1955. The fluid connector 1955 can be used in conjunction with positive and / or negative pressure applied to the pressure side of the chamber to drive liquid in the liquid side between the two chambers, thereby mixing the liquid in each of the chambers. The chamber can be bisected by an elastic material (e.g., an elastic layer or membrane), and deflecting the elastic material within a fixed volume of the chamber can drive any liquid in the liquid into / out of the liquid contact side of the chamber (e.g., between the two chambers).

[0136] Box 1900 may include more than one pair of chambers, any one of which may include a permeable insert. Each pair of chambers may be used for different processes. For example, a first pair of chambers 1953 may be used for the synthesis of RNA. A second pair of chambers 1957, 1957' may be used for purifying synthetic polynucleotides. When pressure is applied to the pressure receiving side 1919 of the corresponding chamber and a valve 1959 between the first pair of chambers 1953 and the second pair of chambers 1957 is opened, the fluid from the first pair of chambers 1953 may be driven to the second pair of chambers. The valve chamber 1959 may be formed by an elastic layer 1907 in the connector channel between the two pairs of chambers.

[0137] like Fig. 10A and Fig. 10B The illustrated cassette 1900 may have a plurality of pressure ports and fluid ports 1923, 1923'. The plurality of pressure ports and fluid ports may be disposed proximate the perimeter of the cassette and configured to connect to the fluid interface assembly 109 as described above.

[0138] The port (eg, sealing valve) may be formed by the elastic layer along the length of the connecting channel 1939 (pressure channel or fluid channel), such as Fig. 10A As shown, valve 1961 can control the timing of delivery of reagents driven from fluid port 1923, but can also allow metering of chambers of the device when placed in series with one or more similarly configured valves. Fig. 10A, three valve chambers are shown (described in more detail below); the first of the three valves can be used as a peristaltic pump, while the middle valve can be a small metering metering chamber (e.g., having a metering volume of about 10nL, about 20nL, about 25nL, about 50nL, about 75nL, about 100nL, etc.). The size of the channel and particularly the size of the chamber connected to the channel can meter out the volume distributed along the fluid connection channel 1939, 1921 and delivered to the chamber 1953 connected to the fluid connection channel 1939, 1921. In some examples, the metering volume can be as small as 50nL. A metering volume of about 100nL, about 1 microliter, about 5 microliters, or more can be input. A variety of valve sizes can be pre-selected to be combined in the microfluidic drive device 1900, and reagents can be connected to the appropriate metering size by user selection.

[0139] Additionally, more than one valve body 1961 may be included in a row along the fluid connection channel 1939. A series of valves 1961 may be used as a peristaltic pump to move fluids, including (but not limited to) viscous fluids. The ability to act as a peristaltic pump for fluids may be particularly advantageous for moving fluids that may be viscous or contain suspended particles such as purification or capture beads.

[0140] As mentioned, the cartridge 1900 may also include a delivery or output reservoir or depot 1963. Fig. 10A In the embodiment of the present invention, the pre-selected volume can be formed similar to the above-mentioned chamber configuration, or only the metering side can be included as needed. In either case, the valve can be used to meter the desired volume entering the reservoir 1963. Valve 1965 can control the delivery of fluid from the reservoir 1963. If a larger volume is required, delivery can be repeated. Alternatively, if the reservoir 1963 is pre-selected as an output reservoir, valve 1965 can be opened and delivered from chamber 1957, while keeping valve 1967 closed, which allows only the fluid of the measured volume to be output to the reservoir 1963. Then the fluid can be output to the fluid vial on the reagent storage rack for further processing or detection. In some examples, the chamber, reservoir or reservoir (e.g., 1963) can be constructed as a metering section of a 1 μL pump formed by three valve structures (1967, 1965, 1967). The chamber can be constructed for example to output waste from the mixing chamber 1957.

[0141] The cartridge 1900 can be a sealed path configuration. When the fluid vials, fluid lines, and microfluidic drive device are connected, the operation of the device can be performed without any material exchange into or out of the system, and specifically without any material exchange into or out of the fluid path of the cartridge for processing, including synthesis of polynucleotides (e.g., RNA) and preparation thereof for biological delivery (as a therapeutic agent, such as a drug, vaccine, etc.). Thus, the entire system can be operated as a closed path and / or a separate microfluidic drive device can be operated as a closed path (protected from the atmosphere) in the system.

[0142] Some examples of box 1900 may also include a concentrator within the chamber, which may be disposed within the thickness of the second plate and may be 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 output to the outside of box 1900 for further processing 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 drive device.

[0143] Cartridge 1900 can be formed of a material that is at least substantially translucent to visible light; in some examples, the material can be substantially transmissive to ultraviolet light (or both visible and UV light). In some examples, microfluidic actuator device 1900 can be formed of a material that is substantially transparent to visible and / or ultraviolet light.

[0144] As described above, the box can be formed by two or more plates stacked on top of each other, wherein the chamber and / or channel is formed between the plates; the elastic material can be sandwiched between the first plate and the second plate. The first plate and / or the second plate can be formed of a rigid material. The plates can be formed of the same material or different materials. For example, the rigid material can be a polymer or glass. The polymer or glass can be biocompatible, for example, not leaching any monomer or soluble small molecule that is toxic to living cells. Any suitable biocompatible polymer can be used, including medical grade polycarbonate-polyurethane, silicone polycarbonate polyurethane, polyether polyurethane, etc. In some examples, the polymer can be a cyclic olefin copolymer.

[0145] Fig. 10B A cross section through a portion of the cartridge is shown, showing the UV measurement chamber 1981 of the UV measurement region 1993. Thus, the microfluidic actuator device may be constructed as a multi-layer structure consisting of two more rigid layers 1903, 1905 and a flexible membrane 1907 sandwiched between the two ridged layers. Fig. 10BA portion of a cross-sectional view (transverse to the plane of the box) through an example of a box with multiple layers is shown, and the multiple layers form a reactor for processing therapeutic agents as described herein. The reactor may include seals, channels, valves and chambers including pumping chambers formed by multiple layers. For example, the box may be formed by two or more rigid or semi-rigid plates 1903, 1905 and at least one elastic layer 1907. The elastic layer 1907 can be a sheet of elastic material that is impermeable to liquid. The elastic layer can be slightly gas permeable, or it can be treated to be more or less gas permeable (including in various regions). Although a single continuous sheet of elastic material can be used, in some examples, multiple sheets of elastic material can be used, or a "sheet" can be formed by sections of multiple sheets. These layers and elastic sheets can be laminated together. Generally speaking, a chamber for holding, valving and / or pumping fluids can be formed in a plate on either side of the elastic layer so that the elastic layer divides the chamber into two equal parts: a liquid-holding side and a pressure (e.g., gas) application side. The total volume of the chamber can be constant and can be formed in both the first (e.g., upper) plate and the second (e.g., lower) plate, but the volume can be divided into a pressure side and a liquid side. By applying positive or negative pressure to the pressure side, the elastic sheet can be deformed to reduce the volume of the liquid containing side (drop to zero, closing the chamber) or increase the volume of the liquid containing side (to a predetermined maximum value). The pressure applying side of the chamber can be connected, for example, via a pressure port in the upper plate 1903 connected to the pressure channel 1947 to apply negative or positive pressure to the pressure receiving side 1919 of one or more chambers. The liquid containing side 1917 opposite the pressure applying side of each chamber can be connected to a fluid port 1923 via a fluid channel 1921. Both the fluid port and the pressure port can be formed by an opening to the upper plate 1903 and the elastic layer 1907, thereby allowing a sealed connection isolated from the atmosphere even when there are multiple different input lines, because the pressure lines are pushed into the elastic layer 1907 supported on the lower side of the port by the relatively rigid or semi-rigid layer 1905.

[0146] exist Fig. 10B , the cartridge 1900 includes a first (e.g., upper) plate 1903 having a first (e.g., top or upper) surface 1911 and a second (bottom or lower) surface 1929 and a thickness therebetween. The first surface 1911 may form an exposed exterior surface. The microfluidic drive device also includes a second plate 1905 having a first (e.g., upper or top) surface 1931 and a second (e.g., lower or bottom) surface 1933 and a thickness therebetween. The elastic layer 1907 is sandwiched between the second surface 1929 of the first plate 1903 and the first surface 1931 of the second plate 1905.

[0147] Fig. 10BThe illustrated cartridge 1900 may also include a plurality of chambers 1915, 1916, 1918, 1920, each having a fixed volume. The chambers are formed by cutout areas (e.g., circular / curved cutouts) in the second (bottom) surface 1929 of the first plate 1903 and the first (upper) surface 1931 of the second plate 1905; the elastic layer 1907 bisects the chambers 1915 so that each chamber includes a liquid-receiving side 1917 and a pressure-receiving (e.g., gas-receiving) side 1919.

[0148] The cartridge 1900 may also include a plurality of liquid (e.g., fluid) channels. Fig. 10B , the 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 the length of the liquid channel 1921 extending parallel to the bottom surface of the third plate.

[0149] With respect to fluid port 1923, the diameter of the opening into first plate 1903 forming fluid port 1923 extending through the thickness of the first plate can be larger than the diameter of fluid channel opening 1925 extending through elastic layer 1907 and into liquid (e.g., fluid) channel 1921. Fluid channel opening 1925 can be centered relative to the bottom of the fluid port opening and can be offset from the wall of the fluid port opening by at least the expected wall thickness of the fluid line or fluid line coupling interface to be connected to the fluid port.

[0150] The fluid channel 1921 is connected to the liquid receiving side 1917 of the first chamber 1915. The first chamber can be configured as a valve, which has a relatively low holding volume (fixed volume), but can be fully opened or closed by the movement of the elastic layer 1907.

[0151] The cartridge 1900 also includes a plurality of pressure channels that can be independently controlled to apply positive and / or negative pressure. Each of the chambers 1915, 1916, 1918 can be connected to a separate pressure port and pressure channel for independently operating and controlling the movement of the portion of the elastic layer 1907 that bisects the chambers to independently valve and / or pump each chamber. In some examples, the pressure port can be shared between multiple chambers.

[0152] Passing through Fig. 10B In the cross-section of the cassette 1900 shown in FIG. 1 , there are multiple connections to other fluid (e.g., liquid) lines, fluid ports, pressure lines, and pressure ports that are not shown because they may be out of the plane shown. For example, in FIG. Fig. 10BIn the embodiment of the present invention, the liquid-receiving side or portion 1917 of the fourth chamber can be connected to additional valves (chambers) and / or channels, including, for example, an outlet channel extending from the liquid-receiving side 1917. Additional chambers (e.g., configured as valves) not shown can be formed as described above. In some examples, the outlet channel can deliver fluid from one or more chambers to a fluid receiving reservoir, such as a vial, tube, etc., through another fluid port (not shown). The receiving reservoir can be maintained in a reagent storage rack.

[0153] exist Fig. 10E In the embodiment, box 1980 includes four PCR chambers 1983, 1983', 1983", 1983'", each of which may include a liquid contact side, a pressure (e.g., gas) side and they are fluidly connected to adjacent PCR chambers. Each PCR chamber has a fixed volume, and as described above for general chambers, these fixed volumes are formed between the first surface of the first plate and the second surface of the second plate; the first plate and the second plate can be connected together 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 contact side in the second surface and a pressure receiving side in the first surface.

