Apparatus and method for measuring DNA / RNA production in biochips

Abstract

Microfluidic devices (e.g., systems, apparatuses, etc.) and microfluidic methods provide for detection of polynucleotide concentrations. These devices can include removable, single-use, or reusable microfluidic components that can 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 components can be microfluidic cartridges that can be inserted, held, and / or seated in a microfluidic driver device that can oversee and control operations within one or more cartridges based in part on concentrations of detected polynucleotides.

Description

[0001] Claiming priority

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

[0003] References merged

[0004] All publications and patent applications mentioned in this specification are incorporated herein by reference in full as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Background Technology

[0005] UV spectrophotometry is a standard technique for nucleic acid quantification. In UV spectrophotometry, the 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 along a specified path length, and the absorbance of the sample at a specific wavelength is measured. The absorbance at 260 nm (A260) is commonly 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 approximately 260 nm. The maximum absorbance of nucleic acids occurs at a wavelength of 260 nm. The intensity of this absorbance is proportional to the concentration of the nucleic acid. Due to these physical properties, the nucleic acid concentration of a sample can be determined. Based on the absorbance reading, the concentration of the sample can be determined. The purity of the sample can also be estimated by comparing it with the absorbance at 280 nm or 230 nm.

[0006] However, traditional UV spectrophotometric assays for polynucleotide quantification typically require sample removal and transfer to a dedicated spectrophotometer. Typically, the minimum sample volume must be at least 1 μL to 75 μL to obtain accurate instrument readings. This can be problematic if the sample is highly diluted or if the extraction technique requires eluting nucleic acids to a low volume. Contamination is also an issue. For example, if measuring DNA, biomolecules from the extraction process such as proteins, RNA, and ionizing salts can erroneously increase the nucleic acid concentration estimate. Buffer salts such as Tris, EDTA, and guanidine isothiocyanate absorb strongly at 230 nm and diffuse into the 260 nm absorbance range. Additionally, free nucleotides present in the sample can affect UV quantification methods. Changes in sample pH also alter UV readings. An alternative to spectrophotometers is to add fluorescent probes and measure the sample in a fluorometer, which requires further processing of the RNA / DNA sample and labeling such molecules with fluorescent molecules, increasing cost and complexity. All of these methods have major drawbacks and may require manual intervention and output measurements not associated with the system's microfluidic control.

[0007] This paper describes a device that can address these shortcomings, specifically a microfluidic device. Summary of the Invention

[0008] This document describes microfluidic devices (e.g., systems, apparatuses, etc.) and methods for microfluidic detection of polynucleotide concentrations. These devices may include removable, single-use, or reusable microfluidic components, which may include one or more channels, chambers, and / or substrates for performing microfluidic manipulation. For example, the microfluidic component may be a microfluidic cassette, a microfluidic chip (“biochip”), etc., and may be referred to herein as a “cassette.” Any of these cassettes may be a generally flat planar structure that can be inserted, held, and / or disposed within a microfluidic actuator device that can monitor and control operation within one or more cassettes. The microfluidic actuator device may be referred to herein as a microfluidic path device and may include actuators, sensors, controllers (including control circuitry, processors, etc.). As will be described in detail below, the cassette may be adapted to include one or more polynucleotide quantification regions adapted for optical sensing and quantification of polynucleotides such as DNA, RNA (e.g., mRNA), etc. This polynucleotide quantification region is configured for spectrophotometric readings via an emitter and detector, which can be part of a microfluidic actuator device. The microfluidic actuator device can also be adapted to control the activity of the microfluidic actuator device and cartridge based on feedback from the spectrophotometric readings.

[0009] For example, the devices and methods described herein can be configured to quantify RNA and / or DNA generated within a microfluidic device (e.g., within a cartridge driven by a microfluidic actuator) via an exponential biochemical reaction. These devices can alter or control and regulate the output to correct for variations caused by reagent, biochip, or temperature defects. Specifically, the methods and devices described herein can microfluidically measure the concentration of RNA and / or DNA generated when the device is operated and can control the operation of the device during the preparation of therapeutic mRNAs (including, but not limited to, mRNA vaccines). These methods and devices enable the confirmation of success of various amplification and synthesis steps, and the normalization of multiple batches of output. This also allows for the reuse and optimization of the cartridge. Repetition of synthesis steps within a cartridge can produce discrete outputs that traditionally require detailed quality analysis for release. Alternatively, the methods and devices described herein can allow output to be pooled into a single batch, thus requiring only a single quality analysis process and also simplifying manual sample collection and sorting. If the concentration of the RNA / DNA product measured in each repetition is within the 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 detection" or "waste" container. This measurement-determined transfer can be operator-driven or automatically determined by the microfluidic system.

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

[0011] A stable output eliminates the measurement and dilution / concentration steps between DNA / RNA generation and the next step in the therapeutic material production process. To stabilize the output, the methods and apparatus described herein allow for the selection of low target concentrations and dilution of the system's output, thus keeping it within a more controlled range. To automate this dilution, we need to know the initial concentration of the DNA / RNA and use it to determine the amount of diluent to be added.

[0012] The methods and apparatus described herein also allow for iterative improvements to automated microfluidic RNA / DNA production for better control. For example, once a process performed by the apparatus (e.g., in a cartridge) has proven to be under tight control with regular and consistent output concentrations, the product can be released without the need for detailed analysis. Instead, a “parameter release” can be performed, where measures such as temperature, reagent consumption, and intermediate RNA / DNA concentration are used to demonstrate that the process has nominally occurred.

[0013] Generally, this document describes a cassette (“biochip”) particularly suitable for allowing automated or semi-automated systems (e.g., microfluidic actuators) to determine the concentration of a therapeutic polynucleotide being processed (e.g., formed, mixed, synthesized) within the cassette, and / or to modify the operation of the microfluidic actuator on the cassette based on the determined concentration. For example, this document describes a cassette (e.g., a cassette device) for processing polynucleotides, comprising: a first layer having a first thickness; a second layer having a second thickness; an elastic material extending between the first and second layers and separating the first and second layers by a separation thickness; and a UV measurement region formed through the first and second layers, 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 the regions of the first and second layers in the UV measurement region to form a UV measurement chamber with separation thicknesses. Therapeutic polynucleotides may refer to solutions of polynucleotides (e.g., aqueous solutions).

[0014] Any of these boxes can be formed from polymeric materials such as cyclic olefin copolymers (COC) or cyclic olefin polymers (COP). Although these materials are not very UV-transmitting and their UV transmission properties can (as surprisingly found herein) change with UV exposure, the methods and apparatus described herein can be adapted or modified to allow for sensitive and accurate estimation of UV absorption, and thus allow for sensitive and accurate estimation of the concentration of polynucleotides (e.g., therapeutic polynucleotides). The first and second layers can be formed from the same or different materials. For example, the second layer may contain COC and / or COP materials.

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

[0016] An elastic material, or multiple sheets of elastic material, may be sandwiched between the first and second layers (including 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, or may be adherently 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 approximately the same as the separation thickness because the UV sensing chamber can be formed in the space between the first and second layers where no elastic material exists. The elastic material can therefore surround the UV sensing chamber (on its lateral sides). The box can have one or more UV sensing chambers. In some examples, the UV sensing chambers are in a consistent location across various box designs or configurations.

[0018] Generally, any of these boxes may include one or more fluid channels in fluid communication with the UV measurement chamber within the second layer. The fluid channels may be formed by milling, cutting, or otherwise machining 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 within the second layer. The second layer may be formed from multiple sublayers.

[0019] The cassette device may also include a plurality of pneumatic valves arranged to control flow into and out of the UV measurement chamber. These pneumatic valves may each be formed from a pneumatic chamber in a first layer and a fluid chamber in a second layer; portions or areas of an elastic material separating the pneumatic chamber from the fluid chamber may move up / down to open / close (or remain open in an intermediate position) the fluid chamber, which may be in fluid communication with the fluid passage and / or other fluid chambers within the cassette. The pneumatic chamber of each valve may be in fluid communication with a pneumatic passage in the first layer, which is configured to communicate with a pressure port on an external area of ​​the device to actuate the pneumatic valve (e.g., to move that area 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 a fluid passage in the second layer (e.g., such that fluid can flow between the fluid chamber and the UV measurement chamber when the pneumatic valve is open).

[0020] Any of these boxes may include an in vitro transcription (IVT) chamber that is at least partially formed within the second layer and is in fluid communication with the UV measurement chamber. These boxes may also include one or more mixing chambers (mixers), reaction chambers, etc. Generally, the microfluidic actuator 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, a cartridge device may include one or more vacuum ports on an external area of ​​the device, which 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 containing a therapeutic polynucleotide), the resulting negative pressure can draw the therapeutic polynucleotide into the UV measurement chamber.

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

[0023] Any of these boxes can also be configured for continuous mixing of solutions. For example, any of these boxes may include a further dilution mixing chamber in fluid communication with the UV measurement chamber, which is configured to dilute the fluid sample.

[0024] In any of these examples, the box can be configured to prevent the presence of air bubbles in the sensing region of the UV measurement chamber (so that they will not interfere with the UV detection of the therapeutic nucleotide absorption signal). 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 thinner regions of the first and second layers). Therefore, the diameter of the UV measurement chamber may be larger than the diameter of the region of the first layer in the UV measurement region (and larger than the diameter of the region of the second layer in the UV measurement region) to prevent air bubbles in the central region of the UV measurement chamber.

[0025] For example, a cassette device for processing polynucleotides may include: a first layer comprising a polymeric material having a first thickness; a second layer comprising a polymeric material having a second thickness; an elastic material extending between the first and second layers; a UV measurement region formed through the first and second layers, wherein a region of the first layer in the UV measurement region has a thickness less than or equal to the first thickness, a region of the second layer in the UV measurement region has a thickness less than or equal to the second thickness, and wherein the elastic material is removed between the region of the first layer in the UV measurement region and the region of the second layer in the UV measurement region to form a UV measurement chamber; and an in vitro transcription (IVT) chamber formed at least partially within the second layer and in fluid communication with the UV measurement chamber.

[0026] This document also describes methods for using any of these cartridges having any of the microfluidic actuator devices described herein. Generally, these methods involve on-cassette (“closed path”) detection and determination of the concentration of the therapeutic polynucleotide. Any of these methods may also include techniques for optimizing or enhancing detection, including adjusting one or more parameters of the microfluidic actuator device based on feedback from the detection concentration of the therapeutic polynucleotide (e.g., from the UV absorption signal of the detection) or from a differential signal between the UV absorption signal of the therapeutic polynucleotide signal and one or more blanks.

[0027] For example, this document describes a method for manufacturing polynucleotides using a microfluidic actuator device operated on a cartridge, the method comprising: forming a therapeutic polynucleotide in a cartridge; and determining the concentration of the therapeutic polynucleotide by the microfluidic actuator device through the following steps: driving a first blank solution into the 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; 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 may include adjusting the operation of the microfluidic driver device based on an estimated concentration of the therapeutic polynucleotide in the processor of the microfluidic driver device. For example, adjustment may include directing the resulting therapeutic polynucleotide into a pool for additional therapeutic polynucleotides. Additional therapeutic polynucleotides may also be formed in the cassette. In some examples, adjusting the operation of the microfluidic driver device may 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 may include comparing an estimated concentration of a therapeutic polynucleotide to a concentration range via a processor of a microfluidic actuator, and based on that comparison, directing the therapeutic polynucleotide for one of the following: collection, disposal, or further analysis with one or more additional batches of therapeutic polynucleotides formed in the cassette.

[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 a microfluidic actuator. Thus, the microfluidic actuator (e.g., a controller of the microfluidic actuator) can use an estimate of the measured concentration and / or volume (which may be, for example, an optically sensed and / or cell-based chamber and / or channel known volume) to add fluid to achieve a 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 the therapeutic polynucleotide formed via a microfluidic actuator based on an 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 volume and / or time, etc.

[0032] Generally, the apparatus and methods described herein can be configured to automatically adjust to allow for accurate concentration determination. For example, the methods and apparatus described herein can be configured to prevent saturation (e.g., where minimal UV light is absorbed by the therapeutic polynucleotide), increase excessive concentration, reduce sensitivity, or decrease background noise, such as the detection limit where noise is absorbed by the walls (first and / or second layers) of the UV measurement chamber. For example, adjusting the light power, detector sensitivity, or, in some methods and apparatus, performing serial dilution of the therapeutic polynucleotide to generate a graph of absorption by the diluent. Therefore, any of these methods can include determining the concentration of the therapeutic polynucleotide, which may include performing serial dilution under the control of a microfluidic actuator in one or more chambers of a cartridge, and repeating the step of driving a sample solution of the therapeutic polynucleotide for each of the one or more diluents formed by the serial dilution to generate a dilution curve. Furthermore, 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 may include altering one or more of the sensitivity of the UV detector and the intensity of the UV emitter (e.g., a UV light source) of the microfluidic actuator in response to a second absorption measurement via the cartridge. The method or apparatus may include automatically adjusting one or more of the UV detector sensitivity and the UV emitter intensity based on an absorption measurement of a sample containing the therapeutic polynucleotide. Therefore, any of these apparatuses and methods may automatically, semi-automatically (e.g., using user input), and / or manually adjust the LED intensity based on detector readings. In some examples, the apparatus of the method may adjust the LED intensity and / or detector sensitivity during measurement (in operation), which may extend the dynamic range.

[0034] Any method described herein may include pretreating the UV measurement chamber with UV light prior to performing the first absorption measurement through the chamber. As described herein, it has been surprisingly found that the absorption profile of the chamber-forming material varies over time with the amount of UV light applied. Surprisingly, the absorption of the chamber material (e.g., COC, COP, and other polymers) can actually decrease initially (e.g., within the first 60 minutes, 55 minutes, 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, etc.), resulting in an increase in the detection signal. Since readings from a particular chamber may be short (e.g., within 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.), pretreating the material to provide readings with a larger detection signal may be beneficial. Furthermore, the absorption of a material can increase with further γ irradiation, so the methods and apparatus described herein may include features that improve or increase transmission through the material, for example, by thinning the material in the UV measurement region to improve UV light transmission.

[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 the IVT reaction in a cassette. In any of these methods, the therapeutic polynucleotide comprises therapeutic mRNA. Any of these methods may further include encapsulating the therapeutic mRNA in a cassette using a delivery vector.

