Method for sensor device sample incubation loading

Abstract

For various sequencing systems, effectively loading template beads into the sensor device is a crucial step among many important steps in providing high-quality sequencing data. Loading various types of template beads into the microwell array of a sensor device can be effectively achieved through incubation loading. The time required for incubation loading of a sensor device can be well within the range of a desired device loading workflow for sequencing, ranging from approximately 10 minutes to approximately 1 hour.

Description

[Background technology]

[0001] For various sequencing systems, effectively loading template beads into the sensor device is a critical step among many important steps in providing high-quality sequencing data. The loading solution can contain hundreds of millions of hydrophilic polymer beads to be loaded into the sensor device's reaction chamber. To ensure that optimal loading is performed within a target timeframe, considering the various sizes of hydrophilic polymer beads related to the cross-sectional area of ​​the reaction chamber opening, instrument-mediated methods for loading template beads into the sensor device are utilized. [Overview of the Initiative] [Means for solving the problem]

[0002] According to this disclosure, optimal loading within a target timeframe can be achieved under selected conditions for the incubation of the sensor device with the bead sample. The time required for the incubation loading of the sensor device may be well within the range of a desired device loading workflow for sequencing, approximately 10 minutes to approximately 1 hour. [Brief explanation of the drawing]

[0003] Novel features of this disclosure are specifically described in the appended claims. A better understanding of the features and advantages of what is disclosed herein will be obtained by referring to the following detailed description, which illustrates illustrative examples in which the principles of this disclosure are used, and to the appended drawings.

[0004] [Figure 1] This is a perspective view illustrating an exemplary sensor device of the present disclosure. [Figure 2] This is a perspective view illustrating an exemplary sensor device of the present disclosure.

[0005] [Figure 3]This is a general overview illustrating a physical-mechanical approach to loading beads into the microwells of a sensor device.

[0006] [Figure 4] This is a schematic description illustrating the general use of centrifugation for loading beads into the microwells of a sensor device.

[0007] [Figure 5] Figures 1 and 2 provide a conceptual schematic illustration of the first step in the incubation loading of a bead sample into a flow cell for a sensor device.

[0008] [Figure 6] This is a general overview illustrating a sensor device loaded with beads.

[0009] [Figure 7] This is a series of micrographs illustrating sensor device loading studies, illustrating bead loading of a sensor device as a function of bead input and temperature.

[0010] [Figure 8] Figure 7 shows graphs and tables of data from the loading study.

[0011] [Figure 9] These graphs and tables illustrate a general overview of sensor device loading studies, showing bead loading of a sensor device as a function of time and temperature.

[0012] [Figure 10] This graph illustrates the general relationship between the bead diameter per unit of the cross-sectional diameter of the microwell opening for various sensor devices described herein.

[0013] [Figure 11]A schematic diagram generally illustrating variables that affect the loading of two or more beads per well.

[0014] [Figure 12] A schematic diagram generally illustrating a sequencing system using the sensor device of the present disclosure.

[0015] [Figure 13A] A schematic diagram generally illustrating another sequencing system using the sensor device of the present disclosure.

[0016] [Figure 13B] A perspective view generally illustrating a sequencing system such as the sequencing system of FIG. 13A.

[0017] [Figure 13C] A perspective view illustrating the container cabinet of the sequencing system of FIG. 13B.

[0018] [Figure 14] A schematic cross-sectional view generally illustrating the flow cell chambers of various sensor devices of the present disclosure.

[0019] [Figure 15] A schematic depiction conceptually illustrating a cross-section of a sensor device including a microwell array across the top of the sensor device.

[0020] [Figure 16] A schematic depiction generally illustrating a method for preparing amplified beads.

[0021] [Figure 17] A schematic depiction generally illustrating a method for preparing single-template beads.

[0022] [Figure 18] A schematic depiction generally illustrating a method for on-device preparation of templated beads from single-template beads. [Modes for carrying out the invention]

[0023] For various sequencing systems, effectively loading template beads into the sensor device is a critical step among many important steps in providing high-quality sequencing data. The loading solution can contain hundreds of millions of beads to be loaded into the microwells of the sensor device. Various sensor devices of this disclosure can have microwells with a cross-sectional diameter of the microwell opening ranging from about 0.65 μm to about 1.30 μm, and the beads can have a diameter ranging from 0.8 μm in the case of single-copy template beads to 1.2 μm in the case of amplified or template beads. As will be described in more detail herein, for example, various devices have utilized templated beads for loading into sensor devices to enhance the loading process in order to ensure that optimal loading is performed within a target time frame. In contrast, according to this disclosure, optimal loading within a target time frame can be achieved under selected conditions of incubation of the sensor device with a sample of beads.

[0024] Figure 1 illustrates an exemplary sensor device of the present disclosure. As depicted in Figure 1, the sensor device 100 includes a substrate 110 on which a sensor die 120 is mounted, the substrate 110 providing wall structures that seat on the substrate 110 and provide electrical contacts between the sensor die 120 of the sensor device and the substrate 110. As will be described in more detail herein, the substrate 110 having the internally mounted sensor die 120 provides the bottom and walls of the flow cell. The sensor die 120 can be fabricated within a semiconductor substrate. The sensor die 120 may include a microwell array formed on a sensor array. In Figure 1, the flow cell 160 includes a flow cell cover 130 that is sealably mounted on the sensor die 120. For example, the flow cell cover 130 can be bonded to the sensor die 120 using an adhesive. The flow cell cover 130 includes fluid ports 133 and 135 that fluidly communicate with the flow cell chamber 140. The flow cell chamber 140 is defined between the flow cell cover 130 and the sensor die 120. Since the fluid ports are functionally interchangeable, one fluid port can be used as an inlet port while the other can be used as an outlet port. Thus, fluid applied to either port can flow through the flow cell chamber 140 and out through the opposite port.

[0025] Figure 2 illustrates an exemplary multi-lane sensor device of the present disclosure. As depicted in Figure 2, the multi-lane sensor device 200 includes a substrate 210 on which a sensor die 220 is mounted. The sensor die 220 may include a microwell array formed on the sensor array. In a manner similar to that described for the sensor device 100 in Figure 1, the flow cell cover 230 in Figure 2 is firmly mounted on the sensor die 220. However, in the case of the multi-lane sensor device 200, the sensor die 220 of the flow cell volume is divided into a plurality of separate flow cells, for example, using a compressible gasket formed between the sensor die 220 and the flow cell cover 230. As a non-limiting example, the multi-lane sensor device 200 may be divided into flow cells 260A to 260D. The flow cells 260A to 260D are also referred to as flow cell lanes 260A to 260, and each flow cell lane is individually fluid-communicated with a set of fluid ports. As illustrated in Figure 2, the flow cell cover 230 of the multi-lane sensor device 200 includes four sets of first fluid ports 233A-233D and four sets of second fluid ports 235A-235D. Either set of fluid ports 233A-D or 235A-D can be used as inlet ports, since the sets are functionally interchangeable, while the other set can be used as outlet ports. Thus, the fluid applied to either set of ports can flow out through each respective flow cell chamber 240A-240B from the opposite set of ports. Finally, alignment pins 260A and 260B are used to mount the multi-lane sensor device 200 within the analysis system.