[0154] exist Fig. 10D In the box shown, the pressure receiving side 1919 of each chamber is further divided by one or more fluid-connected serpentine paths 1985. These serpentine paths in the pressure receiving surface are configured to more evenly distribute the positive and negative pressures (and specifically, negative pressures) applied through the channels over the surface of the relatively large chamber. The subdivision of the pressure receiving surface in each PCR chamber (e.g., in some examples, by one or more serpentine paths) can support the deflectable membrane when negative pressure is applied to pull the deflectable membrane away from the fluid-containing chamber. This can also prevent the formation of bubbles and can maintain a fixed, predictable volume.

[0155] Fig. 10D The illustrated cartridge also includes a plurality of fluid channels that each extend from fluid ports 1923, 1923' through the first plate region and into the second plate region to fluidically connect with the liquid contacting side of one or more of the plurality of chambers (similar to Fig. 10A and Fig. 10B The structure shown). Fig. 10D In the example, a subset of fluid ports are labeled, including those providing access to (outside the device) plasmid 1923, PCR buffer, primers (e.g., T7 primers), oligo dT, enzymes (e.g., polymerase), purification substrates (e.g., Ampure TMBeads), RNase-free air, a source of 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 fluid ports are also included and may be redundant or may not be used. As described, the pressure ports can provide connectivity for applying positive and / or negative pressure to the pressure receiving side of each chamber, channel, vacuum cover, valve, etc. Thus, the controller can control the movement of fluids within the device, including mixing, pumping, valving, etc., by applying positive or negative pressure to a specific pressure port 1943 or a combination of pressure ports. The pressure ports and fluid ports can be arranged on the upper side of the first plate, typically around the periphery of the plate, such as Fig. 10A and Fig.19 As shown in D.

[0156] exist Fig. 10D , the device also includes a plurality of pressure channels 1947, each of which extends from one or more pressure ports, through the first plate region and the elastic layer, into the second plate region, and back through the elastic layer and into the first plate region (similar to Fig. 10B ), wherein each of the plurality of pressure channels extends within the first plate region and is fluidly connected to one or more pressure receiving sides of one or more chambers of the plurality of chambers. Fig. 10B As shown, applying positive or negative pressure (from a pressure port) through a pressure channel by a controller of a system including a fluid reservoir and a pneumatic drive can open / close valves 1915, 1918, and can pump fluid through chambers 1916, 1920, 1983.

[0157] As mentioned, any of the boxes described herein may also 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 therethrough for quantification of the polynucleotides in the UV yield detection chamber. The UV measurement chamber 1990 may also be connected to a buffer source for performing UV detection. The UV detection may measure the absorbance of the buffer in which the DNA is located. A device (system) including a controller that coordinates operations on a microfluidic drive device may be configured to control the operation of the UV yield detection chamber, as will be described in detail below. For example, a controller configured to use a system using a microfluidic drive device may first check the absorbance of a UV buffer without a product, and then may add a predetermined amount of (e.g., purified) product for comparison. The system (e.g., controller) may then automatically or semi-automatically use the determined concentration to warn the user and / or make a decision to discard or mark the sample for further analysis and / or dilute the product before the product is output from the box or moved to another box.

[0158] Fig. 10E Shown with Fig. 10D Another example of a cartridge (eg, a "biochip") 1980' similar to the cartridge shown in FIG. 1 includes a UV yield detection chamber 1990' in fluid communication with a sample and / or blank (eg, buffer) chamber, wherein the Fig. 10E In, the cartridge device also includes a waste port 1923' (eg, a negative pressure port that can be coupled to a waste line).

[0159] Generally speaking, Fig. 10D and Fig. 10E The equipment shown in is similar and may include FIG. 10A to FIG. 10C For example, the port can be formed by the elastic layer along the length of the connecting channel 1939 (pressure channel or fluid channel), such as Fig. 10A One or more valve bodies 1961 may be included in a row along the fluid connection channel 1939 .

[0160] The PCR chamber can be configured to optimize the PCR process described herein. For example, the microfluidic drive device can include a PCR chamber having an area much larger than the height of the liquid contact side. For example, the liquid contact side of each PCR chamber can have a thickness of 1.5 cm or less, such as specifically 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. Typically, the lower the height (e.g., the "thickness" of the chamber), the more efficient the heat transfer due to thermal cycling, but the lower the total volume. The box described herein can be used for PCR in the PCR chamber without the need to add oil / hydrophobic material, because evaporation can be limited by the closed (or closable) structure of the chamber.

[0161] Generally, any of these cassettes can include a purification chamber in fluid communication with a purification substrate (eg, Ampure beads).

[0162] The cartridges described herein can also be configured to provide mixing (e.g., bubble mixing) by applying air (e.g., RNase-free air) from the cartridge through the liquid contacting side of the device and out of the fluid port into a reservoir coupled to the fluid port. Fig. 10D In the embodiment of the present invention, the cartridge can be controlled by a controller (of a system coupled to the cartridge) to mix and resuspend substrate beads (e.g., Ampure TM The substrate beads can be suspended in the reservoir. This can cause bubbling and mixing of the substrate beads within the reservoir. The controller can control mixing by applying RNase-free air (using positive and / or negative pressure applied through the pressure port of the microfluidic actuator device), and after mixing, the resuspended substrate can be directed out of the reservoir and into the purification chamber for purification of the template product.

[0163] As mentioned, any of these devices can be constructed as a removable box that is configured to engage with a fluid reservoir and a pneumatic drive device and can be connected to a system, for example, a microfluidic drive device control system that includes a controller for coordinating the operation of the microfluidic drive device to produce a template.

[0164] Generally speaking, the cassette can be of any suitable size / volume. For example, the cassette can be configured to have a total PCR reactor size between about 3 mL and about 10 mL (e.g., between about 4 mL and about 8 mL, between about 5 mL and 7 mL, etc.). Fig. 10D In the example shown, the total PCR reactor volume (all four PCR chambers combined) is approximately 6.03 mL. Fig. 10DIn the PCR reaction volume is about 3 mL, it has been found to produce about 130 ng / μL of template product (in 3 mL).

[0165] Fig.11A and Fig. 11B An example of a cartridge comprising a UV measurement channel 1181 coupled to one or more valves 1176, 1177 and an input (e.g., a source of polynucleotide material, such as a polynucleotide forming chamber 1179 and / or a source of buffer or other blank solution) is illustrated. Fig.11A In the embodiment, the UV measurement chamber is also connected to a suction (eg, waste) port 1172. The suction can be used to load the UV measurement chamber 1181 through a control valve. Fig. 11B Shows Fig.11A Slightly enlarged view of the UV measurement chamber.

[0166] Fig.13A and Fig. 13B Another example of a UV measurement chamber is shown in . In this example, the UV measurement chamber 1381 is formed by thinned areas of the upper plate and the lower plate (with the UV chamber 1381 formed between them), and an elastic layer may be additionally included in the space. The UV measurement chamber in this example is formed of a COC polymer (from the upper and lower layers) and has a diameter of about 2.5 mm. The elastic layer (e.g., PDMS) has been cut to form a chamber measuring 3 mm×4.5 mm. In this example, the UV chamber includes a fluid inlet 1383 connected to a valve 1376 and a fluid outlet 1385 connected to a second valve 1377. The fluid inlet valve 1376 is in fluid communication with the polynucleotide sample line 1339 and the water (or buffer / blank) fluid line 1338; the flow through each of these lines is also controlled by valves 1378, 1378'. In operation, the valve can be opened and fluid from the polynucleotide sample line 1339 or the water / buffer / blank line 1338 can be pumped into the UV measurement chamber 1381, or fluid can be pulled into the UV measurement chamber by applying negative pressure (suction) from the suction / waste port 1388.

[0167] Fig.12The operation of the above-mentioned device for processing polynucleotides using a microfluidic drive device operated on a box is schematically shown. In this example, the method can be used for (e.g., in a box) therapeutic polynucleotides 1201, as described above. Additional treatment (e.g., purification, concentration, etc.) can be performed on the polynucleotides. In addition, the UV measurement subsystem can be prepared for measurement 1203. For example, the device can pre-irradiate the chamber as described above and / or can form one or more diluents of a polynucleotide solution. The device can then measure the concentration 1205 of the therapeutic polynucleotides. This can include continuously driving the first blank solution into the UV measurement chamber and performing a first absorption measurement 1207, then driving the sample solution (or its diluted version) and performing a second absorption measurement 1209. Optionally, a second blank measurement 1210 can be performed, in which case the first blank measurement and the second blank measurement can be compared and / or combined. For example, two blank measurements can be averaged. Then, the device can estimate the concentration 1211 of the polynucleotides in the sample from the first absorption measurement and the second (and optional third) absorption measurement.

[0168] Thereafter, the method (or a device performing the method) can adjust operation of a device (eg, a microfluidic actuator device) based on the absorption measurement and / or the concentration of the therapeutic polynucleotide 1213 .

[0169] Example

[0170] Fig.14 An example of a portion of an apparatus as described herein that includes a UV detection subsystem is illustrated. For example, the apparatus may include a controller (shown here as a separate processor 1401 for data processing); in some examples, the controller may be integrated into the apparatus. In this example, both the UV light source (shown as LED 1411) and the UV detector 1415 include power supplies 1407, 1407' that can allow them to be adjusted individually. For example, the LED intensity can be increased or decreased (e.g., brightened / dimmed 1405) by adjusting the power supplied by the power supply 1407 to the LED driver 1409, which controls the output of the LED 1411 and thereby controls the illumination of the sample or blank through the UV detection chamber 1413. Similarly, the power supplied by the power supply 1407' to the UV detector 1415 can be adjusted to increase or decrease the sensitivity of the LED detector to sense absorption. As described above and as Fig.14 As illustrated, the resulting UV absorption signal may be stored and / or analyzed by the device. Fig.14In the apparatus, the device includes a signal grabber circuit 1403 to capture, store and / or transmit the resulting absorbance. The controller can also provide feedback based on the detected absorbance to adjust either or both of the intensity of the UV light source 1409 and / or the detector sensitivity of the UV detector 1415.

[0171] As described above, any of the devices configured to use UV light absorption to determine polynucleotide concentrations can be configured to adjust the UV light intensity over a range of values ​​to achieve a linear output range for the absorption of the polynucleotide sample, thereby avoiding saturation of the absorption signal output. Fig.15A , a dimming voltage versus current curve is shown for one example of a UV light source (LED) that can be used. The dimming voltage over this range is linear and can be related to the resulting light intensity in a linear manner, such as Fig. 15B shown. Fig. 15B The voltage readout for different dimming voltages applied is shown. Fig.14 , thus the LED can be adjusted to increase or decrease the intensity (e.g., by adjusting a dimming voltage) based on the absorption signals detected 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 reduced or increased. The same adjustment (e.g., dimming voltage) can be used for both the sample and the blank.