[0036] Generally, a microfluidic actuator can supervise the driving of a first sample (e.g., a blank), a second sample (e.g., the sample of interest), and a third sample (e.g., a second blank) by pneumatically deflecting one or more regions of the membrane of the cartridge to open and / or close the cartridge's valves due to higher pressure on the blank or sample fluid and / or by creating a vacuum in the UV measurement chamber before opening the valve. For example, the method may include causing the microfluidic actuator to pneumatically deflect one or more regions of the 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 that operates on a cartridge may include: forming a therapeutic polynucleotide in a cartridge, wherein the therapeutic polynucleotide comprises therapeutic mRNA; determining the concentration of the therapeutic mRNA by the microfluidic actuator device through the following steps: driving a first blank solution into the 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 first absorption measurement through the cartridge; 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 in a processor of the microfluidic actuator device based on the estimated concentration of the therapeutic polynucleotide.

[0038] This document also describes apparatus (e.g., systems) configured to perform these methods, which may include microfluidic actuators. Any of these systems may include software, hardware, and / or firmware for performing these methods. Specifically, any of these systems may include a controller having control circuitry (including one or more processors) for coordinating the actions described herein (and performing these methods), specifically including estimating the concentration of therapeutic polynucleotides. Any of these controllers may include a non-transitory computer-readable storage medium storing a set of instructions executable by the system.

[0039] For example, this article describes 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 cartridge mount; multiple pressure lines; multiple 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 or outlet port held on a cartridge in the mounting assembly; an ultraviolet (UV) light source; a UV photodetector; and a controller configured to control the application of pressure through the pressure lines to drive fluid through the cartridge, wherein the controller is also configured to guide the formation of a therapeutic polynucleotide in the cartridge and to estimate the concentration of the therapeutic polynucleotide by: driving a first blank solution through the UV measurement chamber of the cartridge and performing a first absorption measurement using a UV light source and a UV photodetector; driving a sample solution containing the therapeutic polynucleotide through the UV measurement chamber of the cartridge and performing a second absorption measurement using a UV light source and a UV photodetector; and driving a second blank solution through the UV measurement chamber of the cartridge and performing a third absorption measurement using a UV light source and a UV photodetector, 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] The controller can also be configured to modify system operation based on the estimated concentration of the therapeutic polynucleotide. For example, the controller can also be configured to compare the estimated concentration of the therapeutic polynucleotide to a concentration range and, based on that comparison, direct the therapeutic polynucleotide for one of the following: collection with one or more additional batches of therapeutic polynucleotide formed in the cassette, disposal, or further analysis. The controller can be configured to dilute the therapeutic polynucleotide to a standard concentration for output. In some examples, the controller is configured to automatically adjust one or more parameters of the therapeutic polynucleotide formation based on the estimated concentration of the therapeutic polynucleotide. These one or more parameters may 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 dilutions in one or more chambers of the cartridge, and to repeat the steps of driving the sample solution of the therapeutic polynucleotide for each of one or more diluents of the sample solution of the therapeutic polynucleotide formed by the serial dilutions to generate a dilution profile. The controller can be configured to estimate the concentration of the therapeutic polynucleotide from the dilution profile. Alternatively or additionally, 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 a second absorption measurement of the cartridge.

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

[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 also configured to generate a template for the IVT reaction within the cassette. Generally, the therapeutic polynucleotide can be a therapeutic mRNA. The controller can be configured to encapsulate all or part of the therapeutic mRNA with a delivery vector.

[0044] The controller can also be configured to pneumatically deflect one or more regions of the membrane of the cell to open and / or close the cell's valves when driving the first blank, the second blank, and the third blank. In some examples, the controller is also configured to pneumatically deflect one or more regions of the membrane of the cell 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 can be applied by providing pressure on the upstream sample and blank lines or by vacuum suction (e.g., "absorbing") the sample material (e.g., the sample of interest and / or the blank sample) in the LTV chamber within the UV chamber.

[0045] Generally, the system described herein can be configured to include one or more cartridges and a microfluidic actuator. For example, the system may include: a cartridge; and a microfluidic actuator comprising: a cartridge mount; multiple pressure lines; multiple fluid lines, each fluid line being 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 fixed to an inlet or outlet port held on a cartridge in the mounting; an ultraviolet (UV) light source; a UV photodetector; and a controller configured to control the application of pressure through the pressure lines to drive fluid through the cartridge, wherein the controller is further configured to guide the formation of therapeutic polynucleotides, further wherein... The controller is configured to estimate the concentration of a therapeutic polynucleotide by: driving a first blank solution through the UV measurement chamber of the cartridge 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 cartridge and performing a second absorption measurement using a UV light source and a UV light receiver; driving a second blank solution through the UV measurement chamber of the cartridge and performing a third absorption measurement using a UV light source and a UV light receiver; and modifying the operation of the microfluidic actuator based on the concentration of the therapeutic polynucleotide determined from the first, second, and third absorption measurements.

[0046] All methods and apparatuses described herein are considered in any combination and can be used to achieve the benefits described herein. Attached Figure Description

[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 of exemplary embodiments and the accompanying drawings, in which:

[0048] Figure 1 The illustration schematically demonstrates UV measurement of polynucleotide material within the UV measurement region of the cartridge using a microfluidic actuator comprising a UV light source and a UV detector.

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

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

[0051] Figures 3A to 3C An example of a cartridge for a microfluidic actuator device as described herein is illustrated.

[0052] Figure 4A It is a cross-section of a portion of an example of a box used with a microfluidic actuator.

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

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

[0055] Figure 6 This is a graph showing the UV transmittance of the material forming the exemplary box before and after γ-irradiation.

[0056] Figure 7 This is a graph showing the change in the detection signal over time for an exemplary material of the box under prolonged UV exposure.

[0057] Figure 8 This is an example of a dilution curve (graph) as described in this article.

[0058] Figure 9 An example of a portion of the box that includes the UV measurement area is illustrated schematically.

[0059] Figure 10A This is a top view of an example of a box that may include a UV measurement area for a microfluidic actuator.

[0060] Figure 10B This is an example of a cross-section of an area passing through a box that includes the UV measurement area.

[0061] Figure 10C An example of a portion of a cartridge for a microfluidic actuator is shown, schematically illustrating a vacuum cover for removing air bubbles.

[0062] Figure 10D This is a top view of an example of a box (“biochip”) including a UV measurement chamber.

[0063] Figure 10E This is another example of a top view of a box that includes a UV measurement chamber.

[0064] Figures 11A to 11B A partial view of the box, including the UV measurement area, is shown.

[0065] Figure 12 B is Figure 11A A magnified view of the UV measurement area.

[0066] Figure 12 The diagram illustrates the treatment of polynucleotides and includes a method using UV absorption measurements, which can be used to estimate the concentration of polynucleotides and / or to modify the treatment of polynucleotides.

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

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

[0069] Figure 15A and Figure 15B This shows the intensity adjustment range of an example UV light source. Figure 15A ) and the resulting light intensity ( Figure 15B The curve graph of ).

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

[0071] Figure 17 A top plan view depicts another example of a box including a UV measurement chamber.

[0072] Figure 18 Depicting Figure 17 Enlarged top plan view of the box area.

[0073] Figure 19 A flowchart illustrating examples of methods that can be used to assess the limit of quantitation is shown.

[0074] Figure 20 A top plan view depicting an example of a backscatter shield.

[0075] Figure 21 Depicting the process during UV measurement, including having Figure 20 A cross-sectional side view of the arrangement of the backscattering shielding box.

[0076] Figure 22 A cross-sectional side view schematic depicts the arrangement of a box including another example of a backscatter shield during a UV measurement process.

[0077] Figure 23 A flowchart illustrating an example of a method that can be used to consider bubbles in a fluid within a UV measurement chamber is shown. Detailed Implementation

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

[0079] These methods and devices can be used as part of an integrated closed-path system to measure the concentration of RNA or DNA, and the resulting concentrations can be used to modify peptide treatment. Light absorption, and specifically ultraviolet (UV) transmission through the UV measurement region of the closed box, can be used to estimate the concentration of polynucleotides within the closed box. The box can be adapted to have a narrow path length and optimize the signal from the polynucleotide sample's absorption of LTV light. Microfluidic actuators for operation on these boxes are also described herein. These systems can control operations within the closed box for the formation and / or treatment (e.g., synthesis) of therapeutic polynucleotides, including determining the concentration of therapeutic polynucleotides at various stages of treatment within the box, and also include using concentration data, directly or based on LTV absorption data proportional to the concentration of the therapeutic polynucleotides, to control the operation of the microfluidic actuator on the box.

[0080] Generally, the methods and apparatus described herein can use UV absorbance techniques to measure the concentration of polynucleotides (e.g., RNA and / or DNA) on a cartridge. These techniques can compare the level (in some examples, ratio) of transmitted UV light at wavelengths between about 250 nm and 270 nm (e.g., centered at about 260 nm) from a polynucleotide sample (e.g., a sample of therapeutic polynucleotides) with 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., the sample or blank), the apparatus or method can determine the concentration of the polynucleotides.

[0081] These devices (and related methods) are particularly compact and can use readily available and easily manufactured materials, including polymeric materials that absorb UV light themselves (e.g., plastics). These methods and devices also allow for tight control over path lengths, and the path lengths (“critical path lengths”) 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.), allowing sampling of very small volumes while achieving high accuracy and extended dynamic range. These methods and devices can also be adapted to a large dynamic range of concentration and noise levels by adjusting sample dilution 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, these methods and devices can use disposable or limited-use cartridges, as well as durable / reusable microfluidic actuators that operate on the cartridges. However, any of these techniques can be adapted for use with reusable cartridges and / or cartridge-free systems that integrate features of the cartridge into the microfluidic actuator device.

[0082] In any of these methods and apparatuses, a sample (e.g., a polynucleotide sample) can be microfluidically driven into a UV measurement chamber that minimizes the amount of UV-absorbing material (e.g., plastic) in the light path while still being made of a relatively UV-absorbing material. The measurement chamber can be configured with a critical path length for light through the sample, which can be fixed. When the chamber is positioned within the microfluidic actuator, the UV light source and detector can be positioned on either side of the measurement chamber. The light path can be perpendicular to the chamber. For example, Figure 1 An example of a microfluidic actuator device 102 operating on a housing 105 is schematically illustrated. The microfluidic actuator device includes a UV light source (e.g., a UV LED 101) positioned above the housing 105 and a UV detector 103 positioned directly below the housing. Alternatively or otherwise, an optical fiber may 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 the measurement chamber 107. In any of these examples, the light source and detector may be spaced apart such that the beam (which is conical in some examples, but may be collimated) interrogates only a defined area of ​​the UV measurement region, allowing the sample volume in a short optical path to be known by the system.

[0083] Device

[0084] The methods described herein can generally be performed using devices that can be used with one or more cartridges (e.g., biochips) and / or include one or more cartridges, as well as systems configured to control operation within the cartridges (e.g., microfluidic control systems). These microfluidic control systems may be referred to herein as microfluidic devices, microfluidic control devices, microfluidic actuator device control systems, microfluidic control systems, or microfluidic systems. The cartridges may be housed within the microfluidic control system and may operate in a closed-path manner, preventing some, or more preferably almost all, or all, components of the fabricated parts within the system from being exposed to the atmosphere. Specifically, the portions of the device that contact the fluid within the system are prevented from being exposed to the atmosphere. Figure 2A An example of a microfluidic actuator control system is shown, comprising: a microfluidic actuator management system 203 (including hardware for holding the cartridge, applying positive / negative pressure to operate the microfluidic actuator within the cartridge, heating / cooling all or a region of the microfluidic actuator, 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 Class 5 cabinet). The system may use or may include one or more cartridges 201. These cartridges may be any of those described herein, wherein any cartridge may include one or more UV measurement areas.

[0085] Microfluidic devices can be used to form therapeutic polynucleotides (e.g., therapeutic mRNA). The device may include: a mount for removably holding a cassette; multiple pressure lines; multiple fluid vials, each vial including or configured to be coupled to a fluid line, wherein each fluid line and at least a subset of the pressure lines are configured to be biased against a cassette held in the mount to form a closed fluid path; and a controller configured to control pressure application through the pressure lines to drive fluid movement within the cassette while the cassette is held in the mount, wherein the controller is configured to guide the synthesis of a synthetic template, guide an in vitro transcription (IVT) reaction using the template to form a therapeutic polynucleotide, and estimate the concentration of the therapeutic polynucleotide, and / or guide the purification of the therapeutic polynucleotide held in one or more cassettes in the mount. Any of these devices may include an optical or UV concentration detection subsystem comprising one or more UV light sources and one or more UV detectors configured to be positioned above the UV detection area of ​​the cartridge when the cartridge is held or placed in the device.

[0086] Microfluidic devices (e.g., microfluidic devices for forming therapeutic polynucleotides such as therapeutic mRNA) may include: a mount (e.g., a placement mount) for removably holding a cassette; multiple pressure lines; multiple fluid vials, each of which includes 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 be biased against the cassette held in the placement mount to form a closed fluid path; and a controller configured to control pressure applied through the pressure lines to drive fluid movement within the cassette while the cassette is held in the placement mount, wherein the controller is configured to determine the contents of the fluid vials, transfer sub-microliter amounts of material from the fluid vials to one or more reactors held in the cassette in the placement mount, guide the synthesis of a synthetic template, use the template to guide an in vitro transcription (IVT) reaction to form a therapeutic polynucleotide, guide the purification of the therapeutic polynucleotide in one or more microfluidic actuators held 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 can be configured to perform any of the methods described herein, specifically 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 fluid in the microfluidic actuator, the temperature (including thermal cycling) of various regions of the microfluidic actuator, flushing / combination, the opening / closing of valves in the microfluidic actuator, detection of the microfluidic actuator, etc. The controller may include one or more microprocessors, communication circuitry, 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 around the mounting bracket and reagent storage rack to monitor fluid levels within the reagent storage rack and fluid movement within the microfluidic actuator when the microfluidic actuator is mounted in the mounting bracket.

[0089] Alternatively or otherwise, an optical sensor may be located on the bottom of the device (e.g., below the mounting bracket) and may be pointed upwards to detect fluid volume, movement, etc.

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

[0091] The described methods and apparatus typically include one or more fluid dynamic loops for moving material (liquid material) between or within a fluid chamber (reservoir, liquid contact side, reactor, etc.) and a channel of a microfluidic actuator, and in some cases, between the microfluidic actuator and a fluid reservoir (vial, bottle, container, etc.) within the apparatus. The fluid dynamic loop can be a hydraulic or pneumatic loop, which may include the microfluidic device and, specifically, one or more pressure channels and pressure receiving sides of the chambers within the microfluidic device. The fluid dynamic loop may also be referred to as a microfluidic loop. A single microfluidic chip may include multiple fluid dynamic loops; the fluid dynamic loop may also include one or more pressure lines and interfaces between the pressure lines of the microfluidic control device and one or more microfluidic chips within the microfluidic actuator. One or more fluid dynamic loops may share components (valves, pressure lines, vacuum caps, etc.) with other overlapping fluid dynamic loops. Furthermore, for convenience, it should be understood that when the term "pneumatic" is used, a general fluid dynamic loop (e.g., hydraulic and / or pneumatic) may be used alternatively or additionally. The fluid material driven by the fluid power pipeline can be any suitable fluid (e.g., gas or liquid, such as air, water, oil, etc.).