[0026] As illustrated in Figure 2, the multi-lane sensor device 200 includes four flow cell lanes, but a multi-lane sensor device can include fewer than four flow cell lanes or five or more flow cell lanes. For example, the multi-lane sensor device 200 can include 2 to 10 flow cell lanes, such as 2 to 8 flow cell lanes or 4 to 6 flow cell lanes. Finally, considering that each lane of the multi-lane sensor device is effectively a sensor device similar to the sensor device 100 in Figure 1, as will be described in more detail herein, flow cell lanes such as lanes 260A to 260C in Figure 2 can be used at different times or simultaneously, depending on a user-defined sequencing execution plan.

[0027] Tables 1 and 2 summarize the attributes of various exemplary sensor devices of this disclosure, such as sensor device 100 in Figure 1 and sensor device 200 in Figure 2. [Table 1] [Table 2]

[0028] As can be seen by examining Tables 1 and 2, there are many similarities among the device types presented in the tables. For example, Type 1-1 devices and Type 2-1 devices share many of the same attributes, but are differentiated by the multi-lane structure of the devices in Figure 2. In addition, there are some differences between the top cross-sectional diameter (TXSD) of the microwell openings of Type 1 devices and Type 2 devices.

[0029] Currently, apparatus and related methods for loading bead samples into microwell arrays are based on minimizing loading time through instrument-mediated facilitation of loading beads into microwell arrays. For example, Figure 3 conceptually illustrates a physicomechanical approach to bead loading using magnetic loading within a sensor device 150, which can be either the sensor device 100 in Figure 1 or the sensor device 200 in Figure 2. As illustrated, the sensor device 150 includes a flow cell cover 130 and a microwell array 300, which border the flow cell chamber 140. In Figure 3, the magnet loads a magnetic loading bead pile 60 from a combination of beads and magnetic loading beads 60 having a radius substantially larger than each bead 50. P Create a magnetic loading bead pile 60 as shown. P This pushes the beads toward the microwell array 300, increasing the probability that the beads will be loaded into the microwells. Although shown in a horizontal configuration, depending on the modeling, magnetic loading, as depicted in Figure 3, can also be performed in a vertical orientation.

[0030] As an additional example, Figure 4 conceptually illustrates the loading of beads into a sensor device 150 using centrifugation. The sensor device 150 in Figure 4 is similar to that described in Figure 3 and includes a flow cell cover 130 and a microwell array 300 bordering the flow cell chamber 140. As illustrated in Figure 4, a bead sample 55 containing a collection of hydrophilic polymer beads 50 is loaded into the flow cell chamber 140. The sensor device 150 can be inserted into a centrifuge, and as a result, the bead sample receives a defined centripetal force for a defined time, driving the movement of the beads onto the surface of the microwell array 300, thereby facilitating the loading of the beads into the microwells.

[0031] The common definition of incubation is maintaining a chemical or biochemical system under specific conditions favorable to development or reaction. Therefore, the specific development in this disclosure is the interaction between hydrogel beads and the microwell array surface of a sensor device, leading to the loading of microwell sites. The inventors performed initial experiments in which the loading of hydrogel beads into the microwell array of a sensor device was carried out without the use of any instrument-mediated method, but even more surprisingly, by incubating a sensor device with a sample of beads. Following these initial experiments, the inventors identified conditions under which the time required for incubation loading of the sensor device could be well within the range of a desired device loading workflow for sequencing, approximately 10 minutes to approximately 1 hour.

[0032] Incubation loading, as conceptually depicted in Figure 5, may, in a first step, involve introducing a bead sample 55 into the flow cell chamber 140 of the sensor device 150. The sensor device can be oriented horizontally or vertically. Each bead 50 is bounded in a first orientation between the flow cell cover 130 and the microwell array 300, and bounded in a second orientation by the flow cell wall (not shown). Beads close to the microwell array 300 may encounter interstitial surfaces of the microarray, such as each interstitial surface 321 depicted in Figure 5, or may encounter a well opening 312 and be loaded into the well. As will be described later herein, hydrogel beads having a diameter of about 60% to about 120% of the cross-sectional diameter of the microwell opening can be loaded into the microwell arrays of various sensor devices of this disclosure via incubation loading without loading two beads into the same well. As illustrated in Figure 5, when aligned with the opening 312 of the microwell 310, instead, for example, the intervening surface 321, the hydrogel beads can be loaded into the microwell 310. Although not bound by theory, modeling of the spatial movement of beads in a flow chamber performed by the inventors suggests that, once a bead reversibly encounters a surface, the bead has a 98% probability of finding the surface again within 5 minutes under typical experimental conditions such as those described herein. Therefore, even if the bead 50 first encounters the intervening space due to its proximity to the microwell array, there is a high probability that the bead 50 will interact within the defined timeframe for loading into the microwell.

[0033] Figure 6 is a schematic, generally illustrative depiction of a bead sample 55 containing a collection of hydrophilic polymer beads, such as beads 50, being loaded into the microwell array 300 of a sensor device 150. All loading methods described begin with introducing the bead sample into a flow cell, as depicted in Figure 5, and end with the loaded sensor device, as depicted in Figure 6; however, incubation loading is the only method described herein, and there is no additional processing between the introduction of the beads into the flow cell and the loaded sensor device. In the case of incubation loading of beads with reduced radius, a solution with appropriately varied ionic strength or dielectric constant can be introduced into the flow cell chamber 140 to provide beads with an extended radius that seat in each microwell.