[0172] In general, the devices and methods described herein can provide highly accurate and reproducible polynucleotide concentration determinations. For example, Fig.16A Examples of the detection of concentrations of various samples (and corresponding blanks) are illustrated. Fig.16A In the present invention, the device described in this article can be used to first apply a blank 1602 and then measure the UV absorption, such as Fig.16A As shown. In this embodiment, the absorption of each polynucleotide sample or blank is measured for 10 seconds, and a stable reading is obtained during this entire time period; in some examples, shorter (e.g., 5 seconds, 1 second, 0.5 seconds, 0.1 seconds, etc.) or longer absorption times may be measured. After measuring the first blank, the (e.g., undiluted) first RNA sample is moved into the UV measurement chamber to replace the blank, and the UV absorption is measured 6104. The sample fluid can then be moved out of the UV measurement chamber, and a second blank can be moved in and measured 1602', followed by a second sample (e.g., a half-diluted RNA sample) 1606. The second sample (e.g., a 10-fold diluted RNA sample) is measured 1608, followed by a third blank 1602". The sample measurements can be of different polynucleotides and / or different dilutions of the same polynucleotide (e.g., Fig.16A ).

[0173] Fig. 16B A comparison between measurements performed as described above and a commercially available concentration detection system requiring a different method and technique for determining analyte concentration is shown, showing very close agreement. In this example, the commercially available analyte concentration detector may be, for example, a Nanodrop TM System (Thermo Fisher). Thus, the methods and apparatus described herein, which are particularly suitable for on-box (eg, "on-chip") UV and therefore for concentration measurements, show similar or even better results than other larger and more expensive systems.

[0174] Figure 17 to Figure 18 Another example of a box (e.g., a "biochip") 2000 is depicted. The box 2000 of this example may be constructed and operated similarly to any other box described herein, with examples of differences being described below. The box 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 the pressure channels 2047 may provide a pressure receiving side for applying positive and / or negative pressure to each chamber, channel, vacuum cover, valve, etc. in the box 2000. Therefore, the controller may control the movement of the fluid in the box 2000, including mixing, pumping, valve adjustment, etc., by applying positive or negative pressure to a specific pressure port 2043 or a combination of pressure ports 2043. The pressure channel 2047 may therefore be understood as a pneumatic channel.

[0175] The pressure port 2043 and the fluid port 2023 may be arranged on the upper side of the box 2000 (eg, around the outer periphery of the box 2000), such as Fig.17 Alternatively, the pressure port 2043 and / or the fluid port 2023 may be arranged on the underside of the cartridge 2000 .

[0176] Each fluid port 2023 is fluidically coupled to a corresponding fluid channel in the 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 coupled to a plasmid, a PCR buffer, a primer (e.g., a T7 primer), an oligo dT, an enzyme (e.g., a polymerase), a purification substrate (e.g., an Ampure TM Beads), RNase-free air, a source of dNTPs, a product output collection receiver, an output from a UV yield detection channel, a buffer for UV detection, water, ethanol (e.g., 70% ethanol rinse), or waste fluidly connected. Fluid ports 2023 (and corresponding channels) can be understood to provide a path for liquid communication. In some cases, one or more fluid ports 2023 are redundant and therefore not used during certain processes.

[0177] The box 2000 of this example also includes PCR chambers 2083, 2083', 2083", 2083'", each of which may include a liquid contact side, a pressure (e.g., gas / pneumatic) side and are fluidly connected to adjacent PCR chambers. Each PCR chamber has a fixed volume, and as described above for general chambers, these fixed volumes are formed between the first surface of the first plate and the second surface of the second plate, and the first plate and the second plate can be connected together with an elastically deformable membrane (e.g., an elastic layer) therebetween to divide the chamber. The elastic layer divides each chamber into a liquid contact side in the second surface and a pressure receiving side in the first surface.

[0178] The pressure receiving side of each PCR chamber 2083, 2083', 2083", 2083'" is divided by one or more fluid-connected serpentine paths 2085. These serpentine paths 2085 in the pressure receiving surface are configured to more evenly distribute the positive and negative pressures (and specifically, negative pressure) applied through the corresponding pressure channels 2047 over the surface of the relatively large chamber. 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 the deflectable membrane away from the fluid-containing chamber. This can also prevent the formation of bubbles and can maintain a fixed, predictable volume.

[0179] The box 2000 of this example also includes a UV measurement chamber 2020, which is fluidly connected to one or more of the PCR chambers 2083, 2083', 2083", 2083"' and / or a blank (e.g., buffer) chamber, so that the UV measurement chamber 2020 can receive an aliquot of liquid from one or more of the PCR chambers 2083, 2083', 2083", 2083"' and / or a blank (e.g., buffer) chamber. The UV measurement chamber 2020 may include a UV yield detection window, which is configured to allow UV light to pass therethrough for quantification of the polynucleotides in the UV measurement chamber 2020 as described herein. Fig.18 As best seen in FIG. 2 , inlet liquid channel 2026 leads to UV measurement chamber 2020 , while outlet liquid channel 2050 leads out of UV measurement chamber 2020 .

[0180] Vacuum cover 2024 is positioned upstream of inlet liquid passage 2026. Another vacuum cover 2052 is positioned downstream of outlet liquid passage 2050. In some versions, vacuum covers 2024, 2052 are similar to those described above with reference to Fig. 10C The vacuum cover 1938 described is constructed and operates like that.

[0181] In this example, vacuum covers 2024, 2052 are operable to remove bubbles from the liquid in UV measurement chamber 2020. Vacuum covers 2024, 2052 are pneumatically coupled together via pneumatic channel 2040, which facilitates simultaneous operation of vacuum covers 2024, 2052. In some versions, one or both of vacuum covers 2024, 2052 can also function as a valve that selectively allows or prevents the transfer of liquid aliquots / samples to or from UV measurement chamber 2020. In this case, vacuum covers 2024, 2052 can be pneumatically actuated like the other valves described herein.

[0182] A pair of liquid passages 2028, 2030 lead to the vacuum cover 2024. A valve 2076 selectively allows or prevents liquid from flowing to the vacuum cover 2024 via the passage 2028. Similarly, a valve 2032 selectively allows or prevents liquid from flowing to the vacuum cover 2024 via the passage 2030. Another vacuum cover 2070 is in fluid communication with the valve 2076; and is pneumatically coupled to the vacuum cover 2024 via the passage 2022. Yet another vacuum cover 2072 is also pneumatically coupled to the vacuum cover 2070; and is in fluid communication with another valve 2074. Thus, the vacuum covers 2024, 2052, 2070, 2072 can be pneumatically coupled together via the passages 2022, 2040 to facilitate simultaneous operation of the vacuum covers 2024, 2052, 2070, 2072.

[0183] In the present example, valve 2076 is fluidly interposed in the path between PCR chamber 2083, 2083', 2083", 2083'" and channel 2028, such that valve 2076 is operable to selectively prevent or allow liquid to pass from PCR chamber 2083, 2083', 2083", 2083'" to channel 2028. And thus to UV measurement chamber 2020 via vacuum cover 2024 and channel 2026. In some versions, PCR chamber 2083, 2083', 2083", 2083'" may be pressurized to push liquid aliquot / sample into UV measurement chamber 2020 when the valve in the path may be in an open state. Alternatively, liquid may be driven into UV measurement chamber 2020 by applying a vacuum through output port 2060 when the valve in the path may be in an open state. In other versions, the main reservoir may be pressurized; and valves 2076, 2052 (and possibly vacuum lids 2024, 2052) may be sequentially actuated to peristaltically pump liquid from PCR chambers 2083, 2083', 2083", 2083'" into UV measurement chamber 2020. Alternatively, any other suitable structural features and / or techniques may be used.

[0184] Channel 2030 provides a path for passing a blank or buffer (e.g., water, etc.) fluid to the UV measurement chamber 2020 via the vacuum cover 2024 and the inlet liquid channel 2026. As described above, valve 2032 selectively allows or prevents liquid from flowing to the vacuum cover 2024 (and thus to the inlet liquid channel 2026 and the UV measurement chamber 2020) via channel 2030. Channel 2034 extends from valve 2030 to pump chamber 2036. Pump chamber 2036 can also be fluidly coupled to a blank or buffer source. And when valve 2032 is in an open state, it can be pneumatically operated to drive a blank or buffer toward the UV measurement chamber 2020 via channels 2034, 2030, 2026. Alternatively, when the valve in the path can be in an open state, a blank or buffer can also be driven into the UV measurement chamber 2020 by applying a vacuum through the output port 2060.

[0185] Another channel 2054 and valve 2056 are also coupled to the vacuum cover 2052. A pneumatic channel 2062 also extends from the valve 2056 and provides a path for pressurizing the valve 2056, thereby switching the valve 2056 between an open state 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 the output port 2060. The output port 2060 can operate similarly to the suction / waste port 1388 described above.

[0186] The box 2000 of this example also includes an aggregation output port 2002. Fig.18 As best seen in the figure, the aggregation output port 2002 is coupled to a liquid communication channel 2004 leading to a valve 2006. The valve 2006 is in fluid communication with a pneumatic channel 2010 through which the valve 2006 can be selectively pressurized to switch the valve 2006 between an open state and a closed state. A liquid communication channel 2008 is also coupled to the valve 2006. The channel 2008 is also fluidly coupled to the PCR chambers 2083, 2083', 2083", 2083'" so that when the valve 2006 is in the open state, fluid can be transferred from the PCR chambers 2083, 2083', 2083", 2083'" to the aggregation output port 2002 via the channels 2004, 2008. The aggregation output port 2002 may thus be used to aggregate the outputs of the PCR chambers 2083, 2083', 2083", 2083'" into a single batch in a receptacle external to the cartridge 2000, as described above.

[0187] Some versions of the box 2000 may also include an additional output port (not shown) that is fluidically connected to the PCR chamber 2083, 2083', 2083", 2083' so that fluid can be transferred from the PCR chamber 2083, 2083', 2083", 2083' to the additional output port (not shown) instead of to the collection output port 2002. In other words, the output fluid from the PCR chamber 2083, 2083', 2083", 2083' can be selectively transferred to the collection output port 2002 or the additional output port so that the PCR chamber 2083, 2083', 2083", 2083' can effectively have two different and separately controlled outputs. In some cases, the fluid transferred from the PCR chamber 2083, 2083', 2083", 2083' to the additional output port (not shown) can be retained for further analysis or can be processed in other ways. In some such cases, fluid from the PCR chambers 2083, 2083', 2083", 2083'" may be delivered to an additional output port (not shown) rather than being delivered to the aggregation output port 2002, due to the concentration measurement data obtained by the UV measurement chamber 2020. It should also be understood that at least some of the fluid output from the PCR chambers 2083, 2083', 2083", 2083'" may be delivered out through the output port 2060. In some such cases, the fluid delivered out through the output port 2060 is disposed of as waste.

[0188] In some cases, the intensity of the UV light source (e.g., UV LED 101) may vary over time. Additionally or in an alternative, the UV transmittance may vary between different boxes. This difference in UV light intensity and / or UV transmittance may affect the limit of quantitation (LoQ) of the UV measurement process. In other words, the variation of UV light intensity, UV transmittance and / or other noise sources may affect the maximum and minimum sample concentrations, which can be reliably detected by UV measurements as described herein with a certain predetermined confidence level. Therefore, it may be desirable to provide a process for establishing the upper LoQ and lower LoQ of a box before using a box to obtain a polynucleotide concentration measurement during a process for forming a therapeutic polynucleotide.

[0189] Fig.19An example of a process 2100 is described that can be used to evaluate the upper LoQ and lower LoQ of a box before using the box to obtain a polynucleotide concentration measurement during the process of forming a therapeutic polynucleotide. The process 2100 can be used with any of the various boxes described herein. The process 2100 can be performed by a controller 221, a processor 1401, and / or any other suitable hardware component. The process 2100 begins by measuring the maximum voltage of a UV detector (e.g., UV detector 103, 1415), as shown in box 2102. Next, the standard deviation of the UV detection signal is measured, as shown in box 2104. Using these measurements, the process 2100 then includes determining the upper LoQ, as shown in box 2106; and determining the lower LoQ, as shown in box 2108.