[0092] This document also describes cassettes (e.g., closed-path microfluidic actuators) for processing therapeutic polynucleotides in closed pathways. As mentioned herein, these cassettes may be referred to herein as microfluidic chips, microfluidic pathway plates, processing chips, biochips, processing plates, etc. Generally, the cassettes can be generally flat, plate-like structures; these structures can be relatively thin (e.g., less than a few millimeters thick, such as between about 0.5 mm and about 20 mm, between about 0.5 mm and about 15 mm, between about 0.5 mm and about 10 mm, etc.). The cassettes described herein are typically at least partially transparent, and specifically, the top of the cassette can be transparent, allowing one or more optical sensors (cameras, CCDs, optical fibers, etc.) to be used to sense, detect, monitor, record, etc., actions, including fluid movement and / or movement of elastic layers, when the microfluidic actuator is used by the microfluidic device described herein.

[0093] Figure 2B This is a schematic diagram of an 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 encapsulates a mounting 215 for holding one or more cartridges 211, which may be a single-use device. The housing may be a chamber, encapsulation, etc., and may include a lid or opening; it may be sealed when closed. The housing may encapsulate a thermal regulator and / or may be configured to be encapsulated in a thermally regulated environment (such as a refrigeration unit). The housing may form a sterile barrier, and in some examples, the housing may form a humid or humidity-controlled environment.

[0094] Mounting element 215 may be configured to secure the box using one or more pins or other components configured to hold the box in a fixed and predefined orientation.

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

[0096] exist Figure 2BIn this device, the fluid interface assembly 209 connects liquid reagents and / or pressure (e.g., gas) to a cartridge 211 held within the mounting bracket 215, and can assist in the delivery of fluid materials, as well as positive / negative gas pressure, from the pressure source 217 into the interior of the cartridge 211. The fluid interface assembly may optionally assist in securing the cartridge, as described in more detail below. The fluid interface assembly may be removably coupled to the device (and may be removed or partially removed) for sterilization between uses.

[0097] The reagent storage rack 207 may be configured to include a plurality of fluid sample holders, each of which may hold a vial configured to hold reagents (e.g., nucleotides, solvents, water, etc.) for delivery to cartridge 211, or alternatively, the vials may be configured to receive products from inside cartridge 211. The reagent storage rack may be referred to as a reagent rack. In some examples, the reagent rack includes multiple pressure lines and / or manifolds configured to branch one or more pressure sources 217 into multiple pressure lines that may be applied to the cartridge and may be controlled independently or collectively (in sub-combinations). Alternatively, the fluid reservoir (vias, etc.) may be configured to be directly secured and sealed against the cartridge.

[0098] The fluid interface assembly may include multiple fluid lines and / or pressure lines and may include biased (e.g., spring-loaded) retainers or ends that individually and independently drive each fluid line and / or pressure line to the cassette when the cassette is held in mounting 215 (or, alternatively, the device may be directly spring-mounted, as mentioned). Conduits (e.g., fluid lines and / or pressure lines) may be part of the fluid interface assembly and / or may be connected to the fluid interface assembly. In some examples, the fluid lines include flexible conduits connected between the reagent storage rack and the cassette via a connector that engages the vial with a locking engagement (e.g., a collar). The ends of the fluid paths (in some examples, the ends of the fluid lines and pressure lines) may be configured to seal against the cassette, for example, at a sealing port formed in the cassette, as described herein. For example, the ends of the fluid lines may be cut or formed flat (vertical internal view). The vial can be pressurized (e.g., >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, the 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, the fluid vial can be driven at a lower pressure than the pneumatic valve, which prevents or reduces 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 can be coded (e.g., by an identifier that can be read by one or more sensors, as described below). The controller can monitor the fluid level and thus the amount of each material in the fluid interface assembly.

[0100] The device may also include a magnetic field applicator 219, which can be configured to generate a magnetic field in a region of the cartridge 211. One or more sensors 205, which may be optical sensors, may be part of the device and may sense one or more of the following: a barcode, fluid level in a vial maintained within a reagent storage rack, and fluid movement within the cartridge 211 when the device is mounted within the mounting 215.

[0101] Any of these devices may include a UV emission / detection subsystem (e.g., concentration estimation subsystem 233) capable of measuring the absorption of UV light (e.g., at approximately 260 nm) and controlling processes on the device based on these measurements, for example, by measuring one or more “blanks” without polynucleotides and comparing them to a sample with processed polynucleotides. In some examples, visual / optical labeling can be used to estimate yield. For example, fluorescence can be used to detect process yield or residual material by labeling with fluorophores. Alternatively or additionally, dynamic light scattering can be used to measure particle size distribution within a portion of a microfluidic actuator device (e.g., a mixing section). In some examples, sensor measurements can be performed using one or two optical fibers to transmit and detect optical signals of light (e.g., laser light). The instrument package may be mounted remotely from the device. This non-contact sensing may be preferred.

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

[0103] The internal portion of the device (e.g., within housing 233) may also be configured to be sterilizable. Specifically, portions of the device may be removed and sterilized individually. Sterilization may 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, may be housed in a high-efficiency particulate air (HEPA) filtered environment. The device, including the housing, may be housed within a temperature-controlled package. Additionally, the device itself may include one or more temperature-controlled areas. In any of the devices described herein, the device may (e.g., within the housing) include a temperature-controlled area for storing reagents and / or for storing mRNA (e.g., therapeutic mRNA) at storage temperatures (e.g., temperatures between about -10°C and about 20°C, such as about 10°C, about 4°C, about -10°C, etc.). Any of these devices may include a library of manufactured mRNAs, which may be synthesized alone 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 controller 221, including applying pressure through housing 211 to at least drive fluid movement. The controller may be wholly or partially external to the housing. The controller can be configured to include user inputs / outputs. For example, the system's user interface 223 may allow for easy operation and guidance of the device and housing.

[0105] Any of the devices described herein may include Figure 2B All or some of the components shown; not all components are required. Figure 2B The diagram shows only some of the connections between components; additional (or alternative) connections may be used.

[0106] The microfluidic actuator control system can support all production activities within the microfluidic actuator, such as reagent supply, fluid control, temperature control, mixing, purification, and process monitoring. Manufacturing activities on the microfluidic actuator control system can be accessed and controlled via application software.

[0107] The cartridge can be configured to include one or more reactors for manufacturing operations performed to precisely prepare therapeutic (e.g., therapeutic mRNA) materials. The same microfluidic actuator can operate on one or more cartridges connected in series and / or parallel without disrupting the continuous path nature of the microfluidic actuator control system. For example, when manufacturing a therapeutic agent using multiple processing operations performed in multiple reactors using multiple cartridges, fluid products (including portions of the product from one cartridge) can be transferred via the device in a closed-path manner to one or more additional cartridges, including by moving fluid containing the cartridge product into a storage reservoir portion of the microfluidic actuator control device.

[0108] Each box can be configured to include one or more reactors for processing during the manufacturing process. For example, Figures 3A to 3C Three examples of boxes are shown. These examples illustrate three different types of boxes: template boxes ( Figure 3A ), In vitro transcription (IVT) cassette ( Figure 3B ) and formulation box ( Figure 3C Each of these box examples can be constructed to include features for performing a set of unit operations in a controlled and highly reproducible manner.

[0109] In some examples, the box can be constructed as a multi-layered structure consisting of two more rigid layers and a flexible membrane sandwiched between the two ridged layers. Figure 4AA cross-sectional view (transverse to the plane of the box) through an example of a box having multiple layers forming a reactor for processing therapeutic agents as described herein is illustrated. The reactor may include seals, channels, valves, and chambers including a pumping chamber formed by multiple layers. For example, the box may be formed of two or more rigid or semi-rigid plates 403, 405 and at least one elastic layer 407. The elastic layer 407 may be a sheet of elastic material that is impermeable to liquids. The elastic layer may be slightly gas-permeable, or may be treated to be more or less gas-permeable (including in various regions). Although a single continuous sheet of elastic material may be used, in some examples, multiple sheets of elastic material may be used, or a “sheet” may be formed from segments of multiple sheets. These layers and elastic sheets may be laminated together. Generally, a chamber for retaining, valving, and / or pumping fluid may be formed in the plate on either side of the elastic layer, such that the elastic layer divides the chamber into a liquid-containing side and a pressure (e.g., gas)-applying side. The total volume of the chambers can be constant and can be formed in both a first (e.g., upper) plate and a second (e.g., lower) plate, but this 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 deform to reduce (decrease 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 the fluid port and the pressure port can be formed by openings leading to the upper plate (upper layer) 403 and the elastic layer 407, thus allowing for an atmospheric-isolated sealed connection even when multiple different input lines are present, as the pressure line is pushed into the elastic layer 407 supported on the underside of the port by the opposing rigid or semi-rigid layer 405.

[0110] exist Figure 4A In this case, 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., a 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 may not be proportional (e.g., the elastic layer 407 may be thinner relative to the plate).

[0111] Figure 4A The illustrated box 400 may further include multiple chambers 415, 416, 420, each with a fixed volume. These chambers are formed by cut-out areas (e.g., circular / curved cuts) in the second (bottom) surface 429 of the first plate 403 and the first (upper) surface 431 of the second plate 405; an elastic layer 407 bifurcates these chambers 415 such that each chamber includes a liquid-containing side 417 and a pressure (e.g., gas-containing) side 419. The box 400 may also include multiple liquid (e.g., fluid) channels. Figure 4A In the diagram, a single fluid channel 421 is shown extending from the thickness of the first plate 403 to the fluid port 423 of the fluid channel opening 425, through the elastic layer 407, and through most of the thickness of the second plate 405, down to the bottom region 433 of the second plate, where the length of the liquid channel 421 extending parallel to the bottom surface of the third plate is formed.

[0112] Regarding fluid port 423, the diameter of the opening of fluid port 423 extending through the thickness of the first plate 403 can be larger than the diameter of fluid channel opening 425 extending through the elastic layer 407 and into liquid (e.g., fluid) channel 421. Fluid channel opening 425 can be centered relative to the bottom of fluid port opening and can be offset from the wall of 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.

[0113] Fluid passage 421 is connected to the liquid-containing 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 the movement of the elastic layer 407.

[0114] The box 400 also includes multiple pressure channels, which can be independently controlled to apply positive and / or negative pressure. Figure 4A The diagram shows a single pressure port 443 connected to a fourth chamber 420, but each of chambers 415, 416 can be connected to a separate pressure port and pressure channel for independent operation and control of the movement of the bisecting portions of the resilient layer 407, for independent valve adjustment and / or pumping of each chamber. In some examples, the pressure port may be shared among multiple chambers. Figure 4AIn this design, a pressure (e.g., gas) port 443 is similar to a fluid (e.g., liquid) port 425 and includes an opening that extends completely through the first plate 403, down to the exposed elastic layer 407, and through the elastic layer to form a pressure (e.g., gas) passage opening 445. The pressure passage opening 445 is connected to a pressure (e.g., gas) passage 447, which extends from the pressure port 443 through a significant portion of the thickness of the first plate 403 and, along a passage in the second plate, returns upward through the second plate and the elastic layer 407 to a region within the first plate that connects to a pressure (e.g., gas) receiving portion 419 of the fourth chamber 420. As described for a similar fluid (e.g., liquid) port, the diameter of the pressure port 443 extending through the thickness of the first plate 403 may be larger than the diameter of the pressure passage opening 445 extending through the elastic layer 407, and the pressure port and the pressure passage opening may be concentric or offset beyond the wall thickness of the pressure line or pressure line connection interface to which the pressure port will be connected.

[0115] In passing Figure 4A In the cross-section of the box 400 shown, there are multiple connections to other fluid (e.g., liquid) lines, fluid ports, pressure lines, and pressure ports, which may be outside the plane shown. For example, in Figure 4A In this configuration, the liquid-containing side or portion 417 of the fourth chamber may be connected to additional valves (chambers) and / or channels, including, for example, an outlet channel extending from the liquid-containing side 417. Additional chambers (e.g., configured as valves), not shown, may be formed as described above. In some examples, the outlet channel may deliver fluid from one or more chambers through another fluid port (not shown) to a fluid receiving reservoir, such as a vial, tube, etc. This receiving reservoir may be held in a reagent storage rack.

[0116] Generally, the microfluidic actuator device and cartridge are constructed in such a way that multiple complex operations can be performed by the device on the cartridge in a fully encapsulated (sealed and protected from atmospheric influences) manner without manual intervention. Fluids can be metered using a fixed-volume chamber and moved, mixed, filtered, etc., by applying pneumatic pressure to deflect regions of the elastic layer.

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

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

[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 is typically 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. Figure 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 in contact with a vacuum via, for example, a vacuum line 1987 in the upper surface or upper plate of the device, such as... Figure 10CAs shown. In operation, the vacuum cover 1938 can remove or reduce air bubbles in the pipeline by holding fluid within the liquid contact side of the chamber and applying negative pressure on the upper (pressure receiving) side of the chamber. The membrane dividing the chamber into the liquid contact side and the pressure receiving side can be gas-permeable, such that the negative pressure can remove gas from the liquid (fluid) side by drawing 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, which is sufficiently gas-permeable to allow gas removal from the liquid side of the membrane. A fluid chamber having a fixed volume (e.g., formed between a first plate and a second plate) as described herein can include or be coupled to one or more bubble removal chambers (vacuum covers) and / or can be configured as a bubble removal chamber. In some examples, the portion of the elastic layer disposed between the first and second surfaces forming the chamber can be deflected only minimally (or not at all), this 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 may be minimally spaced from and / or nearly flush with the relaxed membrane (e.g., flat), while the liquid contact side is concave and extends into the second surface (second plate). The controller may retain fluid within the vacuum cover region, for example, by blocking valves on either side (inlet and outlet) of the vacuum cover, or by applying positive pressure to the pressure receiving side of the valve, and may apply negative pressure to the pressure receiving side of the vacuum cover. The absolute amount of the applied negative pressure (e.g., the magnitude of the negative pressure) may 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 may be configured to deflect against the first surface and / or plate (e.g., upwards), for example, to draw fluid from the input 1989 into the enlarged liquid contact side of the chamber. The membrane may be held on the first upper surface by the applied negative pressure, thereby allowing the removal of gas bubbles (e.g., air bubbles). The controller can 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.). Figure 10C Pressure can be applied via a pressure line 1987 communicating with the pressure receiving side of a chamber formed between the first and second surfaces of the device (e.g., a first plate and a second plate). A vacuum cover 1938 can be valved via one or more valves 1992. Fluid can exit from the liquid contact side via a fluid line 1989 on the opposite side of the vacuum cover.