[0034] Figure 7 is a compilation of micrographs illustrating an investigative sensor device loading study based on initial observations, in which bead loading was investigated as a function of bead input and temperature at regular time intervals. This study was conducted using non-template hydrogel beads. In this study, bead samples were sequentially loaded into each of three multi-lane devices, as described for device 2-1 in Figure 2 and Table 2. Each of the four loading solution preparations varied the amount of hydrogel beads from a loading solution containing 100 million beads per loading solution (100M) to a loading solution containing 400 million beads per loading solution (400M). Each loading solution was prepared by mixing 20 μL of a solution containing the target number of beads with 6 μL of a 0.2 M Tris buffer solution at pH 8.0, also containing 1.0 M potassium chloride, 0.23 M magnesium chloride, and 0.1% Triton® X-100. Under these conditions, the ratio of the bead diameter to the cross-sectional diameter of the well opening was 0.8 μm. Therefore, each lane of the three multi-lane devices was filled using 26 μL of each preparation of the loading solution. Considering that each lane of device 2-1 in Table 2 had a volume of 12 μL, the flow cell was flushed with the excess volume of the prepared solution to ensure that all air bubbles were removed. Each device was then incubated at the specified temperature for 10 minutes, after which any remaining, unloaded beads were flushed out by injecting 100 μL of phosphate-buffered saline (PBS) with 0.2% Tween® into each lane. Although not necessary for effective loading, the beads were seated more deeply in the well by drawing a vacuum over the lane for 10 seconds before and after injecting 50 μL of a mixture of 60 vol% PBS and 40 vol% isopropyl alcohol with 0.2% Tween®.

[0035] As can be seen from a visual examination of Figure 7, bead concentration and temperature have a clear effect on the degree of loading over a set time. Figure 8 provides graphs and tables of data from the loading study in Figure 7. A close examination of the graphs and tables in Figure 8 reveals that bead concentration and temperature over 10 minutes ranged from 14% loading (4°C / 100M beads) to 78% loading (50°C / 400M beads). This initial study demonstrated the feasibility of achieving target loading for sequencing of 65% to 95% within a desired workflow for sequencing.

[0036] Figure 9 is a graph and table illustrating a sensor device loading study, illustrating the bead loading of a sensor device as a function of time and temperature at selected bead concentrations. This study was conducted by sequentially loading each of two multiplane devices, as described for device 2-1 in Figure 2 and Table 2, using single-template hydrogel beads. The study used a loading solution containing 300 million beads (300M) per loading solution. For each lane loaded in the two devices, the solution was prepared by mixing 20 μL of a solution containing 300M beads with 6 μL of a 0.2 M Tris buffer solution at pH 8.0, also containing 1.0 M potassium chloride, 0.23 M magnesium chloride, and 0.1% Triton® X-100. Under these conditions, the ratio of the bead diameter to the cross-sectional diameter of the well opening was 0.8 μm. For each time point, one lane of each device was filled with the loading buffer and then incubated at the specified temperature for the specified time.

[0037] A closer examination of the graphs and tables in Figure 9 reveals that, using a 300M bead loading solution, sensor loading as a function of time and temperature ranged from 71% loading (35°C / 20 min) to 99.5% loading (50°C / 20 min). Generally, a loading of approximately 65% ​​to 95% is desirable to achieve high-quality sequencing execution. As provided in the results presented in Figure 9, these results were easily achieved using incubation loading. Lower boundaries for desired loading are based on achieving the desired number of readings, while higher boundaries for desired loading are based on avoiding overloading. Overloading is defined as loading high enough to negatively impact sequencing performance. For example, overloading may result in the elimination of a desired number of empty wells that could be used as reference wells for signal processing. The threshold for overloading depends on various factors and is typically above 97%. Overloading is a concern with magnetic loading and loading using centrifugal separation, and can be easily avoided by using incubation loading by employing shorter incubation times and / or lower incubation temperatures.

[0038] Figure 10 is a graph illustrating the general relationship between the ratio of bead diameter to the cross-sectional diameter of the microwell opening for various sensor devices of this disclosure. The bead diameter is determined by settling dilution beads on a glass coverslip in a PBS solution. The solution surrounding the beads can then be replaced with the solvent of interest. The bead diameter can then be measured using an optical microscope by comparing their apparent diameter to a calibration curve generated using hard spherical polystyrene particles of known diameter. Taking into account the variability in the diameter of the microwell opening, and therefore the upper cross-sectional diameter of the microwells of various devices, beads can be produced according to the size of the microwell opening and the microwell dimensions. As shown in Tables 1 and 2, factors that may influence the selection of bead diameters for use in any given device include the bead diameter as a result of the template method, the variation in bead diameter as a result of the various buffers and diluents used during loading and sequencing, and the consideration of beads having a diameter large enough to load one bead per microwell.

[0039] For the studies used to generate the graph in Figure 10, the devices in Table 2 were used and loaded with single-template beads. In addition, devices of type 1-3 were used and loaded with fully templated beads. The devices of type 2-1 used in this study had a top cross-sectional diameter of 1.35 μm and were loaded with single-template beads in a first buffer B1 having a composition of 1.6 × PBS + 0.32% Tween® 20, or in a second buffer B2 having a composition of 0.036 mM Tris-HCl, pH 8.0, 180 mM KCl, 41.4 mM MgCl2, and 0.02% Triton® X-100. The average diameter of the beads was 1.06 μm in buffer B1 and 1.04 μm in buffer B2, resulting in ratios of 0.78 and 0.77, respectively. The 2-2 type device used in this study had an upper cross-sectional diameter of 1.08 μm and was loaded with a single template bead in either B1 buffer or B2 buffer. The average diameter of the beads was 0.86 μm in B1 buffer and 0.85 μm in B2 buffer, resulting in ratios of 0.80 and 0.79, respectively. The 3-2 type device used in this study had an upper cross-sectional diameter of 0.65 μm and was loaded with a single template bead in either B1 buffer or B2 buffer. The average diameter of the beads was 0.63 μm in B1 buffer and 0.62 μm in B2 buffer, resulting in ratios of 0.97 and 0.95, respectively.

[0040] Compared to two types of 660-megapixel devices loaded with single template beads, the three types of sensors described in Table 1 and Figure 1 were loaded with fully amplified beads. As will be disclosed later herein, such beads can be prepared using emulsion PCR, resulting in highly negatively charged beads. For the studies used to generate the data presented in Figure 10, fully templated beads with a diameter of 0.80 μM were used in buffer B3 having a composition 0.33 × PBS containing 32 mM TrisHCl, pH 8.0, and further 160 mM KCl, 37 mM MgCl2 containing 0.02% Triton® X-100, and 0.07% Tween® 20. Considering that the top cross-sectional diameter of the three types of chips is 0.65 μm, this results in a bead diameter / MW aperture diameter ratio of 1.2. Therefore, the 120% upper limit for the ratio of bead diameter to microwell opening cross-sectional diameter is clearly demonstrated by the data for the 1-3 types of tips presented in Figure 10.