[0190] By way of example only, the Upper LoQ may be determined by the following equation (I) (block 2106):

[0191] (I)

[0192] Where Vmax = maximum voltage;

[0193] V σ = standard deviation of the signal;

[0194] ε = UV absorption coefficient of the nucleotide; and

[0195] l = sample thickness (ie, path length).

[0196] By way of another example only, the Lower LoQ may be determined by the following equation (II) (block 2108):

[0197] (II)

[0198] Where Vmax = maximum voltage;

[0199] V σ = standard deviation of the signal,

[0200] ε = UV absorption coefficient of the nucleotide; and

[0201] l = sample thickness (ie, path length).

[0202] The determined upper LoQ may be compared to a threshold to determine whether the upper LoQ is acceptable, as shown in block 2110. Similarly, the determined lower LoQ may be compared to a threshold to determine whether the lower LoQ is acceptable, as shown in block 2112. If 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 LoQ or the lower LoQ such that the measured concentration is unreliable; and / or any other suitable message. In some versions, if the upper LoQ or the lower LoQ is found to be unacceptable, the controller 221, the processor 1401, and / or any other suitable hardware component may prevent further use of the cartridge and / or the UV light source unless and until the operator takes corrective action and subsequently finds the upper LoQ and the lower LoQ to be acceptable. In any 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 component may allow the box to be used to form a therapeutic polynucleotide (and / or perform any other suitable process), as shown in box 2118.

[0203] In some cases, particularly when the sample is at a relatively high concentration level (e.g., so that the UV transmittance of the sample is relatively low), when the sample is irradiated with UV light, the sample may tend to cause considerable backscattering. This backscattering of UV light may adversely affect the accuracy of the readings of the UV detector (e.g., UV detector 103, 1415) by adversely affecting the dynamic range. In some cases, this backscattering can be corrected by data processing techniques. However, such data processing techniques may require certain geometric assumptions. Another alternative can include using UV light-blocking materials to suppress scattered light and thereby improve the dynamic range of the UV detector.

[0204] Figure 20 to Figure 21 An example of how UV light blocking materials can be used to suppress scattered light and thereby improve the dynamic range of a UV detector is shown. Specifically, Figure 20 to Figure 21 A UV blocking member 2200 is shown that includes a disc-shaped body 2202 having a central opening 2204 formed therethrough. The body 2202 includes a material configured to prevent UV light from being transmitted through the body 2202 by allowing UV light to be transmitted through the central opening 2204.

[0205] Fig.21Arrangement 2300 is shown in which a UV blocking member 2200 is applied to a box 2310. Box 2310 can be constructed and operated like any other box described herein. Box 2310 of this example includes an upper plate 2320, a lower plate 2330, and an elastic layer 2340 interposed between the plates 2320, 2330. A recess 2322 formed in the upper plate 2320 provides a first UV measurement area; and a recess 2332 formed in the lower plate 2330 provides a second UV measurement area below the recess 2322. UV measurement chamber 2350 is interposed between the recesses 2322, 2332; and can be constructed and operated like other UV measurement chambers described herein.

[0206] UV blocking member 2200 is positioned on top of upper plate 2320 just above recessed portion 2322. In some versions, UV blocking member 2200 is adhered to the upper surface of upper plate 2320. Alternatively, UV blocking member 2200 may be fixed relative to upper plate 2320 in any other suitable manner. Fig.21 As shown, 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 into the recess 2322 so that the UV light 2304 can illuminate the sample in the UV measurement chamber 2350. To the extent that the sample in the UV measurement chamber 2350 provides backscattering of the UV light 2304, the body 2202 will substantially limit such backscattering. The UV light 2360 that leaves the UV measurement chamber 2350 through the recess 2332 will reach the UV detector 2362, which will generate a signal indicative of the concentration of the sample in the UV measurement chamber 2350, as described herein.

[0207] Fig. 22 Another example of an arrangement 2400 that can be used to mitigate backscatter of UV line of sight from a UV measurement chamber is depicted. Similar to the arrangement 2300 described above, the arrangement 2400 of this example includes a UV light source 2402, a box 2410, and a UV detector 2462. Similar to the box 2310 described above (and similar to other boxes described herein), the box 2410 of this example includes an upper plate 2420, a lower plate 2430, and an elastic layer 2440 interposed between the plates 2420, 2430. A recess 2422 formed in the upper plate 2420 provides a first UV measurement area; and a recess 2432 formed in the lower plate 2430 provides a second UV measurement area below the recess 2422. A UV measurement chamber 2450 is interposed between the recesses 2422, 2432; and can be constructed and operated like other LIV measurement chambers described herein.

[0208] Similar to arrangement 2300, arrangement 2400 of this example also includes a UV blocking member 2470; however, UV blocking member 2470 of this example is configured differently from UV blocking member 2200. Specifically, UV blocking member 2470 of this example includes a cylindrical body 2472 defining a central passage 2472 therethrough.

[0209] The body 2472 includes a material configured to prevent UV light from being transmitted through the body 2472 , while the central passage 2472 allows UV light to be transmitted through the central passage 2472 .

[0210] The body 2472 is sized to fit within the recess 2422 so that the outer sidewall of the body 2472 contacts the 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.

[0211] like Fig. 22 As shown, the diameter of the central passage 2472 is smaller than the diameter of the recessed portion 2422. It should be understood that Fig. 22 The diameter of the central passage 2472 shown in FIG. 2 is merely an example; and the diameter of the central passage 2472 may be relative to Fig. 22 In any case, the central passage 2472 allows UV light 2404 from the UV light source 2402 to pass through the UV blocking member 2470 into the recess 2422 so that the UV light 2404 can illuminate the sample in the UV measurement chamber 2450. To the extent that the sample in the UV measurement chamber 2450 provides backscattering of the UV light 2404, the body 2472 will substantially limit such backscattering. The UV light 2460 that leaves the UV measurement chamber 2450 through the recess 2432 will reach the UV detector 2462, which will generate a signal indicative of the concentration of the sample in the UV measurement chamber 2450, as described herein.

[0212] Although Fig.21 and Fig. 22 The UV blocking members 2200, 2470 are shown to be used in separate arrangements 2300, 2400, but some other arrangements may include a combination of UV blocking members 2200, 2470. For example, in Fig. 22In a variation of the arrangement 2400 shown, the UV blocking member 2200 may be positioned on top of the UV blocking member 2470. In some such arrangements, the diameter of the opening 2204 is smaller than the diameter of the central passage 2472. In some such arrangements, the UV blocking member 2200 substantially prevents backscatter that may otherwise occur through the upper opening of the recess 2422; while the UV blocking member 2470 prevents backscatter that may otherwise occur through the sidewalls of the recess 2422. As yet another example of a variation, the sidewalls of the recess 2422 may be laser-charred, coated with a UV blocking material, or otherwise treated to prevent UV light from being transmitted through the sidewalls of the recess 2422.

[0213] In some cases, bubbles may reach the UV measurement chamber, and the presence of such bubbles in the UV measurement chamber may result in an underestimation of the sample concentration in the UV measurement chamber. Therefore, it is desirable to provide a measurement algorithm that takes into account the possibility of inaccurate measurements that may be caused by the presence of bubbles in the UV measurement chamber. An example of such a process 2500 is described in Fig.23 The process 2500 may be used with any of the various cartridges described herein. The process 2500 may be performed by the controller 221, the processor 1401, and / or any other suitable hardware component. Fig.23 As shown in box 2502 of , process 2500 begins with four concentration measurements based on readings from a UV detector as described herein (e.g., UV detectors 103, 1415, 2362, 2462). The average of these four measurements is then calculated, as shown in box 2504. These four measurements are then compared to the calculated average, with outlier measurements discarded, as shown in box 2506. In some versions, outlier measurements are identified as measurements whose values ​​are farthest from the average. In some other versions, outliers are identified based on T-tests. As yet another variation, the measurement that provides the lowest concentration value may be automatically identified as an outlier. In some such versions, the calculation of the average value (box 2504) may be omitted.

[0214] After discarding the outlier measurement (block 2506), the average of the remaining three measurements is then calculated, as shown in block 2508. The updated average is then reported (e.g., as the measured concentration), as shown in block 2510. In some cases, the standard deviation is reported with the average. The updated average (and possible standard deviation) can be reported to an operator and / or control algorithm, which can automatically modify one or more subsequent operations based on the reported average (and possible standard deviation). By way of another example only, the updated average can be used for aggregation as described herein.

[0215] In some cases, the calculation of the average of the first four measurements as shown in box 2504 and the subsequent comparison of the first four measurements with the average can reveal that all four measurements are substantially close to the average. In other words, the standard deviation calculated from the first average may be quite small (e.g., below a predetermined threshold). In some such cases, the process 2500 may stop and report the average (similar to that shown in box 2510 and described above) without discarding outliers (box 2506) and recalculating the average (box 2508). Although four measurements are initially utilized in the process 2500 of this example, other types may provide more or less than four measurements as an initial step. Similarly, although only one outlier is discarded in the process 2500 of this example, other types may discard two or more outliers.

[0216] As use Fig.23 An alternative to the process 2500 shown and described above, a quad detector can be used to account for the presence of any bubbles in the sample. For example, the voltage difference across the axis of the four quadrants can indicate an inhomogeneous sample. This type of method can ensure calibration measurements after each box is loaded to account for optical changes between boxes. Such calibration can be automatic.

[0217] As use Fig.23 As yet another example of an alternative to the process 2500 shown and described above, an imaging detector may be used to account for the presence of any bubbles in the sample. For example, image analysis and / or machine learning techniques may be used to identify bubbles. In the event that bubbles are detected, a vacuum cover (e.g., similar to the vacuum cover 1938 described above) may be used to automatically remove the bubbles in response to optical detection of the bubbles. Alternatively, any concentration measurements captured during bubble detection may be ignored. Further alternatively, optically captured bubble data may be utilized to apply corrections to concentration measurements to effectively eliminate the presence of bubbles in the fluid.

[0218] The following examples pertain to various non-exhaustive ways in which the teachings herein may be combined or applied.

[0219] It should be understood that the following examples are not intended to limit the coverage of any claims that may be raised at any time in this application or a subsequent application of this application. There is no exclusion statement. The following examples are provided for illustrative purposes only. It is contemplated that the various teachings herein may be arranged and applied in a variety of other ways. It is also contemplated that some variations may omit certain features mentioned in the following examples. Therefore, unless the inventor or a successor of the inventor's concern clearly indicates so at a later date, none of the aspects or features mentioned below should be considered critical. If any claims including additional features other than those mentioned below are raised in this application or a subsequent application related to this application, those additional features should not be considered to be added for any reason related to patentability.

[0220] Example 1

[0221] A box device for processing polynucleotides, the box device comprising: a first layer, the first layer having a first thickness; a second layer, the second layer having a second thickness; an elastic material, the 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) measuring region, the ultraviolet (UV) measuring region being formed through the first layer and the second layer, wherein a region of the first layer in the UV measuring region has a thickness equal to or less than the first thickness, and the second layer

[0222] An area in the UV measuring region has a thickness equal to or less than the second thickness, and wherein the elastic material does not exist between the area of ​​the first layer in the UV measuring region and the area of ​​the second layer in the UV measuring region to form a UV measurement chamber having the separation thickness.