[0120] The cartridge can be connected to the microfluidic actuator control system via spring-loaded connectors for both reagents and pneumatic lines for managing fluid movement and valve control. The reagent and gas lines are sealed by pressure applied to an elastomeric layer embedded in the cartridge, creating a completely sealed path from the reagent vial to the cartridge and from the cartridge to the output vial. This sealed path is maintained throughout all reactions within the cartridge, effectively preventing any contact with the atmosphere and minimizing the risk of contamination.

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

[0122] Microfluidic actuator systems control microfluidic actuator devices and can provide one-step connections to all actuators. These control systems can also scan all reagent and cartridge identifiers (e.g., barcodes) and monitor fluid levels. Generally, these microfluidic actuator control systems can automate some or all of the microfluidic actuator functions and generate visual records of all process operations that can be monitored (e.g., for optical quality control analysis of intermediate process outputs), stored, transmitted, or reviewed later.

[0123] As described above, the microfluidic actuator control system may include a microfluidic actuator management system comprising hardware such as a nesting element (microfluidic actuator holder) designed to ensure proper alignment of the cartridge and allow insertion in a single orientation. This can be managed, for example, by two pins and / or a notch in the nesting element that matches the shape of the cartridge. The microfluidic actuator management system (control system) also includes a vial holder for holding reagents and output vials, a downward-facing camera for recording all liquid and valve movements, and product output. A side camera on a guide rail captures barcodes and detects fluid levels, and a robotic arm has magnets for bead manipulation. The cartridge is secured in place with a vacuum chuck that ensures good contact with the Peltier device for temperature management. Once in place, engagement with all connectors is achieved in a single operation by lowering the top portion of the microfluidic actuator management system through a dowelpin guide system.

[0124] Figure 4B An example of a UV measurement region for a box as described herein is illustrated schematically. In this example, as... 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 and second layers. A UV measurement region 493 is formed by reducing the thickness of the first region 495 to form a first UV measurement region 496 and also potentially by reducing the thickness of the second layer 494 to form a second UV measurement region 498. One or more fluid channels 421 allow the UV measurement chamber 481 to be in fluid communication with a source of therapeutic polynucleotides and / or a "blank" solution (e.g., water, buffer, saline, etc.).

[0125] like Figure 4B As shown, the UV measurement chamber 481 is formed in the area between the first and second layers where the elastic material 407 has been removed or is absent. Therefore, the UV measurement chamber has the same thickness as the elastic layer 407. UV light can enter the first UV measurement area (cut area) and pass through the area of ​​the first layer thickness 491, then through any material held in the UV measurement chamber 481 (such as sample material and / or blank material), and then exit the area of ​​the second layer thickness 499 and enter the second UV measurement area 489, where the remaining (unabsorbed) light can be detected.

[0126] exist Figure 4B In the example shown, the UV measurement area is formed by cutout areas in the first plate 496 and the second plate 498, each cutout area having dimensions of approximately 2.5 mm in width and approximately 1.3 mm in depth (within a 1.5 mm thick first layer). In this example, the second layer is a mirror image of the first layer. Therefore, 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, 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 apparatus described herein use techniques for measuring blanks (e.g., solutions identical to those containing polynucleotides, but without polynucleotides), measuring absorbance does not explicitly require measuring the overall transmittance of the cassette. However, the transmittance of the cassette window material can have strong indirect results via signal / noise and measurable sample concentration. UV-opaque windows cannot be used to measure UV transmittance. Most microfluidic materials (e.g., cyclic olefin copolymers COC, cyclic olefin polymers COP, and polydimethylsiloxane PDMS) absorb UV significantly at wavelengths that are the same as those characteristic of DNA / RNA absorption. Therefore, in any of these examples, the measurement region (UV measurement chamber) can be thinned, for example, to between approximately 0.1 mm and 0.5 mm. To control the critical path length, the layered structure of the cassette can be modified. For example, one layer of the cassette (e.g., an elastic layer) can be completely removed from the UV measurement region. This can be advantageous because the layer thickness can generally be controlled more tightly than it is possible through in-layer machining / molding structures. Generally speaking, a finer path length can produce better measurements (or make measurements possible) for higher concentrations of samples compared to lower concentrations of the sample of interest. For example, for measurements at lower concentrations (e.g., when forming template materials for the synthesis of polynucleotides), a chamber with a substrate having a height of about 1 mm (e.g., reflecting a 1 mm PDMS elastic layer) can be used. In some examples, for measurements at 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) may be preferred.

[0128] Figure 5 The outline of an example box is shown, illustrating material thinning and short path length achieved by using the thickness of the elastomeric layer within the box. 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 approximately 0.1 mm (path length of 0.1 mm).

[0129] For sterility, the boxes are typically treated with gamma radiation before use. This has the benefit of destroying contaminants, but it can also alter the material properties of the box. This paper found that gamma treatment alters (e.g., reduces) the UV transmittance of various materials used in some of the sample boxes. For example, the UV transmittance of one component (COC) of the box became significantly more UV opaque after gamma treatment (see [link to sample box]). Figure 6 If it is not Figure 4B and Figure 5 The thinned design shown would be completely opaque to the UV detector.

[0130] The UV transmittance of COC treated with γ can depend on the degree of UV exposure, following an unusual trend of becoming more transparent under moderate exposure and eventually becoming less transparent under prolonged exposure. See, for example... Figure 7 In this example, the transmittance of the polymeric material (e.g., COC) initially and surprisingly increases with exposure, followed by a decrease in admittance over many hours. For example, when the transmittance of the cartridge changes during exposure, in some of the apparatuses and methods described herein, it is advantageous 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 performed before and after "sample" measurements to obtain the trend of transmittance change in the cartridge, and these values ​​can be used to estimate the material response during sample measurements.

[0131] The methods and apparatus described herein can be used with a UV light pretreatment box, thus placing the device in a region of stable curve and high transmittance during measurement. Figure 7 Generally, UV light can be applied only when measurements are required (e.g., by pulse or by using dimming features), thereby minimizing exposure and utilizing dimming features to ensure the LED light is just bright enough to obtain a good signal while avoiding overexposure. LED light adjustment can be done automatically or manually based on detector readings to always ensure a good signal reading from the detector.

[0132] like Figure 8 As shown, any device described herein can be configured to perform measurements on a series of dilutions (serial dilutions). Generally, microfluidic devices can be constructed to perform serial dilutions around a measurement chamber. The process may include microfluidically transferring a reactive aliquot into the measurement chamber, performing a measurement, and then diluting the aliquot for subsequent measurements. The diluted aliquot can be diluted repeatedly, measured each time, thereby producing... Figure 8 The graph shown is illustrated. At the end of the measurement, the chamber was rinsed with pure buffer to collect a "blank" reading and correct for UV exposure effects. Figure 8 The graphs illustrate the mechanics and utility of this process. For example, the sample might initially be too concentrated, making it completely opaque for the UV wavelength of interest (e.g., 260 nm). This is in Figure 8 Point 801 in the upper right corner of the graph is shown. Successive dilutions (described as being at 70%) further reduce the concentration of the measurement, but still effectively keep it opaque. After multiple dilutions, the measurement enters the linear regime, eventually reaching... Figure 8Point 805, located at the lower left corner of the graph, indicates where the aliquot becomes indistinguishable from the blank. UV absorbance can be used to determine concentration directly along the linear range until point 805, at which point UV absorbance measurement may no longer be useful. From the slope of the linear range, the apparatus or method can also determine the dilution rate (i.e., 70% to 73%). The apparatus can use the number of dilution steps, the dilution rate, and the concentration measured at the current dilution to determine the concentration of the original aliquot. Additionally, the linear portion of the curve validates the measurement, as nonlinear components can indicate that the signal is approaching the detector's noise level, or the presence of unexpected time-varying components or dilution errors.

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

[0134] Generally, 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 by the controller to a nominal and acceptable range. If the comparison is favorable, the batch can be collected into a common container. If it is unfavorable, the controller can microfluidically direct the batch to a waste container or for further analysis. In some examples, the device can use the measured concentration to normalize the system output. RNA / DNA concentrations can be measured and directed to product containers. The volume of added product can be read on the product container (e.g., by mass or imaging), and using this concentration and volume, along with a target concentration, the volume of diluent can be calculated and microfluidically added. Alternatively or otherwise, these methods and devices can adjust or control any processing (parameter) element during polynucleotide processing. For example, the measured concentration can be automatically compared by a computer to a nominal and acceptable range. Products can be released for use in relation to parameters, among other process parameters (such as temperature history and reagent dosage). Abnormal products can be classified, and the process can be studied.

[0135] Figures 10A to 10E and Figures 11A to 11B Additional examples of boxes that can be used as described in this article are shown. Figure 10A An example of a housing 1900 is shown. This example also includes a permeable insert 1969 within the liquid-contact side of chamber 1957. Figure 10A In this device, housing 1900 may include at least one pair of chambers 1953, 1957, 1957', each of which may include a liquid contact side 1917, a pressure (e.g., gas) side 1919, a fluid connection, a pressure connection, and fluid / pressure lines that may be formed within the thickness of the microfluidic actuator. In some examples, these chambers are paired, and each chamber in the pair may be connected to each other via a fluid connector 1955. The fluid connector 1955 may 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 chambers may be bisected by an elastic material (e.g., an elastic layer or membrane), and deflection of the elastic material within a fixed volume of the chamber may drive any liquid within the liquid to enter / exit the liquid contact side of the chamber (e.g., between the two chambers).

[0136] The cartridge 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 a different process. For example, the first pair of chambers 1953 may be used for RNA synthesis. The second pair of chambers 1957, 1957' may be used for the purification of synthesized polynucleotides. When pressure is applied to the pressure receiving side 1919 of the respective chamber and the valve 1959 between the first pair of chambers 1953 and the second pair of chambers 1957 is opened, 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 within a connector channel between the two pairs of chambers.

[0137] like Figure 10A and Figure 10B The illustrated housing 1900 may have multiple pressure ports and fluid ports 1923, 1923'. The multiple pressure ports and fluid ports may be disposed adjacent to the periphery of the housing and configured to connect to the fluid interface assembly 109 as described above.

[0138] The port (e.g., a sealing valve) may be formed by an elastic layer along the length of the connection channel 1939 (pressure channel or fluid channel), such as Figure 10A As shown, valve 1961 controls the timing of reagent delivery driven from fluid port 1923, but when placed in series with one or more valves of similar construction, it also allows for metering of the device's chambers. For example, in Figure 10AThe diagram shows three valve chambers (described in more detail below); the first of these three valves can function as a peristaltic pump, while the intermediate valves can be small-metering chambers (e.g., with metering volumes of approximately 10 nL, approximately 20 nL, approximately 25 nL, approximately 50 nL, approximately 75 nL, approximately 100 nL, etc.). The dimensions of the channels, and particularly the dimensions of the chambers connected to the channels, can meter the volume dispensed along fluid connection channels 1939, 1921 and delivered to chamber 1953 connected to fluid connection channels 1939, 1921. In some examples, the metering volume can be as small as 50 nL. Metering volumes of approximately 100 nL, approximately 1 μL, approximately 5 μL, or more can be input. A variety of valve sizes can be pre-selected to be incorporated within the microfluidic actuator 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 the row along the fluid connection passage 1939. A series of valves 1961 can 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 generally be particularly advantageous for moving fluids that may be viscous or contain suspended particles such as purifying or trapping small beads.

[0140] As mentioned, box 1900 may also include a delivery or output storage device or reservoir 1963. Figure 10A In this configuration, the pre-selected volume can be formed similarly to the chamber configuration described above, or may only include the metering side as needed. In either case, a valve can be used to meter the desired volume entering the reservoir 1963. Valve 1965 controls the delivery of fluid from the reservoir 1963. If a larger volume is required, the delivery can be repeated. Alternatively, if the reservoir 1963 is pre-selected as an output reservoir, valve 1965 can open and deliver fluid from chamber 1957 while keeping valve 1967 closed, allowing only the measured volume of fluid to be output to the reservoir 1963. This fluid can then be output to a vial on a reagent storage rack for further processing or detection. In some examples, the chamber, reservoir, or reservoir (e.g., 1963) can be configured as a metering section of, for example, a 1 μL pump formed by three valve configurations (1967, 1965, 1967). The chamber can be configured to, for example, output waste from mixing chamber 1957.

[0141] The cartridge 1900 can be a closed-path configuration. When the fluid vial, fluid lines, and microfluidic actuator are connected, 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 cartridge's fluid path for processing (including the synthesis of polynucleotides (e.g., RNA) and their preparation for biological delivery (as therapeutic agents, such as drugs, vaccines, etc.)). Therefore, the entire system can operate as a closed-path and / or the individual microfluidic actuator can operate as a closed path (protected from atmospheric influence) within the system.

[0142] Some examples of cartridge 1900 may also include an internal concentrator disposed within the thickness of the second plate and in fluid communication with an outlet channel such as 1949. Polynucleotides can be concentrated by removing excess fluid medium, and the concentrated polynucleotide mixture can be output outside cartridge 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 a microfluidic actuator.

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

[0144] As described above, the box can be formed of two or more plates stacked on top of each other, with chambers and / or channels formed between the plates; an elastic material can be sandwiched between the first and second plates. The first and / or second plates 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 monomers or soluble small molecules that are toxic to living cells. Any suitable biocompatible polymer can be used, including medical-grade polycarbonate-polyurethane, silicone polycarbonate-type polyurethane, polyether-type polyurethane, etc. In some examples, the polymer can be a cyclic olefin copolymer.

[0145] Figure 10B A cross-section through a portion of the box is shown, illustrating the UV measurement chamber 1981 of the UV measurement area 1993. Thus, the microfluidic actuator can be constructed as a multilayer structure consisting of two more rigid layers 1903, 1905 and a flexible membrane 1907 sandwiched between the two ridged layers. Figure 10BA portion of a cross-sectional view (transverse to the plane of the box) through an example of a box having multiple layers forming a reactor for processing a therapeutic agent as described herein. The reactor may include seals, channels, valves, and chambers including a pumping chamber formed by multiple layers. For example, the box may be formed of two or more rigid or semi-rigid plates 1903, 1905 and at least one elastic layer 1907. The elastic layer 1907 may be a sheet of elastic material that is impermeable to liquids. The elastic layer may be slightly gas-permeable, or may be treated to be more or less gas-permeable (including in various regions). Although a single continuous sheet of elastic material may be used, in some examples, multiple sheets of elastic material may be used, or a “sheet” may be formed from segments of multiple sheets. These layers and the elastic sheets may be laminated together. Generally, a chamber for holding, valve-regulating, and / or pumping fluid may be formed in the plate on either side of the elastic layer, such that the elastic layer divides the chamber into a liquid-containing side and a pressure (e.g., gas)-applying side. The total volume of the chambers can be constant and can be formed in both a first (e.g., upper) plate and a second (e.g., lower) plate, but this 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 deform to reduce (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 a 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 to 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 openings to the upper plate 1903 and the elastic layer 1907, thereby allowing an atmospheric-isolated sealed connection even when multiple different inlet lines are present, as the pressure line is pushed into the elastic layer 1907 supported on the underside of the port by the opposing rigid or semi-rigid layer 1905.