[0041] The consideration of a lower limit for bead diameter versus microwell opening cross-sectional diameter is based on an analysis of bead diameter size limitations that would exclude loading more than two beads per well. Figure 11 is a schematic diagram illustrating the variables that generally affect loading more than two beads per well. The estimated lower limit can be derived from the following equation, referring to the schematic diagram in Figure 11.

number

[0042] Using Equation 1, for example, with respect to bead 501 in Figure 11, we can estimate the minimum bead diameter that would prevent a second bead, such as bead 502 in Figure 11, from being loaded into a microwell, such as microwell 10 in Figure 10. Table 3 summarizes the results of the analysis. [Table 3]

[0043] Based on the estimates provided by Equation 1, the minimum bead diameter that would result in excluding the second bead from loading is provided in Table 3. Taking into account measurement tolerances, a 60% lower limit for the ratio of the minimum bead diameter to the cross-sectional diameter of the microwell opening is indicated by the analysis performed, as summarized in Table 3.

[0044] In addition, a lower limit based on the estimate provided by Equation 1 can provide the minimum bead diameter to microwell height ratio that would exclude a second bead from loading. Taking into account measurement tolerances, a 65% lower limit for the minimum bead diameter to microwell height ratio is shown by the analysis performed, as summarized in Table 4. [Table 4]

[0045] Regarding the preparation of the loading solution, considering the properties of the bead starting material and the target percentage loading of 65% to 95% of the microwells within the specified loading conditions of time and temperature, the target ratio of 2 to 4 beads per microwell was determined. Table 5 below shows the ratio of the number of beads per microwell, calculated for the target number of beads (in millions) in the loading solution for some exemplary devices in Tables 1 and 2. As can be seen by examining Table 5, the preparation of the loading solution must be adjusted appropriately to provide a lower limit of 2 beads per microwell. For example, to load a single lane of a 2-1 type device, the flow cell volume of the lane is approximately 12 μL. A sufficient volume to process, for example, a preparation of 150 million beads in 26 μL can be prepared. Considering the number of microwells per lane, as provided in Table 2, such a loading solution will provide a ratio of 2 beads per microwell for loading the lane. In contrast, for a Type 1-1 device with a flow cell volume of approximately 45 μL, considering the number of microwells as provided in Table 1, a loading solution prepared using 350 million beads in 50 μL would provide a ratio of 2 beads per microwell for loading the device. [Table 5] Sequencing systems, devices, and reagents

[0046] Figure 12 shows a system used to perform sequencing using the sensor device of the present disclosure. Figure 12 is a block diagram illustrating a chemFET-based analytical system of the present disclosure in general terms, depicting the integration of various elements of a fluid system 1020 having a fluid multiplexer device 1030 and a sensor device 150 which can be the sensor device 100 in Figure 1 or the multi-lane sensor device 200 in Figure 2. As will be described in more detail herein, the sensor device 150 may include a sensor or pixel array or a microwell array cooperatively engaged on a pixel array, where each microwell is capacitively coupled to at least one pixel. Various devices can be fabricated so that each microwell is coupled to 1 to 4 sensors or pixels. As listed herein, the terms “sensor” and “pixel,” as well as the terms “device” and “chip,” and their derivatives, can be used synonymously. In addition, “sensor array” and “chemFET sensor array,” and their derivatives, can be used synonymously.

[0047] As depicted in Figure 12, the reagent and solution containers 1040A-40E of the fluid system 1020 are in fluid communication with the fluid multiplexer device 1030. During use, the sensor device 150 is sealedly attached to the fluid multiplexer device 1030. The microfluidic multiplexer circuit 1032 of the fluid multiplexer device 1030 allows various user-selected reagents and solutions from the reagent and solution containers 1040A-1040E to be controllably delivered to the sensor device 150 via reagent values ​​1043 controlled through the system controller 1010. The selected reagent or solution is positioned in fluid communication with each of the inlet ports, such as the fluid multiplexer circuit inlet port 1034 in Figure 12, and can then be directed to the sensor device 150 via the fluid multiplexer device inlet channel 1036 of the fluid multiplexer device 1030. The effluent flowing from the sensor device 150 is returned to the fluid multiplexer device 1030 via the fluid multiplexer outlet channel 1038, which can be configured to fluidly communicate with any of the fluid manifold waste lines 1041A to 1041B leading to the waste container 1044. Thus, reagents and solutions from the fluid system 1020 can be selectively driven through the fluid multiplexer device 1030 and then driven through the sensor device 150 to the waste container 1044, controlled by the reagent valve 1043 which receives a signal from the system controller 1010.

[0048] The system controller 1010 in Figure 12 provides control of the cleaning fluid valves 1043A and 1043B, as well as the cleaning fluid valves 1045A and 1045B. The reference electrode 1046 is a critical component for providing a stable reference voltage to the sensor device, as each sensor in the sensor device generates an output signal that depends on a stable reference voltage value. As depicted in Figure 12, the reference electrode 1046 is in fluid communication with the cleaning fluid container 1040F through the cleaning fluid fluid line 1047 and is in contact with the sensor device 150 via the fluid multiplexer device 1030. The cleaning fluid container 1040F contains a cleaning fluid of a known electrolyte composition. Thus, a constant electrolyte fluid environment of the reference electrode 1046 provides a constant and stable reference voltage to the sensor device 150. As depicted in Figure 12, the tee in the cleaning fluid line provides a controllable flow path for the cleaning fluid to the microfluidic multiplexer circuit 1032 and to the fluid multiplexer device inlet channel 1036. When a reagent or solution is controllably selected from reagent and solution containers 1040A to 1040E, the washing solution valve 1045B is closed while the reference electrode 1046 is in contact with the washing solution. If the washing solution is the selected solution, the washing solution valve 1045B is opened. During the delivery of reagents or solutions through the system, the reference electrode 46 remains in constant contact with the washing solution, and a stable reference potential is applied to the sensor device 150. According to this disclosure, the reference electrode 1046 can be a hollow cylindrical structure of an inert metal, for example, stainless steel, platinum, or titanium, in non-limiting examples. Such a hollow cylindrical metal structure can provide effective ohmic contact with the fluid in the flow stream. Alternatively, the reference electrode can be a wire or a plate.