[0223] Example 2

[0224] The cartridge device of embodiment 1, wherein the first layer comprises a polymer material.

[0225] Example 3

[0226] The cartridge device according to embodiment 2, wherein the first layer comprises a cyclic olefin copolymer (COC) material.

[0227] Example 4

[0228] The box device according to any one of embodiments 1 to 3, wherein the first thickness is between 1 mm and 5 mm.

[0229] Example 5

[0230] The cartridge device according to any one of embodiments 1 to 4, wherein the second layer comprises a polymer material.

[0231] Example 6

[0232] The cartridge device according to embodiment 5, wherein the second layer comprises a cyclic olefin copolymer (COC) material.

[0233] Example 7

[0234] The box device according to any one of embodiments 1 to 6, wherein the second thickness is between 1 mm and 5 mm.

[0235] Example 8

[0236] The box device according to any one of embodiments 1 to 7, wherein the separation thickness is between 1 mm and 0.1 mm.

[0237] Example 9

[0238] The cartridge apparatus according to any one of embodiments 1 to 8 further comprising a fluid channel in the second layer in fluid communication with the UV measurement chamber.

[0239] Example 10

[0240] According to any one of embodiments 1 to 9, the box device also includes a plurality of pneumatic valves arranged to control the flow in and out of the UV measuring chamber, wherein the plurality of pneumatic valves are each formed by a pneumatic chamber in the first layer and a fluid chamber in the second layer, wherein a portion of the elastic material separates the pneumatic chamber from the fluid chamber, further wherein the pneumatic chamber is fluidly connected to a pneumatic channel in the first layer, the pneumatic channel is configured to be connected to a pressure port on an external area of ​​the box device to actuate the pneumatic valve, and wherein when the pneumatic valve is opened, the fluid chamber is fluidly connected to the UV measuring chamber via the fluid channel in the second layer.

[0241] Embodiment 11

[0242] The cartridge device according to any one of embodiments 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.

[0243] Example 12

[0244] According to any one of embodiments 1 to 11, the box device further comprises one or more vacuum ports on an external area of ​​the box device, and the one or more vacuum ports are configured to be connected to a negative pressure source to draw fluid into the UV measurement chamber.

[0245] Example 13

[0246] According to the box device described in any one of Examples 1 to 12, the box device also includes a first inlet channel within the second layer and a second inlet channel within the second layer, the first inlet channel is connected to a first chamber fluid configured to hold a polynucleotide sample fluid, and the second inlet channel is connected to a second chamber fluid configured to hold a blank sample fluid.

[0247] Embodiment 14

[0248] The cartridge device according to any one of embodiments 1 to 13 further comprises one or more dilution mixing chambers in fluid communication with the UV measurement chamber, the one or more dilution mixing chambers being configured to dilute the fluid sample.

[0249] Embodiment 15

[0250] The cell apparatus according to any one of embodiments 1 to 14, wherein the diameter of the UV measurement chamber is larger than the diameter of the area of ​​the first layer in the UV measurement region to confine bubbles in a central area of ​​the UV measurement chamber.

[0251] Example 16

[0252] A box device for processing polynucleotides, the box device comprising: a first layer, the first layer comprising a polymer material having a first thickness; a second layer, the second layer comprising a polymer material having a second thickness; an elastic material, the elastic material extending between the first layer and the second layer; an ultraviolet (UV) measuring region, the ultraviolet (UV) measuring region being formed through the first layer and the second layer, wherein an area of ​​the first layer in the UV measuring region has a thickness less than or equal to the first thickness, an area of ​​the second layer in the UV measuring region has a thickness less than or equal to the second thickness, and wherein the elastic material is removed between the area of ​​the first layer in the UV measuring region and the area of ​​the second layer in the UV measuring region to form a UV measuring chamber; and an in vitro transcription (IVT) chamber, the in vitro transcription (IVT) chamber being at least partially formed within the second layer and being fluidically connected to the UV measuring chamber.

[0253] Embodiment 17

[0254] A method for manufacturing polynucleotides using a microfluidic actuator device operating on a box, the method comprising: forming a therapeutic polynucleotide in a box; and determining the concentration of the therapeutic polynucleotide by the microfluidic actuator device through the following steps: driving a first blank solution into an ultraviolet (UV) measurement chamber of the box and performing a first absorption measurement through the box; driving a sample solution of the therapeutic polynucleotide into the UV measurement chamber of the box and performing a second absorption measurement through the box; and estimating the concentration of the therapeutic polynucleotide from the first absorption measurement and the second absorption measurement.

[0255] Embodiment 18

[0256] According to the method of Example 17, the method also includes adjusting the operation of the microfluidic drive device based on the estimated concentration of the therapeutic polynucleotide in a processor of the microfluidic drive device.

[0257] Embodiment 19

[0258] According to the method described in any of Examples 17 to 18, the method also includes comparing the estimated concentration of the therapeutic polynucleotide with a concentration range by a processor of the microfluidic drive device, and based on this comparison, directing the therapeutic polynucleotide for one of the following: pooling with one or more additional batches of therapeutic polynucleotides formed in the box, discarding or further analysis.

[0259] Embodiment 20

[0260] The method according to any one of embodiments 17 to 19 further comprises diluting the therapeutic polynucleotide to a standard concentration for output under the control of a processor of the microfluidic actuator device.

[0261] Embodiment 21

[0262] The method of any one of embodiments 17 to 20 further comprising automatically adjusting one or more parameters of formation of the therapeutic polynucleotide by the microfluidic actuator device based on the estimated concentration of the therapeutic polynucleotide.

[0263] Embodiment 22

[0264] A method according to embodiment 21, wherein the one or more parameters include one or more of the following: temperature, reagent volume, reagent concentration, time and mixing.

[0265] Embodiment 23

[0266] A method according to any one of Examples 17 to 22, wherein determining the concentration of the therapeutic polynucleotide also includes performing a serial dilution in one or more chambers of the box under the control of the microfluidic drive device, and repeating the step of driving the sample solution of the therapeutic polynucleotide for each of the one or more dilutions formed by the serial dilution to generate a dilution curve.

[0267] Embodiment 24

[0268] The method according to embodiment 23, wherein estimating the concentration of the therapeutic polynucleotide comprises estimating the concentration of the therapeutic polynucleotide from the dilution curve.

[0269] Embodiment 25

[0270] A method according to any one of embodiments 17 to 24, wherein estimating the concentration of the therapeutic polynucleotide also includes changing one or more of the sensitivity of the UV detector and the intensity of the UV emitter of the microfluidic drive device in response to the second absorption measurement through the box.

[0271] Embodiment 26

[0272] Embodiment 25. The method of any one of Embodiments 17 to 25 further comprising pre-treating the UV measurement chamber with UV light prior to performing the first absorption measurement through the cartridge.

[0273] Embodiment 27

[0274] The method of any one of embodiments 17 to 26, wherein forming the therapeutic polynucleotide comprises performing an in vitro transcription (IVT) reaction to form the therapeutic polynucleotide.

[0275] Embodiment 28

[0276] The method according to Example 27 further includes generating a template for the IVT reaction in the box.

[0277] Embodiment 29

[0278] The method of any one of embodiments 17 to 28, wherein said therapeutic polynucleotide comprises a therapeutic mRNA.

[0279] Embodiment 30

[0280] According to the method described in Example 29, the method also includes encapsulating the therapeutic mRNA with a delivery vehicle.

[0281] Embodiment 31

[0282] A method according to any one of Examples 17 to 30, wherein the microfluidic actuator device supervises the steps of driving the first blank solution and driving the sample solution by pneumatically deflecting one or more regions of the membrane of the box to open and / or close the valve of the box.

[0283] Embodiment 32

[0284] A method according to Example 31, wherein the microfluidic actuator device pneumatically deflects one or more regions of the membrane of the box to drive the first blank solution and / or the sample solution into the UV measurement chamber.

[0285] Embodiment 33

[0286] A method for manufacturing polynucleotides using a microfluidic drive device operating on a box, the method comprising: forming a therapeutic polynucleotide in a box, wherein the therapeutic polynucleotide comprises a therapeutic mRNA; determining the concentration of the therapeutic mRNA by the microfluidic drive device by the following steps: driving a first blank solution into an ultraviolet (UV) measurement chamber of the box and performing a first absorption measurement through the box; driving a sample solution of the therapeutic polynucleotide into the UV measurement chamber of the box and performing a second absorption measurement through the box; driving a second blank solution into the UV measurement chamber of the box and performing a third absorption measurement through the box; and estimating the concentration of the therapeutic polynucleotide from the first absorption measurement, the second absorption measurement, and the third absorption measurement; and adjusting the operation of the microfluidic drive device in a processor of the microfluidic drive device based on the estimated concentration of the therapeutic polynucleotide.

[0287] Embodiment 34

[0288] A system for preparing and / or processing polynucleotides, the system comprising: a box mount; a plurality of pressure lines; a plurality of fluid lines, each fluid line connected to or configured to be connected to a fluid source, wherein each fluid line and at least a subset of the pressure lines are configured to be fixed to an inlet port or an outlet port on a box held in the box mount; an ultraviolet (UV) light source; a UV light detector; and a controller, the controller being configured to control the application of pressure through the pressure lines to drive fluid through the box, wherein the controller is also configured to direct the formation of therapeutic polynucleotides in the box, and to estimate the concentration of the therapeutic polynucleotide by the following steps: driving a first blank solution through a UV measurement chamber of the box and performing 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 box and performing a second absorption measurement using the UV light source and UV light receiver; further, wherein the controller is configured to estimate the concentration of the therapeutic polynucleotide from the first absorption measurement and the second absorption measurement.

[0289] Embodiment 35

[0290]

[00136] The system of embodiment 34, wherein the controller is further configured to modify the operation of the system based on the estimated concentration of the therapeutic polynucleotide.

[0291] Embodiment 36

[0292] A system according to any of embodiments 34 to 35, wherein the controller is further configured to compare the estimated concentration of the therapeutic polynucleotide to a concentration range, and based on this comparison, direct the therapeutic polynucleotide for one of the following: pooling with one or more additional batches of therapeutic polynucleotides formed in the box, discarding, or further analysis.

[0293] Embodiment 37

[0294] The system of any one of embodiments 34 to 36, wherein the controller is further configured to dilute the therapeutic polynucleotide to a standard concentration for output.

[0295] Embodiment 38

[0296]

[00136] The system of any one of embodiments 34 to 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.

[0297] Embodiment 39

[0298] A system as described in Example 38, wherein the one or more parameters include one or more of the following: temperature, reagent volume, reagent concentration, time, and mixing.

[0299] Embodiment 40

[0300] A system according to any one of embodiments 34 to 39, wherein the controller is further configured to perform a serial dilution within one or more chambers of the box, and to 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 generate a dilution curve.

[0301] Embodiment 41

[0302]

[00136] The system of embodiment 40, wherein the controller is further configured to estimate the concentration of the therapeutic polynucleotide from the dilution curve.

[0303] Embodiment 42

[0304] The system of any one of embodiments 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 by the cartridge.