[0146] exist Figure 10B In the device, the housing 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 outer surface. The microfluidic actuator 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. An 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] Figure 10BThe box 1900 shown may also include a plurality of chambers 1915, 1916, 1918, 1920, each having a fixed volume. These chambers are formed by cut-out areas (e.g., circular / curved cuts) in the second (bottom) surface 1929 of the first plate 1903 and the first (upper) surface 1931 of the second plate 1905; an elastic layer 1907 bisects these chambers 1915 such that each chamber includes a liquid-containing side 1917 and a pressure-receiving (e.g., gas-containing) side 1919.

[0148] The cartridge 1900 may also include multiple liquid (e.g., fluid) channels. Figure 10B In the diagram, the fluid channel 1921 is shown extending from the thickness of the first plate 1903 to the fluid port 1923 of the fluid channel opening 1925, through the elastic layer 1907, and through most of the thickness of the second plate 1905, at which the length of the liquid channel 1921 extending parallel to the bottom surface of the third plate is formed.

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

[0150] Fluid passage 1921 is connected to the liquid-containing side 1917 of the first chamber 1915. The first chamber can be configured as a valve having a relatively low holding volume (fixed volume) but can be fully opened or closed by the movement of the elastic layer 1907.

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

[0152] In passing Figure 10B In the cross-section of the box 1900 shown, there are multiple connections to other fluid (e.g., liquid) lines, fluid ports, pressure lines, and pressure ports (not shown), as these may be outside the plane shown. For example, in Figure 10BIn this configuration, the liquid-containing side or portion 1917 of the fourth chamber may be connected to additional valves (chambers) and / or channels, including, for example, an outlet channel extending from the liquid-containing side 1917. Additional chambers (e.g., configured as valves), not shown, may be formed as described above. In some examples, the outlet channel may deliver fluid from one or more chambers through another fluid port (not shown) to a fluid receiving reservoir, such as a vial, tube, etc. This receiving reservoir may be held in a reagent storage rack.

[0153] exist Figure 10E In this case, the box 1980 includes four PCR chambers 1983, 1983', 1983"', and 1983"'. Each of the four PCR chambers may include a liquid contact side and 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 a general chamber, these fixed volumes are formed between a first surface of a first plate and a second surface of a second plate; the first and second plates may be connected together by an elastic deformable membrane (e.g., an elastic layer) between them, thereby dividing the chambers. 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 Figure 10D In the illustrated cassette, the pressure-receiving side 1919 of each chamber is further subdivided by one or more fluid-connected meandering paths 1985. These meandering paths in the pressure-receiving surface are configured to distribute the positive and negative pressures (and specifically, the negative pressures) applied through the channels more evenly across the surface of the relatively large chamber. The subdivision of the pressure-receiving surface in each PCR chamber (e.g., through one or more meandering paths in some examples) can support the deflectable membrane when a negative pressure is applied to pull the deflectable membrane away from the fluid-containing chamber. This also prevents bubble formation and maintains a fixed, predictable volume.

[0155] Figure 10D The illustrated box also includes multiple fluid channels, each extending from fluid ports 1923, 1923' through the first plate region and into the second plate region, to fluidly connect with the liquid contact side of one or more of the multiple chambers (similar to...). Figure 10A and Figure 10B (The structure shown). In Figure 10D The text indicates that a subset of fluid ports are labeled, including those providing (external) plasmid 1923, PCR buffer, primers (e.g., T7 primers), oligodT, enzymes (e.g., polymerase), and purification substrates (e.g., Ampure). TMThose fluidly connected to the following: bead-free air, RNase-free air, dNTP source, 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 not used. As described, pressure ports can provide communication for applying positive and / or negative pressure to the pressure receiving side of each chamber, channel, vacuum cover, valve, etc. Therefore, the controller can control the movement of fluids within the device, including mixing, pumping, valve adjustment, etc., by applying positive or negative pressure to a specific pressure port 1943 or a combination of pressure ports. Pressure ports and fluid ports may be arranged on the upper side of the first plate, typically around the perimeter of the plate, such as... Figure 10A and Figure 19 As shown in D.

[0156] exist Figure 10D The device also includes multiple pressure channels 1947, each extending from one or more pressure ports, passing through the first plate region and the elastic layer, entering the second plate region, and returning through the elastic layer and into the first plate region (similar to...). Figure 10B As shown in the diagram, 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 of the plurality of chambers. Figure 10B As shown, valves 1915 and 1918 can be opened / closed by a controller (from the pressure port) of a system including a fluid reservoir and a pneumatic drive device, which applies positive or negative pressure through a pressure channel, and can pump fluid through chambers 1916, 1920, and 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 PCR chambers in the PCR chamber. The UV yield detection chamber may include a UV yield detection window configured to allow UV light to pass through for quantification of polynucleotides within the UV yield detection chamber. The UV measurement chamber 1990 may also be connected to a buffer source for performing UV detection. UV detection can measure the absorbance of the buffer containing the DNA. A device (system) including a controller coordinating operation on a microfluidic actuator 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 employing a microfluidic actuator may first check the absorbance of a UV buffer containing no product, and then may add a predetermined amount (e.g., purified) of product for comparison. The system (e.g., the controller) may then automatically or semi-automatically use the measured concentration to alert the user and / or make a decision to discard or label the sample for further analysis and / or dilute the product before it is discharged from the box or moved to another box.

[0158] Figure 10E It shows the relationship with Figure 10D Another example of a similar box (e.g., a "biochip") shown in the figure 1980' includes a UV yield detection chamber in fluid communication with a sample and / or blank (e.g., buffer) chamber 1990'. Figure 10E The box device also includes a waste port 1923' (e.g., a negative pressure port, which can be connected to a waste line).

[0159] Generally speaking, Figure 10D and Figure 10E The devices shown are similar and may include Figures 10A to 10C Any of the features shown. For example, the port may be formed by an elastic layer along the length of the connection channel 1939 (pressure channel or fluid channel), such as Figure 10A As shown. One or more valve bodies 1961 may be included in the row along the fluid connection passage 1939.

[0160] PCR chambers can be configured to optimize the PCR process described herein. For example, a microfluidic actuator device may 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 may 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, however, the lower the total volume. The cartridges described herein can be used for PCR within PCR chambers without the need for the addition of oleophobic / hydrophobic materials, as evaporation can be limited by the closed (or sealable) construction of the chamber.

[0161] Generally speaking, any of these boxes may include a purification chamber in fluid communication with the purification substrate (e.g., Ampure beads).

[0162] The cartridge 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 contact side of the device and exiting from the fluid port into a reservoir coupled to the fluid port. For example, in Figure 10D In this system, by driving RNase-free air through a fluid channel connected to a fluid port, the cartridge can be controlled by a controller (of a system coupled to the cartridge) to mix and resuspend substrate beads (e.g., Ampure) within a reservoir coupled to the fluid port. TM (Small beads). This may cause the substrate beads to bubble and mix within the reservoir. The controller can control the mixing by applying RNase-free air (using positive and / or negative pressure applied through the pressure port of the microfluidic actuator), and after mixing, the resuspended substrate can be guided from the reservoir and directed to the purification chamber for purification of the template product.

[0163] As mentioned, any of these devices can be configured as a removable cartridge that is configured to engage with a fluid reservoir and a pneumatic drive and can be coupled to a system, such as a microfluidic actuator control system, which includes a controller for coordinating the operation of the microfluidic actuator to generate a template.

[0164] Generally, the cassette can be any suitable size / volume. For example, the cassette can be constructed 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.). Figure 10D In the example shown, the total PCR reactor volume (the combination of all four PCR chambers) is approximately 6.03 mL. Therefore, in Figure 10DIn this study, the PCR reaction volume was approximately 3 mL, and it was found to produce approximately 130 ng / μL of template product (within 3 mL).

[0165] Figure 11A and Figure 11B An example of a housing is illustrated, comprising a UV measurement channel 1181 coupled to one or more valves 1176, 1177 and an input (e.g., a polynucleotide material source, such as a polynucleotide forming chamber 1179 and / or a buffer or other blank solution source). Figure 11A In this configuration, the UV measurement chamber is also connected to a suction (e.g., waste) port 1172. Suction can be used to load the UV measurement chamber 1181 via a control valve. Figure 11B It shows Figure 11A A slightly magnified view of the UV measurement chamber.

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

[0167] Figure 12The operation of the apparatus described above, using a microfluidic actuator operating on a cartridge, is schematically illustrated. In this example, the method can be used for (e.g., in a cartridge) a therapeutic polynucleotide 1201, as described above. Additional processing (e.g., purification, concentration, etc.) can be performed on the polynucleotide. Additionally, a UV measurement subsystem can be prepared for measurement 1203. For example, the apparatus can pre-irradiate the chamber and / or one or more diluents that can form a polynucleotide solution, as described above. The apparatus can then determine the concentration of the therapeutic polynucleotide 1205. This can include continuously driving a first blank solution into the UV measurement chamber and performing a first absorbance measurement 1207, and then driving the sample solution (or a diluted version thereof) and performing a second absorbance 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, the two blank measurements can be averaged. The apparatus can then estimate the concentration of the polynucleotide in the sample 1211 from the first absorbance measurement and the second (and optionally a third) absorbance measurement.

[0168] Subsequently, the operation of the device (e.g., a microfluidic actuator device) can be adjusted based on absorption measurements and / or the concentration of the therapeutic polynucleotide 1213.

[0169] Example

[0170] Figure 14 Examples of portions of an apparatus as described herein, including a UV detection subsystem, are illustrated. For example, the apparatus may include a controller (shown herein 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 allow them to be adjusted individually. For example, the LED intensity (e.g., brightening / dimening 1405) can be increased or decreased by adjusting the power supplied from power supply 1407 to LED driver 1409, which controls the output of LED 1411 and thus controls the illumination of the sample or blank through the UV detection chamber 1413. Similarly, the power supplied from power supply 1407' to UV detector 1415 can be adjusted to increase or decrease the sensitivity of the LED detector in sensing absorption. As described above and as... Figure 14 As illustrated, the obtained UV absorption signal can be stored and / or analyzed by the device. Figure 14The device includes a signal capture circuit 1403 to capture, store, and / or transmit the obtained absorbance. The controller may 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 determine polynucleotide concentration using UV light absorption can be configured to adjust the UV light intensity over a certain range to achieve a linear output range of absorption for the polynucleotide sample, thereby avoiding saturation of the absorption signal output. Figure 15A The example shown illustrates the relationship between dimming voltage and current for a usable UV light source (LED). The dimming voltage is linear across this range and can be linearly correlated with the resulting light intensity, such as... Figure 15B As shown. Figure 15B The voltage readouts for the different applied dimming voltages are shown. (Re-reference) Figure 14 Therefore, the LED can be adjusted to increase or decrease its intensity based on the absorption signals detected from the sample and blank (e.g., by adjusting the dimming voltage). 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 otherwise, the sensitivity of the UV detector can be decreased or increased. The same adjustment (e.g., dimming voltage) can be applied to both the sample and blank.

[0172] Generally speaking, the apparatus and methods described herein can provide highly accurate and reproducible determinations of polynucleotide concentrations. For example, Figure 16A Examples of concentration detection for various samples (and corresponding blanks) are shown. Figure 16A In this context, the apparatus described herein can be used to first apply blank 1602 and then measure UV absorption, such as... Figure 16A As shown. In this embodiment, the absorption of each polynucleotide sample or blank is performed for 10 seconds, and a stable reading is obtained throughout this entire time period; in some examples, shorter (e.g., 5 seconds, 1 second, 0.5 seconds, 0.1 seconds, etc.) or longer absorption periods can be measured. After measuring the first blank, a first RNA sample (e.g., undiluted) is transferred into the UV measurement chamber to replace the blank, and the UV absorption is measured at 6104. The sample fluid can then be removed from the UV measurement chamber, and a second blank can be transferred in and measured at 1602', followed by a second sample (e.g., a half-diluted RNA sample) at 1606. The second sample (e.g., a 10-fold diluted RNA sample) is measured at 1608, followed by a third blank at 1602". Sample measurements can be performed on different polynucleotides and / or different dilutions of the same polynucleotide (e.g., 1602'). Figure 16A (as shown in the image).

[0173] Figure 16B A comparison is shown between the measurements performed as described above and commercially available concentration detection systems that require different methods and techniques for determining analyte concentrations, demonstrating very close agreement. In this example, a commercially available analyte concentration detector could be, for example, Nanodrop. TM The system (Thermo Fisher). Therefore, the methods and apparatus described herein, particularly applicable to on-chip (e.g., “on-chip”) UV and thus to concentration measurements, show similar or even better results compared to other larger and more expensive systems.

[0174] Figures 17 to 18 Another example of a cartridge (e.g., a "biochip") 2000 is depicted. This example cartridge 2000 may be similar in construction and operation to any other cartridge described herein, with examples of differences as described below. This example cartridge 2000 includes a plurality of pressure ports 2043 and a plurality of fluid ports 2023. A plurality of pressure channels 2047 extend from the pressure ports 2043. The pressure ports 2043 and pressure channels 2047 provide a pressure receiving side for applying positive and / or negative pressure to each chamber, channel, vacuum cover, valve, etc., within the cartridge 2000. Therefore, a controller can control the movement of fluid within the cartridge 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. Pressure channels 2047 can therefore be understood as pneumatic channels.

[0175] Pressure port 2043 and fluid port 2023 may be arranged on the upper side of box 2000 (e.g., around the outer perimeter of box 2000), such as Figure 17 As shown. Alternatively, pressure port 2043 and / or fluid port 2023 may be arranged on the underside of housing 2000.