[0049] Figure 13A is a block diagram illustrating the sequencing system of this instruction in general, which may be a sequencing system incorporating a sample preparation platform. As depicted in Figure 13A, the sequencing system 2000 may include a sample preparation deck 2004, a loading station 2006, and a controller 2002 that communicates with the sequencing station 2008. The sample preparation deck 2004 may include a pipetting robot 2012, which may be a three-axis pipetting robot. The pipetting robot 2012 can access samples 2014, reagents and solutions 2016, a thermocycle 2018, and other devices 2020 such as a magnetic separator or centrifuge. Target sequences of samples to be analyzed on the sequencing system 2000 can be prepared on the sample preparation deck 2004 and then provided to the loading station 2006. For example, the sample preparation deck 2004 can provide library preparation of the sample to be analyzed, as well as preparation of target sequences from the library that can be used to prepare particle or bead samples. Such particle or bead samples can then be provided to the loading station 2006 to be loaded into a sensor device, such as the sensor device 200 in Figure 2. Loading can be performed using magnetic loading, as described with respect to Figure 3. Alternatively, loading can be performed using incubation loading, as described herein with respect to Figures 5 to 11.

[0050] Once loaded, the sensor device can be transferred to the sequencing station 2008 using a sliding mechanism 2007 that can move the sensor device from the loading position to the sequencing position. The sequencing station 2008 may include both fluid and electronic interfaces to automatically process the sample loaded into the sensor device during sequencing. The container cabinet 2010 can accommodate containers for holding various reagents and solutions used in sequencing, and can also accommodate various waste liquid containers. Data collected from the sensor device can be provided to the sequencing computer 2022, which can perform base calling, read alignment, and variant calling.

[0051] The controller 2002 can further communicate with user interfaces such as a monitor, keyboard, mouse, touchscreen, or any combination thereof, among other interfaces such as user interface 2024 in Figure 13A. Furthermore, the controller 2002 can communicate with a network interface that can access a local area network, a wide area network, or a global network. The network interface 2026 can be a wired or wireless interface using various standard communication protocols. The sequencing system 2000 can be powered by a power supply 2028.

[0052] Figure 13B is a perspective view illustrating the sequencing system of this instruction in general. The sequencing system 2500 in Figure 13B can be a sequencing system having various components, as described for the sequencing system 2000 in Figure 13A. The sequencing system 2500 may include an upper section 2502 and a container cabinet 2510, such as the container cabinet 2010 in Figure 13A. The upper section 2502 may include a door 2506 for accessing a sample preparation deck 2504, where samples to be analyzed, reagent containers, and other consumables, such as those described for Figure 13A, can be placed. In addition, various examples of sequencing systems, such as the sequencing system 2500, may include a user interface, such as a touchscreen display 2508.

[0053] Figure 13C illustrates in general terms a container cabinet 2510 that can be a component of a sequencing system, such as the sequencing system 2000 in Figure 13A and the sequencing system 2500 in Figure 13B. The container cabinet 2510 may be useful for managing fluid handling for the sequencing system. For example, as depicted in Figure 13C, the container cabinet 2510 includes a reagent cartridge loading interface 2512 for loading reagent concentrate cartridges. Furthermore, the container cabinet 2510 can accommodate various containers for holding reagents and solutions. For example, washing and cleaning solutions can be held in the containers of the first container assembly 2514 in Figure 13A. Furthermore, bulk nucleotide reagents, as well as bulk calibration solutions, can be held in the containers of the second container assembly 2516 in Figure 13C. In addition, the container cabinet can accommodate a main waste container for collecting various sample preparation wastes, sensor wastes, and effluents. For example, the first waste container 2518A can collect effluent generated during sample preparation, such as effluent produced from the sample preparation deck 2004 of the sequencing system 2000 in Figure 13A and the sample preparation deck 2504 in Figure 13B. In addition, the second waste container 2518B in Figure 13C can collect effluent generated from the system fluid system during sequencing execution, for example.

[0054] Figure 14 illustrates a general cross-sectional view of sensor device 150, which may be a cross-section of sensor device 100 in Figure 1 or of sensors 1-1, 1-2, and 1-3 in Table 1. In addition to providing a wall structure for mounting the sensor die 20, sensor device 150 may include a substrate 10 that can include wire bond elements, including wire bond pads 26 and wire bonds 22. Each wire is connected to the sensor die 20, and as a result, the substrate 10 provides an electrical interconnection between the sensor device and one or more interface boards that are part of an analysis system such as a PLC. The wire bonds are protected by an encapsulating agent 24. A flow cell cover 30 is sealedly mounted on top of sensor device 150 to form a flow cell chamber 40. The height of the flow cell chamber (H FC ) is the height of the flow cell chamber at the center (H FC-C ) is the height of the flow cell chamber at the edge (H FC-E The flow cell cover 30 is formed with a concave surface so that it is smaller than the height (H) of the flow cell chamber in the center, which can be adapted to provide a uniform flow through the flow cell. For example, in various sensor devices, the height (H) of the flow cell chamber in the center can be adapted to provide a uniform flow through the flow cell. FC-C ) can be approximately 60 μm, while the height of the flow cell chamber at the edge (H FC-E The height is 100 μm. For sensor device 200 in Figure 2 or sensors 2-1, 2-2, and 2-3 in Table 2, the height of the flow cell chamber is uniform and can be approximately 125 μm for each lane.

[0055] Figure 15 is a schematic diagram conceptually illustrating a cross-section of a sensor device loaded with beads 50, which may be a cross-section of the sensor device 100 in Figure 1 or a cross-section of the multi-lane sensor device 200 in Figure 2. Note that the sizes of the features depicted in Figure 15 are not drawn to scale, and furthermore, the features of sensor devices having significantly different sizes are depicted in a way that dynamically visualizes them. For example, each microwell 310 of the microwell array 300 includes an opening such as an opening 312. Each opening has a diameter of 312 in the microwell 310 of Figure 15.D such as, having an upper cross-sectional diameter of the microwell. In the case of the sensor device 100 of FIG. 1 as provided in Table 1, the upper cross-sectional diameter of the microwell can range from about 0.65 μm to about 1.30 μm, while the height of the flow cell chamber at the center ( FC-C ) can be about 60 μm, and the height of the flow cell chamber at the edge (H FC-E ) can be about 100 μm. In the case of the sensor device 200 of FIG. 2 as provided in Table 2, the upper cross-sectional diameter of the microwell can range from about 0.65 μm to about 1.35 μm, while for each lane, the height of the flow cell is uniform at about 125 μm.

[0056] Regarding the conceptual cross-section of the sensor device of FIG. 15, for either the sensor die 120 of the sensor device 100 of FIG. 1 or the sensor die 220 of the sensor device 200 of FIG. 2, an array of microwells such as the microwell array 300 of FIG. 15 can be formed on top of an array sensor such as the sensor array 330 of FIG. 15. In FIG. 15, the sensor array 330 is the upper structure of an array of chemically-sensitive field effect transistor (chemFET) sensors, and each microwell can be capacitively coupled to at least one sensor. Various devices can be fabricated such that each microwell is coupled to 1 to 4 sensors. Thus, the microwell array 300 and the sensor array 330 of FIG. 15 are at the top of the sensor die 120 of FIG. 1 or the sensor die 220 of FIG. 2.