[0305] Embodiment 43

[0306] Embodiment 34. The system of any one of Embodiments 34 to 42, wherein the controller is further configured to pre-treat the UV measurement chamber with UV light prior to performing the first absorption measurement through the cartridge.

[0307] Embodiment 44

[0308] The system of any one of embodiments 34 to 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.

[0309] Embodiment 45

[0310] The system of embodiment 44, wherein the controller is further configured to generate a template for the IVT reaction in the box.

[0311] Embodiment 46

[0312] Embodiment 34. The system of any one of embodiments 34 to 45, wherein said therapeutic polynucleotide comprises a therapeutic mRNA.

[0313] Embodiment 47

[0314] The system of Example 46, wherein the controller is further configured to encapsulate the therapeutic mRNA with a delivery vehicle.

[0315] Embodiment 48

[0316] A system according to any one of embodiments 34 to 47, wherein the controller is further configured to pneumatically deflect one or more regions of the membrane of the box to open and / or close the valve of the box when driving the first blank solution, driving the second blank solution, and driving the sample solution.

[0317] Embodiment 49

[0318]

[00136] The system of Embodiment 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.

[0319] Embodiment 50

[0320] A system for preparing and / or processing polynucleotides, the system comprising: a box; and a microfluidic drive device, the microfluidic drive device comprising: a box mount; a plurality of pressure lines; a plurality of fluid lines, each fluid line coupled to or configured to be coupled 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 box held in the box 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 box, wherein the controller is also configured to direct the formation of a therapeutic polynucleotide, and further wherein the controller The device is configured to estimate the concentration of the therapeutic polynucleotide by the following steps: driving a first blank solution through the UV measurement chamber of the box and performing a first absorption measurement using the UV light source and the UV light receiver; driving a sample solution containing the therapeutic polynucleotide through the UV measurement chamber of the box and performing a second absorption measurement using the UV light source and the UV light receiver; and driving a second blank solution through the UV measurement chamber of the box and performing a third absorption measurement using the UV light source and the UV light receiver; modifying the operation of the microfluidic drive device based on the concentration of the therapeutic polynucleotide determined from the first absorption measurement, the second absorption measurement, and the third absorption measurement.

[0321] Embodiment 51

[0322] A method for manufacturing polynucleotides using a microfluidic drive device operating on a box, the method comprising: forming a therapeutic polynucleotide in a box; and determining the concentration of the therapeutic polynucleotide by the microfluidic drive device by the following steps: driving a first blank solution into a concentration measurement chamber of the box and performing a first measurement through the box; driving a sample solution of the therapeutic polynucleotide into the concentration measurement chamber of the box and performing a second measurement through the box; and estimating the concentration of the therapeutic polynucleotide from the first measurement and the second measurement; and comparing the estimated concentration of the therapeutic polynucleotide with a concentration range by a processor of the microfluidic drive device, and based on this comparison, directing the therapeutic polynucleotide for one of the following: pooling with one or more additional batches of therapeutic polynucleotides formed in the box, discarding, or further analysis.

[0323] Embodiment 52

[0324] According to the method of embodiment 51, the concentration measurement chamber includes an ultraviolet (UV) measurement chamber, the first measurement includes a first absorption measurement, and the second measurement includes a second absorption measurement.

[0325] Embodiment 53

[0326] A method according to Example 52, wherein estimating the concentration of the therapeutic polynucleotide also includes changing one or more of the sensitivity of the UV detector and the intensity of the UV emitter of the microfluidic drive device in response to the second absorption measurement by the box.

[0327] Embodiment 54

[0328] Embodiment 52: The method of any one of Embodiments 52-53 further comprising pre-treating the UV measurement chamber with UV light prior to performing the first absorption measurement through the cartridge.

[0329] Embodiment 55

[0330] The method of any one of Embodiments 51 to 54, further comprising adjusting, in a processor of the microfluidic drive device, the operation of the microfluidic drive device based on the estimated concentration of the therapeutic polynucleotide.

[0331] Embodiment 56

[0332] According to any one of the methods of embodiments 51 to 55, the method further comprises diluting the therapeutic polynucleotide to a standard concentration for output under the control of a processor of the microfluidic actuator device.

[0333] Embodiment 57

[0334] According to the method of any one of embodiments 51 to 55, the method also includes automatically adjusting one or more parameters of the formation of the therapeutic polynucleotide based on the estimated concentration of the therapeutic polynucleotide by the microfluidic actuator device.

[0335] Embodiment 58

[0336] A method according to Example 57, wherein the one or more parameters include one or more of the following: temperature, reagent volume, reagent concentration, time and mixing.

[0337] Embodiment 59

[0338] A method according to any one of Examples 51 to 58, wherein determining the concentration of the therapeutic polynucleotide also includes performing a serial dilution in one or more chambers of the box under the control of the microfluidic drive device, and repeating the step of driving the sample solution of the therapeutic polynucleotide for each of the one or more dilutions formed by the serial dilution to generate a dilution curve.

[0339] Embodiment 60

[0340] The method of embodiment 59, wherein estimating the concentration of the therapeutic polynucleotide comprises estimating the concentration of the therapeutic polynucleotide from the dilution curve.

[0341] Embodiment 61

[0342] The method of any one of embodiments 51 to 60, wherein forming the therapeutic polynucleotide comprises performing an in vitro transcription (IVT) reaction to form the therapeutic polynucleotide.

[0343] Embodiment 62

[0344] According to the method of embodiment 61, the method also includes generating a template for the IVT reaction in the box.

[0345] Embodiment 63

[0346] The method of any one of embodiments 51 to 62, wherein said therapeutic polynucleotide comprises a therapeutic mRNA.

[0347] Embodiment 64

[0348] According to the method described in Example 63, the method also includes encapsulating the therapeutic mRNA with a delivery vehicle.

[0349] Embodiment 65

[0350] A method according to any one of Examples 51 to 64, wherein the microfluidic actuator device oversees the steps of driving the first blank solution and driving the sample solution by pneumatically deflecting one or more regions of the membrane of the box to open and / or close the valve of the box.

[0351] Embodiment 66

[0352] A method according to Example 65, wherein the microfluidic actuator device pneumatically deflects one or more regions of the membrane of the box to drive the first blank solution and / or the sample solution into the measurement chamber.

[0353] Embodiment 67

[0354] A system for preparing and / or processing polynucleotides, the system comprising: a box mount; a plurality of pressure lines; a plurality of fluid lines, each fluid line connected to or configured to be connected to a fluid source, wherein each fluid line and at least a subset of the pressure lines are configured to be fixed to an inlet port or an outlet port on a box held in the box mount; and a controller, the controller being configured to control the application of pressure through the pressure lines to drive fluid through the box, wherein the controller is also configured to guide the formation of therapeutic polynucleotides in the box, and to estimate the concentration of the therapeutic polynucleotide by the following steps: driving a first blank solution through a measurement chamber of the box and performing a first measurement of the first blank sample in the measurement chamber; driving a sample solution containing the therapeutic polynucleotide through the measurement chamber of the box and performing a second measurement of the sample solution in the measurement chamber; further, wherein the controller is configured to estimate the concentration of the therapeutic polynucleotide from the first measurement and the second measurement.

[0355] Embodiment 68

[0356] According to the system of embodiment 67, the system also includes: an ultraviolet (UV) light source; and a UV light detector, wherein the controller is configured to use the UV light source and the UV light receiver to perform the first measurement in the form of a first absorption measurement; wherein the controller is configured to use the UV light source and the UV light receiver to perform the second measurement in the form of a second absorption measurement; wherein the controller is configured to estimate the concentration of the therapeutic polynucleotide from the first absorption measurement and the second absorption measurement.

[0357] Embodiment 69

[0358] A system as 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 by the box.

[0359] Embodiment 70

[0360] Embodiment 65. The system of any one of Embodiments 68 to 69, wherein the controller is further configured to pre-treat the UV measurement chamber with UV light prior to performing the first absorption measurement through the cartridge.

[0361] Embodiment 71

[0362]

[00136] The system of any one of embodiments 67 to 70, wherein the controller is further configured to modify the operation of the system based on the estimated concentration of the therapeutic polynucleotide.

[0363] Embodiment 72

[0364] The system of any one of embodiments 67 to 71, wherein the controller is further configured to dilute the therapeutic polynucleotide to a standard concentration for output.

[0365] Embodiment 73

[0366]

[00136] The system of any one of embodiments 67 to 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.

[0367] Embodiment 74

[0368] A system as described in Example 73, wherein the one or more parameters include one or more of the following: temperature, reagent volume, reagent concentration, time, and mixing.

[0369] Embodiment 75

[0370] A system according to any one of embodiments 67 to 74, wherein the controller is further configured to perform a serial dilution within one or more chambers of the box, and to 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 generate a dilution curve.

[0371] Embodiment 76

[0372]

[00136] The system of embodiment 75, wherein the controller is further configured to estimate the concentration of the therapeutic polynucleotide from the dilution curve.

[0373] Embodiment 78

[0374]

[00136] The system of any one of embodiments 67 to 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.

[0375] Embodiment 79

[0376] The system of embodiment 78, wherein the controller is further configured to generate a template for the IVT reaction in the box.

[0377] Embodiment 80

[0378]

[0136] The system of any one of embodiments 67-79, wherein said therapeutic polynucleotide comprises a therapeutic mRNA.

[0379] Embodiment 81

[0380] The system of Example 80, wherein the controller is further configured to encapsulate the therapeutic mRNA with a delivery vehicle.

[0381] Embodiment 82

[0382] A system according to any one of embodiments 67 to 81, wherein the controller is also configured to pneumatically deflect one or more regions of the membrane of the box to open and / or close the valve of the box when driving the first blank solution, driving the second blank solution, and driving the sample solution.

[0383] Embodiment 83

[0384]

[00136] 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.

[0385] Embodiment 84

[0386] A method, the method comprising: emitting UV light through an ultraviolet (UV) measurement chamber of a box, the UV measurement chamber being configured to receive a fluid; receiving the UV light at a UV light detector, the received UV light having passed through the UV measurement chamber of the box; measuring a maximum voltage of the UV light detector; measuring a standard deviation of a signal from the UV light detector; 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 any of the following: allowing the box to be used to generate therapeutic polynucleotides if the upper LoQ and the lower LoQ are acceptable; or providing a warning if one or both of the upper LoQ or the lower LoQ are unacceptable.

[0387] Embodiment 85

[0388] According to the method of embodiment 84, the method also includes forming or processing a therapeutic polynucleotide on the box.

[0389] Embodiment 86

[0390] A box device for processing polynucleotides, the box device comprising: a first layer, the first layer defining a first recessed portion providing a first ultraviolet (UV) measurement area; a second layer, the second layer defining a second recessed portion providing a second UV measurement area; an elastic material, the elastic material extending between the first layer and the second layer and separating the first layer from the second layer; a UV measurement chamber, the UV measurement chamber formed between the first UV measurement area and the second UV measurement area, the UV measurement chamber being configured to receive a fluid, the first layer and the second layer being configured to allow UV light to pass through the UV measurement chamber; and a UV blocking feature, the UV blocking feature being positioned on or in the first layer, the UV blocking feature being configured to limit UV light from passing through the first layer or backscattering from the first layer.

[0391] Embodiment 87

[0392] According to the box device of embodiment 86, the elastic material is not present along the UV measurement chamber.