[0176] Each fluid port 2023 is fluidly coupled to a corresponding fluid channel in the cartridge 2000, such that each fluid channel extends from the corresponding fluid port 2023. By way of example only, one or more fluid ports 2023 may be coupled to plasmids, PCR buffers, primers (e.g., T7 primers), oligodT, enzymes (e.g., polymerases), purification substrates (e.g., Ampure). TM (Small beads), RNase-free air, dNTP source, product output collection receiver, output from UV yield detection channel, buffer for UV detection, water, ethanol (e.g., 70% ethanol rinse), or waste fluidly connected. Fluid ports 2023 (and corresponding channels) can be understood as providing pathways for liquid communication. In some cases, one or more fluid ports 2023 are redundant and therefore not used during certain processes.

[0177] The example box 2000 also includes PCR chambers 2083, 2083', 2083”, and 2083”', each of which may include a liquid contact side, a pressure (e.g., gas / pneumatic) side, and they are fluidly connected to adjacent PCR chambers. Each PCR chamber has a fixed volume, and as described above for a general chamber, these fixed volumes are formed between a first surface of a first plate and a second surface of a second plate, the first and second plates being connected together by an elastic deformable membrane (e.g., an elastic layer) between them, thereby dividing the chambers. 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] Each PCR chamber 2083, 2083', 2083"' ...

[0179] The example cartridge 2000 also includes a UV measurement chamber 2020, which is in fluid communication with one or more of the PCR chambers 2083, 2083', 2083"', and 2083"' and / or a blank (e.g., buffer) chamber, such that the UV measurement chamber 2020 can receive aliquots of liquid from one or more of the PCR chambers 2083, 2083', 2083"', and / or a blank (e.g., buffer) chamber. The UV measurement chamber 2020 may include a UV yield detection window configured to allow UV light to pass through for quantification of polynucleotides within the UV measurement chamber 2020 as described herein. Figure 18 As best seen, the inlet liquid channel 2026 leads to the UV measurement chamber 2020, while the outlet liquid channel 2050 leads out of the UV measurement chamber 2020.

[0180] Vacuum cover 2024 is positioned upstream of inlet liquid channel 2026. Another vacuum cover 2052 is positioned downstream of outlet liquid channel 2050. In some configurations, vacuum covers 2024 and 2052 are positioned as shown in the reference above. Figure 10C The vacuum cover described is constructed and operated as in 1938.

[0181] In this example, vacuum covers 2024 and 2052 are operable to remove air bubbles from the liquid in the UV measurement chamber 2020. Vacuum covers 2024 and 2052 are pneumatically connected together via a pneumatic channel 2040, facilitating simultaneous operation of both covers. In some configurations, one or both of vacuum covers 2024 and 2052 may also function as valves that selectively allow or prevent the transfer of liquid aliquots / samples to or from the UV measurement chamber 2020. In this case, vacuum covers 2024 and 2052 can be pneumatically actuated like other valves described herein.

[0182] A pair of liquid channels 2028 and 2030 lead to a vacuum cover 2024. Valve 2076 selectively allows or prevents liquid from flowing through channel 2028 to vacuum cover 2024. Similarly, valve 2032 selectively allows or prevents liquid from flowing through channel 2030 to vacuum cover 2024. Another vacuum cover 2070 is in fluid communication with valve 2076 and is pneumatically connected to vacuum cover 2024 via channel 2022. Yet another vacuum cover 2072 is also pneumatically connected to vacuum cover 2070 and is in fluid communication with another valve 2074. Therefore, vacuum covers 2024, 2052, 2070, and 2072 can be pneumatically connected together via channels 2022 and 2040 to allow simultaneous operation of vacuum covers 2024, 2052, 2070, and 2072.

[0183] In this example, valve 2076 is fluidly inserted in the path between PCR chambers 2083, 2083', 2083"', 2083"' and channel 2028, such that valve 2076 is operable to selectively prevent or allow liquid from flowing from PCR chambers 2083, 2083', 2083"' to channel 2028. And thus, liquid flows to UV measurement chamber 2020 via vacuum cover 2024 and channel 2026. In some configurations, with the valve in the path open, PCR chambers 2083, 2083', 2083"' can be pressurized to push liquid aliquots / samples into UV measurement chamber 2020. Alternatively, with the valve in the path open, liquid can be driven into UV measurement chamber 2020 by applying a vacuum through output port 2060. In other configurations, the main reservoir may be pressurized; and valves 2076, 2052 (and possibly vacuum covers 2024, 2052) may be sequentially actuated to peristally pump liquid from PCR chambers 2083, 2083', 2083"', 2083"' to the UV measurement chamber 2020. Alternatively, any other suitable structural features and / or techniques may be used.

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

[0185] Another passage 2054 and valve 2056 are also connected to vacuum cover 2052. Pneumatic passage 2062 also extends from valve 2056 and provides a path for pressurizing valve 2056, thereby switching valve 2056 between an open and closed state. Another passage 2058 also extends from valve 2056 and provides a path for fluid communication from valve 2056 to output port 2060. Output port 2060 may operate similarly to the suction / waste port 1388 described above.

[0186] The example box 2000 also includes a collection output port 2002. For example... Figure 18 As best seen in the image, the collection output port 2002 is connected to a liquid communication channel 2004 leading to valve 2006. Valve 2006 is in fluid communication with pneumatic channel 2010, through which valve 2006 can be selectively pressurized, thereby switching valve 2006 between an open and closed state. Liquid communication channel 2008 is also connected to valve 2006. Channel 2008 is also in fluid communication with PCR chambers 2083, 2083', 2083"', and 2083"', so that when valve 2006 is in the open state, fluid can be transferred from PCR chambers 2083, 2083', 2083"' to collection output port 2002 via channels 2004 and 2008. The collection output port 2002 can therefore be used to collect the outputs of PCR chambers 2083, 2083', 2083”, 2083”' into a single batch in the receiver outside the box 2000, as described above.

[0187] Some versions of cartridge 2000 may also include an additional output port (not shown) fluidly connected to PCR chambers 2083, 2083', 2083"', 2083"', allowing fluid to be transferred from PCR chambers 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 PCR chambers 2083, 2083', 2083"' can be selectively transferred to the collection output port 2002 or another output port, effectively allowing PCR chambers 2083, 2083', 2083"' to have two distinct and individually controlled outputs. In some cases, fluid transferred from PCR chambers 2083, 2083', 2083"' to another output port (not shown) can be retained for further analysis or can be otherwise processed. In some such cases, due to concentration measurement data obtained via the UV measurement chamber 2020, fluid from PCR chambers 2083, 2083', 2083"', and 2083"' can be transferred to an additional output port (not shown) instead of to the collection output port 2002. It should also be understood that at least some of the fluid output from PCR chambers 2083, 2083', 2083"' can be transferred through output port 2060. In some such cases, the fluid transferred through 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 alternative embodiments, UV transmittance may vary between different cells. Such differences in UV light intensity and / or UV transmittance can affect the limit of quantitation (LoQ) of the UV measurement process. In other words, variations in UV light intensity, UV transmittance, and / or other noise sources can affect the maximum and minimum sample concentrations, which can be reliably detected with some predetermined confidence level by UV measurements as described herein. Therefore, it may be desirable to provide a process for establishing the upper and lower LoQs of the cell before using it to obtain polynucleotide concentration measurements during the process for forming therapeutic polynucleotides.

[0189] Figure 19An example of process 2100 is described, which can be used to evaluate the upper LoQ and lower LoQ of a cartridge before obtaining a polynucleotide concentration measurement during the formation of a therapeutic polynucleotide. Process 2100 can be used with any of the various cartridges described herein. Process 2100 can be performed by controller 221, processor 1401, and / or any other suitable hardware component. Process 2100 begins by measuring the maximum voltage of a UV detector (e.g., UV detectors 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, 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 can be determined by the following equation (I) (box 2106):

[0191] (I)

[0192] Where Vmax = maximum voltage;

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

[0194] ε = UV absorption coefficient of nucleotides; and

[0195] l = Sample thickness (i.e., path length).

[0196] By way of just another example, the lower LoQ can be determined by the following equation (II) (box 2108):

[0197] (II)

[0198] Where Vmax = maximum voltage;

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

[0200] ε = UV absorption coefficient of nucleotides; and

[0201] l = Sample thickness (i.e., path length).

[0202] The determined upper LoQ can be compared to a threshold to determine whether the upper LoQ is acceptable, as shown in box 2110. Similarly, the determined lower LoQ can be compared to a threshold to determine whether the lower LoQ is acceptable, as shown in box 2112. If the upper LoQ or lower LoQ is unacceptable, a warning can be provided to the operator, as shown in box 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 either the upper LoQ or the lower LoQ, making the measured concentration unreliable; and / or any other suitable message. In some configurations, if the upper LoQ or lower LoQ is found to be unacceptable, the controller 221, processor 1401, and / or any other suitable hardware component may prevent further use of the box and / or UV light source unless and until the operator takes corrective action and subsequently finds the upper LoQ and 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, the processor 1401 and / or any other suitable hardware component may allow the box to be used to form therapeutic polynucleotides (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., making the sample's UV transmittance quite low), the sample may tend to induce considerable backscattering when irradiated with UV light. This backscattering of UV light can adversely affect the accuracy of UV detector readings (e.g., UV detectors 103, 1415) by negatively impacting the dynamic range. In some cases, this backscattering can be corrected using data processing techniques. However, such data processing techniques may require certain geometric assumptions. Another alternative may include using UV photoresist materials to suppress scattered light and thereby improve the dynamic range of the UV detector.

[0204] Figures 20 to 21 An example is shown of how UV photoresist materials can be used to suppress scattered light and thereby improve the dynamic range of a UV detector. Specifically, Figures 20 to 21 A UV blocking member 2200 is shown, which 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 transmitting through the body 2202, while allowing UV light to transmit through the central opening 2204.

[0205] Figure 21An arrangement 2300 is shown, in which a UV blocking member 2200 is applied to a housing 2310. The housing 2310 can be constructed and operated like any other housing described herein. This example housing 2310 includes an upper plate 2320, a lower plate 2330, and an elastic layer 2340 inserted between the plates 2320 and 2330. A recess 2322 formed in the upper plate 2320 provides a first UV measurement area; while a recess 2332 formed in the lower plate 2330 provides a second UV measurement area below the recess 2322. A UV measurement chamber 2350 is inserted between the recesses 2322 and 2332; and can be constructed and operated like other UV measurement chambers described herein.

[0206] The UV blocking member 2200 is positioned on the top of the upper plate 2320 directly above the recess 2322. In some configurations, the UV blocking member 2200 is adhered to the upper surface of the upper plate 2320. Alternatively, the UV blocking member 2200 may be fixed relative to the upper plate 2320 in any other suitable manner. Figure 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 and enter the recess 2322, so that the UV light 2304 can illuminate the sample in the UV measurement chamber 2350. The body 2202 will substantially limit the backscattering of UV light 2304 provided by the sample in the UV measurement chamber 2350. The UV light 2360 exiting the UV measurement chamber 2350 through the recess 2332 will reach the UV detector 2362, which will generate a signal indicating the concentration of the sample in the UV measurement chamber 2350, as described herein.

[0207] Figure 22 Another example of an arrangement 2400 for mitigating backscattering of UV line of sight from a UV measurement chamber is depicted. Similar to the arrangement 2300 described above, this example arrangement 2400 includes a UV light source 2402, a housing 2410, and a UV detector 2462. Similar to the housing 2310 described above (and other housings similar to those described herein), this example housing 2410 includes an upper plate 2420, a lower plate 2430, and an elastic layer 2440 inserted between the plates 2420 and 2430. A recess 2422 formed in the upper plate 2420 provides a first UV measurement area; while 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 inserted between the recesses 2422 and 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, the UV blocking member 2470 of this example is configured differently from the UV blocking member 2200. Specifically, the UV blocking member 2470 of this example includes a cylindrical body 2472 defining a central passage 2472 therethrough.

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

[0210] The body 2472 is dimensioned to fit within the recess 2422 such that the outer wall of the body 2472 contacts the inner wall of the recess 2422. In some embodiments, the body 2472 is secured within the recess 2422 by friction fit, adhesive, or any other suitable technique. In this example, the inner wall 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 embodiments, the central passage 2472 is tapered. For example, some variations may provide an opening at the top of the body 2472 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 larger than the opening at the bottom of the body 2472. Alternatively, the central passage 2472 may have any other suitable configuration.

[0211] like Figure 22 As shown, the diameter of the central passage 2472 is smaller than the diameter of the recess 2422. It should be understood that... Figure 22 The diameter of the central passage 2472 shown is merely an example; and the diameter of the central passage 2472 can be relative to... Figure 22 The increase or decrease is shown in the diagram. 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. The body 2472 will substantially limit the backscattering of UV light 2404 provided by the sample in the UV measurement chamber 2450. The UV light 2460 exiting the UV measurement chamber 2450 through the recess 2432 will reach the UV detector 2462, which will generate a signal indicating the concentration of the sample in the UV measurement chamber 2450, as described herein.

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

[0213] In some cases, air bubbles may reach the UV measurement chamber, and the presence of such bubbles in the UV measurement chamber may lead to an underestimation of the sample concentration within the chamber. Therefore, it is desirable to provide a measurement algorithm that takes into account the possibility of inaccurate measurements due to the presence of air bubbles in the UV measurement chamber. An example of such a process 2500 is... Figure 23 As shown in the diagram. Process 2500 can be used with any of the various boxes described herein. Process 2500 can be executed by controller 221, processor 1401, and / or any other suitable hardware component. Figure 23 As shown in box 2502, process 2500 begins with four concentration measurements based on readings from UV detectors 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 types, outlier measurements are identified as those whose values ​​are furthest from the average. In some other types, outliers are identified based on a T-test. As yet another variation, the measurement providing the lowest concentration value may be automatically identified as an outlier. In some of these types, the calculation of the average may be omitted (box 2504).

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

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

[0216] As a use Figure 23 An alternative to the process 2500 shown and described above can use a quadrant detector to account for the presence of any bubbles in the sample. For example, the voltage difference across the axes of the four quadrants can indicate a non-uniform sample. This type of calibration can guarantee a measurement after each cell is loaded to account for optical variations between cells. Such calibration can be automated.

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

[0218] The following examples illustrate various non-exhaustive ways in which the teachings of this article can be combined or applied.

[0219] It should be understood that the following examples are not intended to limit the scope of any claims that may be made at any time in this application or subsequent applications. No exclusions are made. The following examples are provided merely for illustrative purposes. 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, the aspects or features mentioned below should not be considered critical unless explicitly stated by the inventor or a successor of interest to the inventor at a later date. If any claim in this application or a related subsequent application includes additional features in addition to those mentioned below, those additional features should not be considered added for any reason related to patentability.

[0220] Example 1

[0221] A cassette device for processing polynucleotides, the cassette 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 thickness; and a UV measurement region formed through the first layer and the second layer, wherein the region of the first layer in the UV measurement region has a thickness equal to or less than the first thickness, and the second layer...