[0057] As depicted in Figure 15, the sensor array 330 includes an upper metal layer 342 of the floating gate 340. The upper metal layer 342 is also a sensing plate. The floating gate 340 also includes metal vias 341 that bond the upper metal layer 342 to a metal layer 344. As will be described in more detail herein, the floating gate of the chemFET of this disclosure may include 4 to 5 metal layers connected by metal vias that overlay the channel region (not shown) of the chemFET. The floating gate 340 is formed within a dielectric layer 332. Dielectric layers 320 and 322 formed on the dielectric layer 332 of the sensor array 330 are dielectric layers on which microwell arrays such as the microwell array 300 of Figure 15 are formed. Each microwell 310 of the microwell array 300 includes an opening such as an opening 312, a side wall such as a side wall 314, and a bottom or floor such as a bottom or floor 316. The microwell sidewalls 312 and microwell bottom 316 form a continuous surface on which a metal layer, such as a metal layer 318, is formed. Thus, the microwell bottom is a conductive metal layer formed on the sensor plate 342, while the microwell sidewalls 314 are part of the continuous metal layer 318, effectively providing a sensing surface such as a sensing surface 315. The metal layer 318 can be selected from, for example, titanium, zirconium, ruthenium, vanadium, and tantalum, but is not limited to the following, and a thin film of metal oxide is formed naturally under atmospheric conditions.

[0058] Reactions carried out within the microwell 310 can be analytical reactions for identifying or determining the properties or characteristics of an analyte of interest. Such reactions may directly or indirectly produce byproducts that affect the amount of charge adjacent to the sensor plate 342. If such byproducts are produced in small amounts, rapidly decay, or react with other components, multiple copies of the same analyte may be analyzed simultaneously within the microwell 310 to increase the output signal generated in response to changes in the amount of charge adjacent to a sensor plate, such as the sensor plate 342 in Figure 15. According to this disclosure, multiple copies of the analyte may be attached to the beads 50 either before or after deposition in the microwell 301. Where used herein, the terms “beads,” “solid support,” or “particles” and their derivatives may be used interchangeably. Briefly, the various beads of this disclosure are hydrophilic polymer solid supports used to provide multiple copies of the same analyte to increase the output signal. In particular, the solid support may contain copies of polynucleotides. For example, such hydrophilic particles can immobilize multiple copies of polynucleotides for sequencing using a sequencing system like the one shown in Figure 12.

[0059] Generally, bead samples can be processed to contain biomolecules, such as nucleosides, nucleotides, nucleic acids (oligonucleotides and polynucleotides), polypeptides, sugars, polysaccharides, lipids, or derivatives or analogs thereof. The ends or any internal parts of the biomolecules can be bound to or attached to polymer particles. The polymer particles can be bound to or attached to biomolecules using linking chemistry. Linking chemistry includes covalent or non-covalent bonds, including ionic bonds, hydrogen bonds, affinity bonds, dipole-dipole bonds, van der Waals bonds, and hydrophobic bonds. Linking chemistry can include, for example, affinities between biorecognition complements, such as between avidin and biotin moieties, between antigen epitopes and antibodies or their immunologically reactive fragments or haptens, between lectins and polysaccharides, and between enzymes and substrates.

[0060] Regarding the preparation of amplification beads, multiple polymer particles 404 can be arranged in solution together with multiple polynucleotides 402, as illustrated in method 400 of Figure 16. The multiple particles 404 may be activated to bind to the polynucleotides 402, or otherwise prepared. For example, particle 404 may contain one oligonucleotide complementary to a portion of one of the polynucleotides 402. In another example, polymer particles 404 can be modified with a target polynucleotide 404 using techniques such as biotin-streptavidin conjugation.

[0061] Hydrophilic particles and polynucleotides are subjected to polymerase chain reaction (PCR) amplification or recombinase polymerase amplification (RPA). For example, dispersed phase droplets 406 or 408 may be formed as part of an emulsion and may contain hydrophilic particles or polynucleotides. In the example of method 400 in Figure 16, polynucleotides 402 and hydrophilic particles 404 are provided at low concentrations and ratios related to each other so that a single polynucleotide 402 is likely to be present in the same dispersed phase droplet as a single hydrophilic particle 404. Other droplets, such as droplet 408, may contain a single hydrophilic particle and not contain polynucleotides. Each droplet 406 or 408 may contain enough enzymes, nucleotides, salts, or other components to facilitate the replication of polynucleotides. Enzymes such as polymerases are present bound to the hydrophilic particles or hydrogel particles of the dispersed phase droplet, or in very close proximity to the hydrophilic particles or hydrogel particles of the dispersed phase droplet. In the example in Figure 16, the polymerase is present in a dispersed phase droplet, such as droplet 408, and catalyzes the replication of polynucleotides. The polymerase enzyme used may be a naturally occurring polymerase, a recombinant polymerase, a mutant polymerase, a variant polymerase, a polymerase that has been fused or otherwise manipulated, a chemically modified polymerase, a synthetic polymerase, or an analogue, derivative, fragment, or subunit thereof.

[0062] Following PCR or RPA, amplification or template particles are formed, such as the amplification or template beads 410 shown in Figure 16, which may contain hydrophilic particles 412 and multiple copies 414 of polynucleotides. In particular, the amplification beads 410 have a monoclonal population of the target polynucleotide. Although the polynucleotides 414 are illustrated as being on the surface of the hydrophilic particles 412, the polynucleotides can extend within the hydrophilic particles 412. Hydrogels and hydrophilic particles having a low concentration of polymer relative to water may contain polynucleotide segments inside and throughout the amplification beads 410, or the polynucleotides may be present in pores and other openings. In particular, the amplification beads 410 can allow the diffusion of enzymes, nucleotides, primers, and reaction products used to monitor the reaction. A larger number of polynucleotides per particle produces a better signal.

[0063] Various polymer particles from the emulsion disruption procedure can be collected and washed in preparation for sequencing. Collection can be carried out by contacting the biotin moiety (e.g., linked to an amplified polynucleotide template attached to the polymer particle) with the avidin moiety and separating it from polymer particles lacking the biotinylated template. The collected polymer particles supporting the double-stranded template polynucleotide can be denatured to produce single-stranded template polynucleotides for sequencing. The denaturation step may include treatment with a base (e.g., NaOH), formamide, or pyrrolidone.