[0393] Embodiment 88

[0394] According to the box device of any one of embodiments 86 to 87, the first recessed portion has a first diameter, and the UV blocking feature has an opening, and the opening has a diameter smaller than the first diameter of the first recessed portion.

[0395] Embodiment 89

[0396] According to the box device of any one of embodiments 86 to 88, the UV blocking feature has a body positioned on the upper surface of the first layer, and the first recessed portion is formed through the upper surface of the first layer.

[0397] Embodiment 90

[0398] According to the box device described in embodiment 89, the main body has a disc shape.

[0399] Embodiment 91

[0400] According to the box device of any one of embodiments 86 to 90, the UV blocking feature has a cylindrical body positioned in the first recess.

[0401] Embodiment 92

[0402] According to the box device of any one of embodiments 86 to 91, the UV blocking feature is configured to block UV light from being transmitted through the side walls of the first recessed portion.

[0403] Embodiment 93

[0404] A method, the method comprising: emitting UV light through an ultraviolet (UV) measurement chamber of a box, the UV measurement chamber containing a fluid; receiving the UV light at a UV light detector; the received UV light having passed through the UV measurement chamber of the box; obtaining a first plurality of concentration measurements, each concentration measurement in the first plurality of concentration measurements indicating a corresponding concentration of the fluid in the UV measurement chamber; calculating an average of the first plurality of concentration measurements; comparing each concentration measurement in the first plurality of concentration measurements with 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 with the identified outliers removed; calculating an average of the second plurality of concentration measurements; and reporting the average of the second plurality of concentration measurements.

[0405] Embodiment 94

[0406] According to the method described in Example 93, the first plurality of concentration measurements includes four concentration measurements.

[0407] Embodiment 95

[0408] According to the method of any one of embodiments 93 to 94, the second plurality of concentration measurements includes three concentration measurements.

[0409] Embodiment 96

[0410] According to the method of any one of embodiments 93 to 95, the identified outlier provides a concentration measurement that deviates significantly from the mean of the first plurality of measurements due to the presence of bubbles in the fluid in the UV measurement chamber.

[0411] Embodiment 97

[0412] According to the method of embodiment 96, the method also includes executing a bubble removal algorithm in response to the identified outlier value deviating from the mean value of the first plurality of measurements by a predetermined degree.

[0413] It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail below (provided such concepts are not mutually inconsistent) are considered to be part of the inventive subject matter disclosed herein and can be used to achieve the benefits described herein.

[0414] The process parameters and step sequences described and / or illustrated herein are given by way of example only and may vary as desired. For example, although the steps illustrated and / or described herein may be shown or discussed in a particular order, the steps do not necessarily need to be performed in the order illustrated or discussed. The various example methods described and / or illustrated herein may also omit one or more of the steps described or illustrated herein, or include additional steps in addition to those disclosed.

[0415] Any of the methods described herein (including user interfaces) may be implemented as software, hardware, or firmware, and may be described as a non-transitory computer-readable storage medium storing a set of instructions executable by a processor (e.g., a computer, tablet computer, smart phone, etc.), which, when executed by the processor, causes the processor to control the execution of any of the steps, including but not limited to: display, communication with a user, analysis, modification of parameters (including timing, frequency, intensity, etc.), determination, warning, etc. For example, any of the methods described herein may be performed at least in part by a device including one or more processors having a memory storing a non-transitory computer-readable storage medium storing a set of instructions for the process of the method.

[0416] Although various embodiments have been described and / or illustrated herein in the context of a fully functional computing system, one or more of these example embodiments may be distributed as various forms of program products, regardless of the specific type of computer-readable medium used to actually perform the distribution. The embodiments disclosed herein may also be implemented using software modules that perform specific tasks. These software modules may include scripts, batches, or other executable files that may be stored on a computer-readable storage medium or in a computing system. In some embodiments, these software modules may configure a computing system to perform one or more of the example embodiments disclosed herein.

[0417] Although illustrated as separate elements, the method steps described and / or illustrated herein may represent portions of a single application. Additionally, in some embodiments, one or more of the steps may represent or correspond to one or more software applications or programs that, when executed by a computing device, may cause the computing device to perform one or more tasks, such as method steps.

[0418] In addition, one or more of the devices described herein may transform data, physical devices, and / or representations of physical devices from one form to another. Alternatively or in addition, one or more of the modules described herein 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 by executing on a computing device, storing data on a computing device, and / or otherwise interacting with a computing device.

[0419] The term "computer-readable medium" as used herein 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 media such as magnetic storage media (e.g., hard drives, tape drives, and floppy disks), optical storage media (e.g., compact disks (CDs), digital video disks (DVDs), and BLU-RAY disks), electronic storage media (e.g., solid-state drives and flash memory media), and other distribution systems.

[0420] When a feature or element is referred to as "on another feature or element" in this article, it may be directly located on other features or elements, or there may also be intermediate features and / or elements. On the contrary, when a feature or element is referred to as "directly on another feature or element", there are no intermediate features or elements. It should be understood that when a feature or element is referred to as "connected", "attached" or "coupled" to another feature or element, it may be directly connected, attached or coupled to another feature or element, or there may be intermediate features or elements. On the contrary, when a feature or element is referred to as "directly connected", "directly attached" or "directly coupled" to another feature or element, there are no intermediate features or elements. Although described or shown with respect to one embodiment, the features and elements described or shown in this way may be applied to other embodiments. It will also be understood by those skilled in the art that a reference to a structure or feature that is set to be "adjacent" to another feature may have a portion that overlaps or is located below the adjacent feature.

[0421] The terms used herein are only used to describe specific embodiments and are not intended to limit the present invention. For example, as used herein, the singular forms "one", "a kind of" and "the" are intended to also include plural forms unless the context clearly indicates otherwise. It should also be understood that when used in this specification, the terms "include" and / or "comprise" specify the presence of stated features, steps, operations, elements and / or parts, but do not exclude the presence or addition of one or more other features, steps, operations, elements, parts and / or their groups. As used herein, the term "and / or" includes any and all combinations of one or more associated listed items and can be abbreviated as " / ".

[0422] Spatially related terms, such as "below", "under", "down", "above", "on", etc., may be used herein for ease of description to describe the relationship between an element or feature and another element or feature as illustrated in the figure. It should be understood that spatially related terms are intended to cover different orientations of the device in use or operation except for the orientation depicted in the figure. For example, if the device in the figure is inverted, the elements described as "below" or "below" other elements or features will be oriented to be "above" other elements or features. Therefore, the exemplary term "below" may include both above and below orientations. The device can be oriented in other ways (rotated 90 degrees or in other orientations) and the spatially related descriptors used herein are interpreted accordingly. Similarly, the terms "upward", "downward", "vertical", "horizontal", etc. are used herein only for explanation purposes unless otherwise explicitly indicated.

[0423] Although the terms "first" and "second" may be used herein to describe various features / elements (including steps), these features / elements should not be limited by these terms unless the context indicates otherwise. These terms can be used to distinguish one feature / element from another feature / element. Therefore, without departing from the teachings of the present invention, the first feature / element discussed below may be referred to as the second feature / element, and similarly, the second feature / element discussed below may be referred to as the first feature / element.

[0424] In general, any of the devices and methods described herein should be understood to be inclusive, but all or a subset of the components and / or steps may alternatively be exclusive and may be expressed as "consisting of various components, steps, sub-components or sub-steps" or alternatively "consisting essentially of various components, steps, sub-components or sub-steps."

[0425] As used herein in the specification and claims, including as used in the examples and unless otherwise expressly stated, all numbers can be interpreted as beginning with the word "about" or "approximately", even if the term does not appear explicitly. When describing a value and / or position, the phrase "about" or "approximately" can be used to indicate that the value and / or position described are within the reasonable expected range of the value and / or position. For example, a numerical value may have a value of + / -0.1% of the value (or range of values), + / -1% of the value (or range of values), + / -2% of the value (or range of values), + / -5% of the value (or range of values), + / -10% of the value (or range of values), etc. Unless the context indicates otherwise, any numerical value given herein should also be understood to include about or approximately the value. For example, if the value "10" is disclosed, "about 10" is also disclosed. Any numerical range described herein is intended to include all subranges contained therein. It should also be understood that when a value is disclosed, "less than or equal to" the value, "greater than or equal to the value," and possible ranges between values ​​are also disclosed, as appropriately understood by those skilled in the art. For example, if the value "X" is disclosed, "less than or equal to X" and "greater than or equal to X" are also disclosed (e.g., where X is a numerical value). It should also be understood that throughout the application, data is provided in a variety of different formats, and that the data represents endpoints and starting points and ranges for any combination of data points. For example, if a specific data point "10" and a specific data point "15" are disclosed, it should be understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15, as well as between 10 and 15, are considered disclosed. It should also be understood that every unit between two specific units is also disclosed. For example, if 10 and 15 are disclosed, 11, 12, 13, and 14 are also disclosed.

[0426] Although various exemplary embodiments are described above, any of a number of changes may be made to the various embodiments without departing from the scope of the invention as described in the claims. For example, in alternative embodiments, the order in which the various described method steps are performed may generally be changed, and in other alternative embodiments, one or more method steps may be skipped entirely. Optional features of the various device and system embodiments may be included in some embodiments but not in other embodiments. Therefore, the foregoing description is provided primarily for exemplary purposes and should not be construed as limiting the scope of the invention set forth in the claims.

[0427] The examples and illustrations included herein show specific embodiments that can practice this theme in an illustrative and non-limiting manner. As mentioned, other embodiments can be used and derived therefrom, so that structural and logical substitutions and changes can be made without departing from the scope of the present invention. These embodiments of the subject matter of the present invention can be referred to individually or collectively by the term "invention" herein, which is only for convenience, rather than actively limiting the scope of the present application to any single invention or inventive concept when more than one invention or inventive concept is actually disclosed. Therefore, although specific embodiments have been illustrated and described herein, any arrangement calculated to achieve the same purpose can replace the specific embodiments shown. The present disclosure is intended to cover any and all modifications or changes of various embodiments. After reviewing the above description, the combination of the above embodiments and other embodiments not specifically described herein will be apparent to those skilled in the art.

Claims

1. A box device for processing polynucleotides, the box device 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) measuring region, the ultraviolet (UV) measuring region being formed through the first layer and the second layer, wherein a region of the first layer in the UV measuring region has a thickness equal to or less than the first thickness, a region of the second layer in the UV measuring region has a thickness equal to or less than the second thickness, and wherein the elastic material does not exist between the region of the first layer in the UV measuring region and the region of the second layer in the UV measuring region, so as to form a UV measuring chamber having the separation thickness.

2. The cartridge device of claim 1, wherein the first layer comprises a polymer 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 said first thickness is between 1 mm and 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. A cartridge device according to any one of claims 1 to 6, wherein said second thickness is between 1 mm and 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 according to any one of claims 1 to 8, further comprising a fluid channel in the second layer in fluid communication with the UV measurement chamber.

10. A box device according to any one of claims 1 to 9, wherein the box device also includes a plurality of pneumatic valves arranged to control the flow in and out of the UV measuring chamber, wherein the plurality of pneumatic valves are each formed by a pneumatic chamber in the first layer and a fluid chamber in the second layer, wherein a portion of the elastic material separates the pneumatic chamber from the fluid chamber, further wherein the pneumatic chamber is fluidly connected to a pneumatic channel in the first layer, the pneumatic channel is configured to be connected to a pressure port on an external area of ​​the box device to actuate the pneumatic valve, and wherein when the pneumatic valve is opened, the fluid chamber is fluidly connected to the UV measuring chamber through the fluid channel in the second layer.