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

[0223] Example 2

[0224] According to the box device of Embodiment 1, the first layer comprises a polymer material.

[0225] Example 3

[0226] According to the box device of Example 2, the first layer comprises a cyclic olefin copolymer (COC) material.

[0227] Example 4

[0228] According to any one of embodiments 1 to 3, the first thickness is between 1 mm and 5 mm.

[0229] Example 5

[0230] According to any one of Examples 1 to 4, the second layer comprises a polymer material.

[0231] Example 6

[0232] According to the box device of Example 5, the second layer comprises a cyclic olefin copolymer (COC) material.

[0233] Example 7

[0234] According to any one of embodiments 1 to 6, the second thickness is between 1 mm and 5 mm.

[0235] Example 8

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

[0237] Example 9

[0238] According to any one of Embodiments 1 to 8, the box device further includes a fluid channel in a second layer that is in fluid communication with the UV measurement chamber.

[0239] Example 10

[0240] According to any one of embodiments 1 to 9, the cartridge device further includes a plurality of pneumatic valves arranged to control the flow into and out of the UV measurement chamber, wherein each of the plurality of pneumatic valves is formed from 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 in fluid communication with a pneumatic channel in the first layer, the pneumatic channel being configured to communicate with a pressure port on an external region of the cartridge device to actuate the pneumatic valve, and wherein when the pneumatic valve is open, the fluid chamber is in fluid communication with the UV measurement chamber through the fluid channel in the second layer.

[0241] Example 11

[0242] The box device according to any one of Embodiments 1 to 10 further includes an in vitro transcription (IVT) chamber that is at least partially formed within the second layer and is 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 includes one or more vacuum ports on an external region of the box device, the one or more vacuum ports being configured to be coupled to a negative pressure source to draw fluid into the UV measurement chamber.

[0245] Example 13

[0246] According to any one of Embodiments 1 to 12, the cassette device further includes a first inlet channel within the second layer and a second inlet channel within the second layer, the first inlet channel being in fluid communication with a first chamber configured to hold a polynucleotide sample fluid, and the second inlet channel being in fluid communication with a second chamber configured to hold a blank sample fluid.

[0247] Example 14

[0248] According to any one of Embodiments 1 to 13, the box device further includes 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] Example 15

[0250] According to any one of Embodiments 1 to 14, the diameter of the UV measurement chamber is larger than the diameter of the area of ​​the first layer in the UV measurement region, so as to limit air bubbles in the central region of the UV measurement chamber.

[0251] Example 16

[0252] A cassette device for processing polynucleotides, the cassette device comprising: a first layer comprising a polymeric material having a first thickness; a second layer comprising a polymeric material having a second thickness; an elastic material extending between the first layer and the second layer; 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 or equal to the first thickness, a region of the second layer in the UV measurement region has a thickness less than or equal to the second thickness, and wherein the elastic material is removed from the region of the first layer in the UV measurement region and the region of the second layer in the UV measurement region to form a UV measurement chamber; and an in vitro transcription (IVT) chamber at least partially formed within the second layer and in fluid communication with the UV measurement chamber.

[0253] Example 17

[0254] A method for manufacturing a polynucleotide using a microfluidic actuator device operated on a cartridge, the method comprising: forming a therapeutic polynucleotide in a cartridge; and determining the concentration of the therapeutic polynucleotide by the microfluidic actuator device through the following steps: driving a first blank solution into the 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; and estimating the concentration of the therapeutic polynucleotide from the first absorption measurement and the second absorption measurement.

[0255] Example 18

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

[0257] Example 19

[0258] The method according to any one of Examples 17 to 18 further includes comparing the estimated concentration of the therapeutic polynucleotide with a concentration range via a processor of the microfluidic actuator, and based on this comparison, directing the therapeutic polynucleotide for one of the following: collection, disposal, or further analysis with one or more additional batches of therapeutic polynucleotides formed in the cassette.

[0259] Example 20

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

[0261] Example 21

[0262] The method according to any one of Examples 17 to 20 further includes automatically adjusting one or more parameters of the formation of the therapeutic polynucleotide via the microfluidic actuator based on the estimated concentration of the therapeutic polynucleotide.

[0263] Example 22

[0264] According to the method described in Example 21, the one or more parameters include one or more of the following: temperature, reagent volume, reagent concentration, time, and mixing.

[0265] Example 23

[0266] According to any one of Examples 17 to 22, determining the concentration of the therapeutic polynucleotide further includes performing a series dilution in one or more chambers of the cartridge under the control of the microfluidic actuator, and repeating the step of driving the sample solution of the therapeutic polynucleotide for each of the one or more diluents formed by the series dilution to generate a dilution profile.

[0267] Example 24

[0268] According to the method of Example 23, estimating the concentration of the therapeutic polynucleotide includes estimating the concentration of the therapeutic polynucleotide from the dilution curve.

[0269] Example 25

[0270] According to any one of Examples 17 to 24, estimating the concentration of the therapeutic polynucleotide further includes changing one or more of the sensitivity of the UV detector and the intensity of the UV emitter of the microfluidic actuator in response to the second absorption measurement via the cartridge.

[0271] Example 26

[0272] According to any one of Examples 17 to 25, the method further includes pretreating the UV measurement chamber with UV light prior to performing the first absorption measurement through the chamber.

[0273] Example 27

[0274] According to any one of Examples 17 to 26, the method for forming the therapeutic polynucleotide includes performing an in vitro transcription (IVT) reaction to form the therapeutic polynucleotide.

[0275] Example 28

[0276] According to the method of Example 27, the method further includes generating a template for the IVT reaction in the box.

[0277] Example 29

[0278] According to the method of any one of Examples 17 to 28, the therapeutic polynucleotide comprises therapeutic mRNA.

[0279] Example 30

[0280] According to the method of Example 29, the method further includes encapsulating the therapeutic mRNA with a delivery vector.

[0281] Example 31

[0282] According to any one of Examples 17 to 30, the microfluidic actuator monitors 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 cartridge to open and / or close the valve of the cartridge.

[0283] Example 32

[0284] According to the method of Example 31, the microfluidic actuator 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.

[0285] Example 33

[0286] A method for manufacturing a polynucleotide using a microfluidic actuator device operated on a cartridge, the method comprising: forming a therapeutic polynucleotide in a cartridge, wherein the therapeutic polynucleotide comprises 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 the 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; 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 in a processor of the microfluidic actuator device based on the estimated concentration of the therapeutic polynucleotide.

[0287] Example 34

[0288] A system for preparing and / or processing polynucleotides, the system comprising: a cassette mount; multiple pressure lines; multiple fluid lines, each fluid line being 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 fixed to an inlet or outlet port on a cassette held in the cassette mount; an ultraviolet (UV) light source; a UV photodetector; and a controller configured to control pressure applied through the pressure lines to drive fluid through the cassette, wherein the controller is further configured to guide the formation of a therapeutic polynucleotide in the cassette and to estimate the concentration of the therapeutic polynucleotide by: driving a first blank solution through a UV measurement chamber of the cassette and performing a first absorption measurement using the UV light source and a UV photodetector; driving a sample solution containing the therapeutic polynucleotide through the UV measurement chamber of the cassette and performing a second absorption measurement using the UV light source and a UV photodetector; 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] Example 35

[0290] According to the system of embodiment 34, the controller is further configured to modify the operation of the system based on the estimated concentration of the therapeutic polynucleotide.

[0291] Example 36

[0292] According to any one of Examples 34 to 35, the controller is further configured to compare the estimated concentration of the therapeutic polynucleotide with a concentration range, and based on this comparison, direct the therapeutic polynucleotide for one of the following: collection, disposal, or further analysis with one or more additional batches of therapeutic polynucleotides formed in the cassette.

[0293] Example 37

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

[0295] Example 38

[0296] According to any one of Examples 34 to 37, 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] Example 39

[0298] According to the system of Example 38, the one or more parameters include one or more of the following: temperature, reagent volume, reagent concentration, time, and mixing.

[0299] Example 40

[0300] According to any one of Examples 34 to 39, the system wherein the controller is further configured to perform serial dilution in one or more chambers of the cartridge, and to repeat the steps of driving the sample solution of the therapeutic polynucleotide for each of one or more diluents of the sample solution of the therapeutic polynucleotide formed by the serial dilution to generate a dilution profile.

[0301] Example 41

[0302] According to the system of Example 40, the controller is further configured to estimate the concentration of the therapeutic polynucleotide from the dilution curve.

[0303] Example 42

[0304] According to any one of embodiments 34 to 41, 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 of the cell.

[0305] Example 43

[0306] According to any one of embodiments 34 to 42, the controller is further configured to pre-treat the UV measurement chamber with UV light prior to performing the first absorption measurement through the chamber.

[0307] Example 44

[0308] According to any one of Examples 34 to 43, the controller is further configured to form the therapeutic polynucleotide, including performing an in vitro transcription (IVT) reaction to form the therapeutic polynucleotide.

[0309] Example 45

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

[0311] Example 46

[0312] The system according to any one of Examples 34 to 45, wherein the therapeutic polynucleotide comprises therapeutic mRNA.

[0313] Example 47

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

[0315] Example 48

[0316] According to any one of Examples 34 to 47, the controller is further configured to pneumatically deflect one or more regions of the membrane of the cartridge to open and / or close the valve of the cartridge when driving the first blank solution, driving the second blank solution, and driving the sample solution.

[0317] Example 49

[0318] According to the system of embodiment 48, 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] Example 50

[0320] A system for preparing and / or processing polynucleotides, the system comprising: a cassette; and a microfluidic actuator comprising: a cassette mount; multiple pressure lines; multiple 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 fixed to an inlet or outlet port held on a cassette held in the cassette mount; an ultraviolet (UV) light source; a UV photodetector; and a controller configured to control the application of pressure through the pressure lines to drive fluid through the cassette, wherein the controller is further configured to guide the formation of therapeutic polynucleotides, further wherein the control... The device is configured to estimate the concentration of the therapeutic polynucleotide by: driving a first blank solution through the 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; 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; and modifying the operation of the microfluidic actuator based on the concentration of the therapeutic polynucleotide determined from the first absorption measurement, the second absorption measurement, and the third absorption measurement.

[0321] Example 51

[0322] A method for manufacturing a polynucleotide using a microfluidic actuator device operated on a cartridge, the method comprising: forming a therapeutic polynucleotide in a cartridge; and determining the concentration of the therapeutic polynucleotide by the microfluidic actuator device through the following steps: driving a first blank solution into a concentration measurement chamber of the cartridge and performing 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; 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 actuator device, and based on this comparison, directing the therapeutic polynucleotide for one of the following: collection with one or more additional batches of therapeutic polynucleotides formed in the cartridge, discarding, or further analysis.

[0323] Example 52

[0324] According to the method described in Example 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] Example 53

[0326] According to the method of Example 52, estimating the concentration of the therapeutic polynucleotide further includes changing one or more of the sensitivity of the UV detector and the intensity of the UV emitter of the microfluidic actuator in response to the second absorption measurement via the cartridge.

[0327] Example 54

[0328] According to any one of Examples 52 to 53, the method further includes pretreating the UV measurement chamber with UV light prior to performing the first absorption measurement through the chamber.

[0329] Example 55

[0330] The method according to any one of Examples 51 to 54 further includes adjusting the operation of the microfluidic actuator device in the processor of the microfluidic actuator device based on the estimated concentration of the therapeutic polynucleotide.

[0331] Example 56

[0332] The method according to any one of Examples 51 to 55 further includes diluting the therapeutic polynucleotide to a standard concentration for output under the control of the processor of the microfluidic actuator.

[0333] Example 57

[0334] The method according to any one of Examples 51 to 55 further includes automatically adjusting one or more parameters of the formation of the therapeutic polynucleotide via the microfluidic actuator based on the estimated concentration of the therapeutic polynucleotide.

[0335] Example 58

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

[0337] Example 59

[0338] According to any one of Examples 51 to 58, determining the concentration of the therapeutic polynucleotide further includes performing a serial dilution in one or more chambers of the cartridge under the control of the microfluidic actuator, and repeating the step of driving the sample solution of the therapeutic polynucleotide for each of the one or more diluents formed by the serial dilution to generate a dilution profile.

[0339] Example 60

[0340] According to the method of Example 59, estimating the concentration of the therapeutic polynucleotide includes estimating the concentration of the therapeutic polynucleotide from the dilution curve.

[0341] Example 61

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

[0343] Example 62

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

[0345] Example 63

[0346] According to any one of Examples 51 to 62, the therapeutic polynucleotide comprises therapeutic mRNA.

[0347] Example 64

[0348] According to the method of Example 63, the method further includes encapsulating the therapeutic mRNA with a delivery vector.

[0349] Example 65

[0350] According to any one of Examples 51 to 64, the microfluidic actuator monitors 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 cartridge to open and / or close the valve of the cartridge.

[0351] Example 66

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

[0353] Example 67

[0354] A system for preparing and / or processing polynucleotides, the system comprising: a cassette mount; multiple pressure lines; multiple 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 fixed to an inlet or outlet port held on a cassette held in the cassette mount; and a controller configured to control the application of pressure through the pressure lines to drive fluid through the cassette, wherein the controller is further configured to guide the formation of a therapeutic polynucleotide in the cassette, and to estimate the concentration of the therapeutic polynucleotide by: driving a first blank solution through a measurement chamber of the cassette 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 cassette 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] Example 68

[0356] According to the system of embodiment 67, the system further includes: an ultraviolet (UV) light source; and a 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.

[0357] Example 69

[0358] According to the system of embodiment 68, 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 of the cell.

[0359] Example 70

[0360] According to any one of embodiments 68 to 69, the controller is further configured to pre-treat the UV measurement chamber with UV light prior to performing the first absorption measurement through the chamber.

[0361] Example 71

[0362] According to any one of Examples 67 to 70, the controller is further configured to modify the operation of the system based on the estimated concentration of the therapeutic polynucleotide.

[0363] Example 72

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

[0365] Example 73

[0366] According to any one of Examples 67 to 73, 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] Example 74

[0368] According to the system of Example 73, the one or more parameters include one or more of the following: temperature, reagent volume, reagent concentration, time, and mixing.

[0369] Example 75

[0370] According to any one of Examples 67 to 74, the system wherein the controller is further configured to perform serial dilution in one or more chambers of the cartridge, and to repeat the steps of driving the sample solution of the therapeutic polynucleotide for each of one or more diluents of the sample solution of the therapeutic polynucleotide formed by the serial dilution to generate a dilution profile.

[0371] Example 76

[0372] According to the system of Example 75, the controller is further configured to estimate the concentration of the therapeutic polynucleotide from the dilution curve.