[0064] The amplification beads 410 in Figure 16 can be loaded into a sensor device 150, which may be the sensor device 100 in Figure 1 or the sensor device 200 in Figure 2, and which may include microwells 310 as previously described herein. For example, the amplification beads 410 can be loaded into the sensor device 150 using incubation loading. Primers can be added to the wells of a microwell array, such as the microwell array 300 in Figure 15, or the amplification beads 410 can be pre-exposed to the primers before being placed in the microwells 310. In particular, the amplification beads 410 may include binding primers. The primers and polynucleotides form a nucleic acid double helix containing a polynucleotide (e.g., template nucleic acid) hybridized to the primer. The nucleic acid double helix is ​​at least partially a double-stranded polynucleotide. Enzymes and nucleotides can be provided to the microwells 310 to facilitate detectable reactions such as nucleotide uptake.

[0065] Sequencing can be performed by detecting nucleotide addition. For example, referring to the sequencing system 1000 in Figure 12, as previously stated herein, reagents and solutions from containers 1040A-1040E can be controllably delivered to the sensor device 150 via a reagent value 1043 controlled through the system controller 1010. Thus, selected reagents or solutions can be sequentially directed to the sensor device 150 via the fluid multiplexer device inlet channel 1036 of the fluid multiplexer device 1030. During the sequencing experiment, various deoxynucleotide triphosphates (dNTPs) can be sequentially flowed onto the sensor device 150 in a defined order. In response to nucleotide addition, the pH in the local environment of the microwell 310 may change. Such a change in pH can be detected by an ion-sensitive field-effect transistor (ISFET) sensor, a type of chemFET sensor such as a chemFET. Therefore, by using a change in pH, it is possible to generate an output signal that indicates the incorporation of dNTPs complementary to the target polynucleotide in the amplification bead 410.

[0066] Regarding the preparation of single-template beads, as illustrated in Method 500 of Figure 17, multiple bead supports 504 can be placed in solution together with multiple polynucleotides 502 (target or template polynucleotides). The multiple bead supports 504 can be activated or otherwise prepared to bind to the polynucleotides 502. For example, the particles 504 may contain oligonucleotides complementary to a portion of the polynucleotides among the multiple polynucleotides 502 (capturing primers). In another example, the bead supports 504 can be modified with the target polynucleotide 502 using techniques such as biotin-streptavidin conjugation.

[0067] With regard to seeding particles to arrive at the preparation of single-template beads, the target polynucleotide can be subjected to polymerase chain reaction (PCR) amplification or recombinase polymerase amplification (RPA). In an example, particle 504 contains a capture primer complementary to a portion of the template polynucleotide 502. The template polynucleotide can hybridize to the capture primer. The capture primer can be extended to form a single-template bead 506 containing a single target polynucleotide bound to it. Other beads may remain unbound to the target nucleic acid, such as the untemplated bead 508 in Figure 17, and other template polynucleotides may be freely suspended in solution, as depicted in Figure 17. As further discussed herein, the preparation of monoclonal single-template beads 506 is carried out to minimize the formation of polyclonal beads having two or more target polynucleotides.

[0068] In method 500 of Figure 17, a single template bead 506 containing a target polynucleotide can be bound to a magnetic bead 510 to form a bead assembly 512. In particular, the magnetic bead 510 can be bound to the single template bead 506 by double-stranded polynucleotide binding. In one example, a further probe including a linker moiety can be hybridized to a portion of the target polynucleotide on the single template bead 506. The linker moiety can be bound to a complementary linker moiety on the magnetic bead 510. In another example, a target nucleic acid can be formed using a template polynucleotide bound to the single template bead 506, and may include a linker moiety that binds to the magnetic bead 510. In yet another example, a template polynucleotide complementary to the target polynucleotide bound to the single template bead 506 can be generated from a primer modified with a linker that binds to the magnetic bead 510. The linker moiety attached to the polynucleotide and the linker moiety attached to the magnetic bead are complementary to each other and can bind to each other. In one example, the linker portion may have affinity and include an avidin moiety and a biotin moiety, or other biorecognition pairs as described herein with respect to Figure 16.

[0069] As illustrated in Figure 17, the bead assembly 512 can be purified by separating elements such as the non-template beads 508 and free target polynucleotides 502 from the bead assembly 12. In Figure 17, the bead assembly 12 can be immobilized by applying a magnetic field to the reaction vessel in which method 500 is being prepared, for example, by means of a test tube, microfuse tube, or microtiter plate, but not limited to the following. Thus, the bead assembly 12 is held on the wall of the reaction vessel, while elements such as the non-template beads 508 and free target polynucleotides 502 remain in the supernatant. The supernatant can be removed while holding the bead assembly 12, and the immobilized bead assembly can be washed, thereby forming a concentrated and purified population of the bead assembly. To prepare a purified solution of single template beads, the immobilized bead assembly can be further subjected to conditions in which the single template beads 506 are released from the magnetic beads 510, for example, by mechanical stirring by vortexing or sonication. Thus, the purified preparation of single template beads can be recovered from the supernatant.

[0070] As illustrated in Figure 18, a purified sample 610 of a single template bead 606 can be loaded into a sensor device 150, which may be the sensor device 100 in Figure 1 or the sensor device 200 in Figure 2, which may include a microwell 310, as previously described herein. For example, a purified sample 610 of a single template bead 606 can be loaded into the device 150 using incubation loading. The target polynucleotide of the single template bead 606 can be amplified (referred to herein as templating) while in the well 310 to provide an amplified or template bead 614, which is depicted having multiple copies 616 of the target polynucleotide. In particular, the amplified bead 614 has a monoclonal population of the target polynucleotide. Such amplification reactions can be carried out using polymerase chain reaction (PCR) amplification, recombinant polymerase amplification (RPA), isothermal amplification, or a combination thereof. Enzymes such as polymerases exist bound to hydrophilic particles or hydrogel particles, or in very close proximity to hydrophilic particles or hydrogel particles. In the example in Figure 18, the polymerase is present in solution or in microwells to facilitate the replication of polynucleotides. The polymerase enzyme used may be a naturally occurring polymerase, a recombinant polymerase, a mutant polymerase, a variant polymerase, a polymerase that has been fused or otherwise manipulated, a chemically modified polymerase, a synthetic polymerase, or an analogue, derivative, fragment, or subunit thereof.