11. The cartridge device of any one of claims 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.

12. The box device according to any one of claims 1 to 11, further comprising one or more vacuum ports on an external area of ​​the box device, the one or more vacuum ports being configured to be connected to a negative pressure source to draw fluid into the UV measurement chamber.

13. The box device according to any one of claims 1 to 12, further comprising a first inlet channel within the second layer and a second inlet channel within the second layer, the first inlet channel being connected to a first chamber fluid configured to hold a polynucleotide sample fluid, and the second inlet channel being connected to a second chamber fluid configured to hold a blank sample fluid.

14. The cartridge device according to any one of claims 1 to 13, further comprising one or more dilution mixing chambers in fluid communication with the UV measurement chamber, the one or more dilution mixing chambers being configured to dilute the fluid sample.

15. The cell apparatus according to any one of claims 1 to 14, wherein the diameter of the UV measurement chamber is larger than the diameter of the area of ​​the first layer in the UV measurement region to confine air bubbles in a central area of ​​the UV measurement chamber.

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

17. A method for producing a polynucleotide using a microfluidic actuator device operated on a cartridge, the method comprising: forming a therapeutic polynucleotide in a cassette; as well as The concentration of the therapeutic polynucleotide is determined by the microfluidic actuator device by the following steps: driving a first blank solution into an ultraviolet (UV) measurement chamber of the cartridge and performing a first absorption measurement through the cartridge; driving a sample solution of the therapeutic polynucleotide into the UV measurement chamber of the cartridge and performing a second absorption measurement through the cartridge; as well as The concentration of the therapeutic polynucleotide is estimated from the first absorption measurement and the second absorption measurement.

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

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

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

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

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. The method of any one 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 box under the control of the microfluidic drive device, and repeating the step of driving the sample solution of the therapeutic polynucleotide for each of the one or more dilutions formed by the serial dilution to generate a dilution curve.

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. The method of any one of claims 17 to 24, wherein estimating the concentration of the therapeutic polynucleotide further comprises changing one or more of the sensitivity of a UV detector and the intensity of a UV emitter of the microfluidic drive device in response to the second absorption measurement by the box.

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

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

28. The method of claim 27, further comprising generating 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. The method of claim 29, further comprising encapsulating the therapeutic mRNA with a delivery vehicle.

31. A method according to any one of claims 17 to 30, wherein the microfluidic drive device supervises the steps of driving the first blank solution and driving the sample solution by pneumatically deflecting one or more regions of the membrane of the box to open and / or close the valve of the box.

32. The method of claim 31, wherein the microfluidic actuator 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. A method of manufacturing a polynucleotide using a microfluidic actuator device operated on a cartridge, the method comprising: forming a therapeutic polynucleotide in a cassette, wherein the therapeutic polynucleotide comprises a therapeutic mRNA; The concentration of the therapeutic mRNA is determined by the microfluidic actuator device by the following steps: driving a first blank solution into an ultraviolet (UV) measurement chamber of the cartridge and performing a first absorption measurement through the cartridge; driving a sample solution of the therapeutic polynucleotide into the UV measurement chamber of the cartridge and performing a second absorption measurement through the cartridge; driving a second blank solution into the UV measurement chamber of the cartridge and performing a third absorption measurement through the cartridge; as well as estimating a concentration of the therapeutic polynucleotide from the first absorption measurement, the second absorption measurement, and the third absorption measurement; as well as Operation of the microfluidic actuator device is adjusted in a processor of the microfluidic actuator device based on the estimated concentration of the therapeutic polynucleotide.

34. A system for preparing and / or processing polynucleotides, the system comprising: Box mounting parts; 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 port or an outlet port on a cassette retained in the cassette mount; Ultraviolet (UV) light source; UV light detector; and A controller configured to control the application of pressure through the pressure line to drive fluid through the box, wherein the controller is also configured to guide the formation of the therapeutic polynucleotide in the box, and to estimate the concentration of the therapeutic polynucleotide by the following steps: driving a first blank solution through the UV measurement chamber of the box and performing a first absorption measurement using the UV light source and the UV light receiver; driving a sample solution containing the therapeutic polynucleotide through the UV measurement chamber of the box and performing a second absorption measurement using the UV light source and the UV light receiver; further, wherein the controller is configured to estimate the concentration of the therapeutic polynucleotide from the first absorption measurement and the second absorption measurement.

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

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

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

38. The system of any one of claims 34 to 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. 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. A system according to 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 box, and to 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 generate a dilution curve.

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

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

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

44. The system of any one of claims 34 to 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. The system of claim 44, wherein the controller is further configured to generate a template for the IVT reaction in the cartridge.

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

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

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 the membrane of the box to open and / or close the valve of the box when driving the first blank solution, driving the second blank solution, and driving the sample solution.

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. A system for preparing and / or processing polynucleotides, the system comprising: box; and A microfluidic actuator device, the microfluidic actuator device comprising: Box mounting parts; 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 port or an outlet port on a cassette retained in the cassette mount; Ultraviolet (UV) light source; UV light detector; and 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 further wherein the controller is configured to estimate the concentration of the therapeutic polynucleotide by: driving a first blank solution through a UV measurement chamber of the cartridge and performing 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 performing 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 performing a third absorption measurement using the UV light source and UV light receiver; The operation of the microfluidic actuator device is modified based on the concentration of the therapeutic polynucleotide determined from the first absorption measurement, the second absorption measurement, and the third absorption measurement.

51. A method for producing a polynucleotide using a microfluidic actuator device operated on a cartridge, the method comprising: forming a therapeutic polynucleotide in a cassette; as well as The concentration of the therapeutic polynucleotide is determined by the microfluidic actuator device by the following steps: 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 performing a second measurement through the cartridge; as well as estimating a concentration of the therapeutic polynucleotide from the first measurement and the second measurement; as well as The estimated concentration of the therapeutic polynucleotide is compared to a concentration range by a processor of the microfluidic drive device, and based on this comparison, the therapeutic polynucleotide is directed for one of the following: pooling with one or more additional batches of therapeutic polynucleotides formed in the box, discarding, or further analysis.

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

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

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

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

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

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

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. A method according to any one of claims 51 to 58, wherein determining the concentration of the therapeutic polynucleotide also includes performing a serial dilution in one or more chambers of the box under the control of the microfluidic drive device, and repeating the step of driving the sample solution of the therapeutic polynucleotide for each of the one or more dilutions formed by the serial dilution to generate a dilution curve.

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

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

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

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

65. A method according to any one of claims 51 to 64, wherein the microfluidic drive device supervises the steps of driving the first blank solution and driving the sample solution by pneumatically deflecting one or more regions of the membrane of the box to open and / or close the valve of the box.

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

67. A system for preparing and / or processing polynucleotides, the system comprising: Box mounting parts; 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 port or an outlet port on a cassette retained in the cassette mount; and A controller configured to control the application of pressure through the pressure line to drive fluid through the box, wherein the controller is also configured to guide the formation of the therapeutic polynucleotide in the box, and to estimate the concentration of the therapeutic polynucleotide by the following steps: driving a first blank solution through a measuring chamber of the box and performing a first measurement of the first blank sample in the measuring chamber; driving a sample solution containing the therapeutic polynucleotide through the measuring chamber of the box and performing a second measurement of the sample solution in the measuring chamber; further, wherein the controller is configured to estimate the concentration of the therapeutic polynucleotide from the first measurement and the second measurement.

68. The system of claim 67, further comprising: Ultraviolet (UV) light source; and UV light detector; wherein the controller is configured to perform the first measurement in the form of a first absorption measurement using the UV light source and the UV light receiver; wherein the controller is configured to perform the second measurement in the form of a second absorption measurement using the UV light source and the UV light receiver; Wherein the controller is configured to estimate the concentration of the therapeutic polynucleotide from the first absorption measurement and the second absorption measurement.

69. The system of claim 68, wherein the controller is further configured to change the sensitivity of the UV detector and the One or more of the intensities of the UV light sources.

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

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

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

73. The system of any one of claims 67 to 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. 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. A system according to any one of claims 67 to 74, wherein the controller is further configured to perform a serial dilution within one or more chambers of the box, and to 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 generate a dilution curve.

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

77. The system of any one of claims 67 to 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.

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

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

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

81. A system according to any one of claims 67 to 81, wherein the controller is also configured to pneumatically deflect one or more regions of the membrane of the box to open and / or close the valve of the box when driving the first blank solution, driving the second blank solution and driving the sample solution.

82. 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.

83. A method comprising: emitting ultraviolet (UV) light through an ultraviolet (UV) measurement chamber of the cartridge, the UV measurement chamber being configured to receive a fluid; receiving the UV light at a UV light detector, the received UV light having passed through the UV measurement chamber of the cartridge; measuring a maximum voltage of the UV light detector; measuring a standard deviation of a signal from the UV light detector; determining an upper limit of quantitation (LoQ) based on the maximum voltage measured 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 Any of the following: where the upper LoQ and the lower LoQ are acceptable, allowing the cassette to be used to produce a therapeutic polynucleotide; or In the event that one or both of the upper LoQ or the lower LoQ are unacceptable, a warning is provided.

84. The method of claim 84, further comprising forming or processing a therapeutic polynucleotide on the box.

85. A box device for processing polynucleotides, the box device comprising: a first layer defining a first recessed portion providing a first ultraviolet (UV) measurement region; a second layer defining a second recessed portion 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 being configured to receive a fluid, the first layer and the second layer being configured to allow UV light to pass through the UV measurement chamber; and UV blocking features are positioned on or in the first layer, the UV blocking features being configured to limit the transmission of UV light through or backscattering from the first layer.

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

87. The box device of any one of claims 86 to 87, the first recessed portion having a first diameter, the UV blocking feature having an opening, the opening having a diameter smaller than the first diameter of the first recessed portion.

88. The box device of any one of claims 86 to 88, the UV blocking feature having a body positioned on the upper surface of the first layer, the first recess being formed through the upper surface of the first layer.

89. The box device according to claim 89, wherein the body has a disc shape.

90. The cartridge apparatus of any one of claims 86 to 90, said UV blocking feature having a cylindrical body positioned in said first recess.

91. The box device of any one of claims 86 to 91, wherein the UV blocking feature is configured to block UV light from being transmitted through the side walls of the first recess.

92. A method comprising: emitting ultraviolet (UV) light through an ultraviolet (UV) measurement chamber of the cartridge, the UV measurement chamber containing a fluid; receiving the UV light at a UV light detector, the received UV light having passed through the UV measurement chamber of the cartridge; obtaining a first plurality of concentration measurements, each concentration measurement of the first plurality of concentration measurements indicating a respective concentration of a 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, from among the first plurality of concentration measurements, outliers relative to the mean of the first plurality of concentration measurements; establishing a second plurality of concentration measurements, the second plurality of concentration measurements comprising the first plurality of concentration measurements with the identified outliers removed; calculating an average of the second plurality of concentration measurements; as well as The average of the second plurality of concentration measurements is reported.

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

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

95. The method of any one of claims 93 to 95, the identified outliers providing concentration measurements that deviate significantly from the mean of the first plurality of measurements due to the presence of bubbles in the fluid in the UV measurement chamber.

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

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