[0373] Example 78

[0374] According to any one of Examples 67 to 76, the system wherein the controller is further configured to form the therapeutic polynucleotide, including performing an in vitro transcription (IVT) reaction to form the therapeutic polynucleotide.

[0375] Example 79

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

[0377] Example 80

[0378] The system according to any one of Examples 67 to 79, wherein the therapeutic polynucleotide comprises therapeutic mRNA.

[0379] Example 81

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

[0381] Example 82

[0382] According to any one of Examples 67 to 81, the controller is further configured to pneumatically deflect one or more regions of the membrane of the cartridge to open and / or close the valve of the cartridge when driving the first blank solution, driving the second blank solution, and driving the sample solution.

[0383] Example 83

[0384] According to the system of embodiment 82, 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] Example 84

[0386] A method comprising: emitting UV light through a UV measurement chamber of a cartridge, the UV measurement chamber being configured to receive fluid; receiving the UV light at a UV photodetector, the received UV light having passed through the UV measurement chamber of the cartridge; measuring a maximum voltage of the UV photodetector; measuring a standard deviation of a signal from the UV photodetector; determining an upper limit of quantitation (LoQ) based on the measured maximum voltage and based on the measured standard deviation; determining a lower LoQ based on the measured maximum voltage and based on the measured standard deviation; determining whether the upper LoQ and the lower LoQ are acceptable; and allowing the cartridge 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 and the lower LoQ are unacceptable.

[0387] Example 85

[0388] According to the method of Example 84, the method further includes forming or treating a therapeutic polynucleotide on the cassette.

[0389] Example 86

[0390] A cassette device for processing polynucleotides, the cassette device comprising: a first layer defining a first recess providing a first ultraviolet (UV) measurement region; a second layer defining a second recess providing a second UV measurement region; 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 region and the second UV measurement region, the UV measurement chamber being configured to receive 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 positioned on or within the first layer, the UV blocking feature being configured to restrict UV light from passing through or backscattering from the first layer.

[0391] Example 87

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

[0393] Example 88

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

[0395] Example 89

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

[0397] Example 90

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

[0399] Example 91

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

[0401] Example 92

[0402] According to any one of embodiments 86 to 91, the UV blocking feature is configured to block UV light from passing through the sidewall of the first recess.

[0403] Example 93

[0404] A method comprising: emitting UV light through a UV measurement chamber of a cartridge containing a fluid; receiving the UV light at a UV light detector; the received UV light having passed through the UV measurement chamber of the cartridge; obtaining a first plurality of concentration measurements, each of the first plurality of concentration measurements indicating a corresponding concentration of fluid in the UV measurement chamber; calculating an average of the first plurality of concentration measurements; comparing each of the first plurality of concentration measurements to the average of the first plurality of concentration measurements; identifying outliers from the first plurality of concentration measurements relative to the average of the first plurality of concentration measurements; establishing a second plurality of concentration measurements, the second plurality of concentration measurements including the first plurality of concentration measurements after removing the identified outliers; calculating an average of the second plurality of concentration measurements; and reporting the average of the second plurality of concentration measurements.

[0405] Example 94

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

[0407] Example 95

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

[0409] Example 96

[0410] According to the method of any one of Examples 93 to 95, due to the presence of bubbles in the fluid in the UV measurement chamber, the identified outlier values ​​provide concentration measurements that deviate significantly from the average of the first plurality of measurements.

[0411] Example 97

[0412] According to the method of embodiment 96, the method further includes performing a bubble removal algorithm in response to a predetermined degree by which the identified outlier deviates from the average of the first plurality of measurements.

[0413] It should be understood that all combinations of the foregoing concepts and other concepts discussed in more detail below (assuming that such concepts are not contradictory) are considered to be part of the inventive subject matter disclosed herein and can be used to achieve the beneficial effects described herein.

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

[0415] Any of the methods (including user interfaces) described herein can be implemented as software, hardware, or firmware, and can be described as a non-transitory computer-readable storage medium storing a set of instructions executable by a processor (e.g., a computer, tablet, smartphone, etc.), which, when executed by the processor, causes the processor to control any of the execution steps, including but not limited to: display, communicating with the user, analysis, modifying parameters (including timing, frequency, intensity, etc.), determining, warning, etc. For example, any of the methods described herein can be executed 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 procedures of the method.

[0416] While various embodiments have been described and / or illustrated herein in the context of a full-featured computing system, one or more of these example embodiments may be distributed as program artifacts of various forms, 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 can be stored on computer-readable storage media or in a computing system. In some embodiments, these software modules may configure the computing system to execute 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 parts of a single application. Additionally, in some embodiments, one or more of these steps may represent or correspond to one or more software applications or programs that, when executed by a computing device, cause the computing device to perform one or more tasks, such as the method steps.

[0418] Additionally, one or more of the devices described herein can transform data, physical devices, and / or representations of physical devices from one form to another. Alternatively or otherwise, one or more modules described herein can transform a processor, volatile memory, non-volatile memory, and / or any other part of the physical computing device from one form of computing device to another by executing on a computing device, storing data on a computing device, and / or otherwise interacting with a computing device.

[0419] As used herein, the term "computer-readable medium" generally refers to any form of device, carrier, or medium capable of storing or carrying computer-readable instructions. Examples of computer-readable media include, but are not limited to, transmissive media such as carrier waves, and non-transitory media such as magnetic storage media (e.g., hard disk drives, magnetic tape drives, and floppy disks), optical storage media (e.g., optical discs (CDs), digital video discs (DVDs), and Blu-ray discs), 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 herein as “on another feature or element,” it may be directly located on the other feature or element, or there may be intermediate features and / or elements present. Conversely, 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 the other feature or element, or there may be intermediate features or elements present. Conversely, 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 illustrated with respect to one embodiment, the features and elements so described or illustrated are applicable to other embodiments. Those skilled in the art will also understand that references to structures or features configured to be “adjacent” to another feature may have portions overlapping with or below that adjacent feature.

[0421] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. For example, as used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It should also be understood that, when used in this specification, the terms “comprising” and / or “including” specify the presence of the stated features, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof. As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items and may be abbreviated to “ / ”.

[0422] Spatial terms, such as “below,” “under,” “down,” “above,” “up,” etc., are used herein for ease of description to describe the relationship between one element or feature and another element or feature illustrated in the figures. It should be understood that spatial terms are intended to cover different orientations of the device in use or operation other than those depicted in the figures. For example, if the device in the figure is inverted, an element described as “below” or “under” other elements or features would be oriented “above” other elements or features. Thus, the exemplary term “below” can include both above and below orientations. The device may be oriented in other ways (rotated 90 degrees or otherwise) and the spatial descriptors used herein are interpreted accordingly. Similarly, the terms “up,” “down,” “vertical,” “horizontal,” etc., are used herein for illustrative purposes only unless otherwise expressly indicated.

[0423] Although the terms “first” and “second” are used herein to describe various features / elements (including steps), these features / elements should not be limited by these terms unless the context otherwise indicates. These terms are used to distinguish one feature / element from another. Therefore, without departing from the teachings of the 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] Generally speaking, any of the apparatuses and methods described herein should be understood as 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 substantially of various components, steps, sub-components or sub-steps”.

[0425] As used herein in the specification and claims, including as in the examples and unless otherwise expressly stated, all figures are to be interpreted as if they begin with the words “about” or “approximately”, even if the term is not explicitly stated. When describing values ​​and / or locations, the phrases “about” or “approximately” may be used to indicate that the described value and / or location is within a reasonably expected range of the value and / or location. For example, a numerical value may have values ​​of + / -0.1%, + / -1%, + / -2%, + / -5%, + / -10%, etc., of the value (or range of values). Unless the context otherwise requires, any numerical value given herein should also be understood to include approximately or approximately that value. For example, if the value “10” is disclosed, “about 10” is also disclosed. Any numerical ranges described herein are intended to include all subranges contained therein. It should also be understood that when a value is disclosed, the terms "less than or equal to" that value, "greater than or equal to" that value, and the possible range between values ​​are also disclosed, as properly understood by one of skill in the art. For example, if the value "X" is disclosed, "less than or equal to X" and "greater than or equal to X" (e.g., where X is a numerical value) are also disclosed. It should also be understood that throughout the application, data is provided in a variety of different formats, and that the data represents a range of endpoints and start points, as well as any combination of data points. For example, if specific data point "10" and 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 values ​​between 10 and 15, are all considered disclosed. It should also be understood that each 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 have been described above, any of the many changes to the various embodiments may be made without departing from the scope of the invention as described in the claims. For example, in alternative embodiments, the order in which the various 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 others. Therefore, the foregoing description is provided primarily for illustrative purposes and should not be construed as limiting the scope of the invention as set forth in the claims.

[0427] The examples and illustrations included herein are shown by way of illustration, not limitation, of specific embodiments in which the subject matter may be practiced. As mentioned, other embodiments, and those derived therefrom, may be utilized, allowing for structural and logical substitutions and changes without departing from the scope of the invention. These embodiments of the subject matter may be referred to herein individually or collectively by the term “invention” for convenience only, and not to intentionally limit the scope of this application to any single invention or inventive concept, given that more than one invention or inventive concept has actually been disclosed. Therefore, although particular embodiments have been illustrated and described herein, any arrangement calculated to achieve the same purpose may replace the particular embodiments shown. This disclosure is intended to cover any and all modifications or variations of the various embodiments. After reviewing the above description, combinations of the above embodiments, as well as other embodiments not specifically described herein, will be apparent to those skilled in the art.

Claims

1. A method for manufacturing polynucleotides using a microfluidic actuator that operates on a cartridge, the method comprising: A therapeutic polynucleotide is formed in the box, wherein the therapeutic polynucleotide comprises a therapeutic mRNA; The concentration of the therapeutic mRNA is determined by the microfluidic actuator device through the following steps: A first blank solution is driven into the ultraviolet (UV) measurement chamber of the box and a first absorption measurement is performed through the box; The sample solution of the therapeutic polynucleotide is driven into the UV measurement chamber of the cartridge and a second absorption measurement is performed through the cartridge; The second blank solution is driven into the UV measurement chamber of the box and a third absorption measurement is performed through the box; as well as The concentration of the therapeutic polynucleotide is estimated from the first absorption measurement, the second absorption measurement, and the third absorption measurement; as well as The operation of the microfluidic actuator is adjusted in the processor of the microfluidic actuator based on the estimated concentration of the therapeutic polynucleotide.

2. A system for preparing and / or processing polynucleotides, said system comprising: Box mounting components; Multiple pressure lines; Multiple fluid lines, each fluid line being 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 or outlet port held on a box in the box 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 guide the formation of a therapeutic polynucleotide in the cartridge and to estimate the concentration of the therapeutic polynucleotide by: driving a first blank solution through the 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; further, wherein the controller is configured to estimate the concentration of the therapeutic polynucleotide from the first absorption measurement and the second absorption measurement.

3. The system of claim 2, wherein the controller is further configured to modify the operation of the system based on the estimated concentration of the therapeutic polynucleotide.

4. The system according to any one of claims 2 to 3, wherein the controller is further configured to compare the estimated concentration of the therapeutic polynucleotide with a concentration range, and based on this comparison, direct the therapeutic polynucleotide for one of the following: collection, disposal, or further analysis with one or more additional batches of therapeutic polynucleotides formed in the cassette.

5. A system for preparing and / or processing polynucleotides, said system comprising: box; and A microfluidic actuator device, the microfluidic actuator device comprising: Box mounting components; Multiple pressure lines; Multiple fluid lines, each fluid line being 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 or outlet port held on a box in the box 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 guide 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 the 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; 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; and modifying the operation of the microfluidic actuator based on the concentration of the therapeutic polynucleotide determined from the first absorption measurement, the second absorption measurement, and the third absorption measurement.

6. A method for manufacturing polynucleotides using a microfluidic actuator that operates on a cartridge, the method comprising: Therapeutic polynucleotides are formed in the box; as well as The concentration of the therapeutic polynucleotide is determined by the microfluidic actuator using the following steps: The first blank solution is driven into the concentration measurement chamber of the box and a first measurement is performed through the box; The sample solution of the therapeutic polynucleotide is driven into the concentration measurement chamber of the cartridge and a second measurement is performed through the cartridge; as well as The concentration of the therapeutic polynucleotide is estimated from the first and second measurements; as well as The processor of the microfluidic actuator compares the estimated concentration of the therapeutic polynucleotide with a concentration range, and based on this comparison, directs the therapeutic polynucleotide for one of the following: collection with one or more additional batches of therapeutic polynucleotides formed in the cassette, disposal, or further analysis.

7. The method according to claim 6, 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.

8. The method of claim 7, wherein estimating the concentration of the therapeutic polynucleotide further comprises altering one or more of the sensitivity of the UV detector and the intensity of the UV emitter of the microfluidic actuator in response to the second absorption measurement via the cartridge.

9. The method according to any one of claims 7 to 8, the method further comprising pretreating the UV measurement chamber with UV light prior to performing the first absorption measurement through the chamber.

10. A system for preparing and / or processing polynucleotides, said system comprising: Box mounting components; Multiple pressure lines; Multiple fluid lines, each fluid line being 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 or outlet port held on a box in the box mount. 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 guide the formation of a therapeutic polynucleotide in the cartridge and to estimate the concentration of the therapeutic polynucleotide by: driving a first blank solution through the measurement chamber of the cartridge 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 cartridge 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.

11. The system of claim 10, further comprising: Ultraviolet (UV) light source; and UV light detector; 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; 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; The controller is configured to estimate the concentration of the therapeutic polynucleotide from the first absorption measurement and the second absorption measurement.

12. The system of claim 11, 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 of the cell.

13. The system according to any one of claims 11 to 12, 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 chamber.

14. A method, the method comprising: UV light is emitted through the ultraviolet (UV) measurement chamber of the box, which contains a fluid; The UV light is received at the UV light detector, and the received UV light has passed through the UV measurement chamber of the box; A first plurality of concentration measurements are obtained, each of the first plurality of concentration measurements indicating the corresponding concentration of the fluid in the UV measurement chamber; Calculate the average value of the first plurality of concentration measurements; Each of the first plurality of concentration measurements is compared with the average value of the first plurality of concentration measurements; Identify outliers from the first plurality of concentration measurements relative to the average value of the first plurality of concentration measurements; Establish a second plurality of concentration measurements, the second plurality of concentration measurements including the first plurality of concentration measurements after removing the identified outliers; Calculate the average value of the second plurality of concentration measurements; as well as The average value of the second plurality of concentration measurements is reported.

15. The method of claim 14, wherein the first plurality of concentration measurements comprises four concentration measurements.

16. The method according to any one of claims 14 to 15, wherein the second plurality of concentration measurements comprises three concentration measurements.

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