[0071] Although multiple copies of polynucleotide 616 of amplification beads 614 are illustrated as being on the surface, the polynucleotide can extend within the amplification beads 614. Hydrogels and hydrophilic particles having low concentrations of polymer relative to water can contain polynucleotide segments inside and throughout the amplification beads 614, or the polynucleotide can be present in pores and other openings. In particular, amplification beads 614 can allow the diffusion of enzymes, nucleotides, primers, and reaction products used to monitor the reaction. A larger number of polynucleotides per particle produces a better signal.

[0072] Sequencing can be performed on a sensor device 150 loaded with the amplification beads 614 shown in Figure 18. For example, the sequencing system 1000 shown in Figure 12 can be used to sequence the amplification beads 614 in Figure 18, as described above for the amplification beads 410 in Figure 16. [Examples]

[0073] Examples: Loading was performed using incubation loading with a GX5® multi-lane chip, as described for sensor device 200 in Figure 2, used in conjunction with a Genexus® chip coupler (A40269). Sequencing was performed using Genexus® equipment, as described for sequencing system 2500 in Figures 13B and 13C. Standard Genexus® single-lane library execution was set up using the Genexus® Control Library from the Ion Torrent Genexus® Control Kit (A40267) according to the user guide. The Genexus® Control Library from the Ion Torrent Genexus® Control Kit can be used, for example, to verify the functionality of Genexus equipment, to evaluate sample performance, and for troubleshooting.

[0074] Before starting the run, Genexus® strip 3 of the Genexus® template strips (A40263) for GX5® was manually denatured. The magnetic loading beads were first removed from well 7 (counting from right to left, starting from 1) using a pipette. Well 7 was then rinsed three times with 600 μL of nuclease-free water to completely remove all magnetic bead residue. 208 μL of nuclease-free water was deposited into the empty well, and the denatured strip 3 was loaded into the Genexus instrument as prompted. The Genexus instrument was operated with the deck door open to allow the user to change the loading method from magnetic loading to incubation loading. This was achieved by removing the magnetic separation plate and replacing it with an empty dummy plate immediately before the contents of well 2 of strip 3 were added to the loading solution in well A9 of the magnetic separation plate. Simultaneously, a 6 μL aqueous solution consisting of 0.2 M Tris-HCl (pH 8.0), 1 M KCl, 0.23 M MgCl2, and 0.10% Triton® X-100 was added to the loading solution in well A9 of the original magnetic separation plate. Immediately before the loading solution was injected into the chip lane, the dummy plate was removed and replaced with the original magnetic separation plate containing the loading solution. The rest of the loading module was started, and the loading solution was incubated in the chip at 35°C for 54 minutes. Template, sequencing, and analysis were started identically to a typical Genexus sequencing run.

[0075] Table 6 shows a comparison between the Genexus® Control Library performance certification specifications and incubation loading runs. As can be seen from a close examination of Table 6, incubation loading sequencing runs met or exceeded the performance certification specifications for AQ20 average read length, average base coverage uniformity, raw read accuracy, and total read count. [Table 6]

[0076] Accordingly, according to the present disclosure, in the first embodiment, a method for preparing a sensor device for analysis includes introducing a loading solution onto a sensor device, wherein the sensor device comprises a microwell array formed on a sensor array, and the loading solution comprises a number of beads providing a ratio of at least two beads per microwell; then selecting incubation conditions suitable for loading the loading solution into about 65% to about 95% of the microwells of the microwell array; and loading the sensor device by incubating the loading solution in the sensor device using the selected incubation conditions.

[0077] A second embodiment includes the inventive features of the first embodiment, further comprising incubation of the sensor device having a loading solution for about 10 minutes to about 1 hour.

[0078] A third embodiment includes the inventive features of Example 1 or 2, further comprising the incubation of the sensor device having a loading solution within a temperature range of about 4°C to about 60°C.

[0079] A fourth embodiment includes the inventive features of any one of Examples 1 to 3, further comprising the ratio of the bead diameter to the cross-sectional diameter of the microwell opening being about 60% to about 120%.

[0080] A fifth embodiment includes the inventive features of any one of Examples 1 to 4, further comprising the bead diameter being approximately 65% ​​or more of the microwell height and approximately 120% or less of the cross-sectional area of ​​the microwell opening.

[0081] A sixth embodiment includes an inventive feature of any one of Examples 1 to 6, and further comprises performing a sequencing assay on a sensor device loaded with a bead sample.

[0082] The seventh embodiment includes the inventive features of Example 6, further specifying that the bead sample includes a sample of hydrophilic polymer beads.

[0083] The eighth embodiment includes the inventive features of Example 7, further specifying that the hydrophilic polymer beads include hydrogel beads.

[0084] The ninth embodiment includes the inventive features of the sixth embodiment, further specifying that the sample of beads loaded onto the sensor device includes a sample of single-copy templated beads.

[0085] The tenth embodiment includes the inventive features of Embodiment 6, further specifying that the sample of beads loaded into the sensor device includes a sample of amplification beads.

[0086] The eleventh embodiment includes the inventive features of Example 6, wherein the sequencing includes controllingly flowing the sequence of a deoxynucleotide triphosphate (dNTP) reagent onto the device.

[0087] The twelfth embodiment includes the inventive features of Example 11, wherein the flow of the deoxynucleotide triphosphate (dNTP) reagent sequence onto the device is performed in a defined order.

[0088] A 13th embodiment includes the inventive features of Example 11 and further comprises generating an output signal indicating the incorporation of a dNTP complementary to a target polynucleotide on a template bead.

[0089] The 14th embodiment includes an inventive feature from any one of Examples 1 to 13, further specifying that the sensor device comprises a chemisensitive field-effect transistor (chemFET) sensor device.

[0090] The 15th embodiment includes the inventive features of the 14th embodiment and further specifies that the chemFET sensor device is an ion-selective field-effect transistor (ISFET) sensor device.

[0091] The 16th embodiment includes the inventive features of the 15th embodiment and further specifies that the ISFET sensor device is selective for hydrogen ions.

[0092] The 17th embodiment includes the inventive features of the 16th embodiment, wherein the sensor device has at least 10 7 ~10 9 Further specify that it is equipped with individual sensors.

[0093] The 18th embodiment includes an inventive feature of any one of Examples 1 to 17, further specifying that the device is a multi-lane sensor device, and further includes a method for loading the multi-lane sensor device by introducing a loading solution across selected lanes of the multi-lane device and incubating the loading solution in the selected lanes of the multi-lane device.

[0094] The 19th embodiment includes the inventive features of Example 18 and further comprises performing a sequencing assay on a sensor device loaded with a bead sample.

[0095] The 20th embodiment includes the inventive features of Example 19, further specifying that the bead sample includes a single copy of a molded bead sample.

[0096] The 21st embodiment includes the inventive features of Example 18, further specifying that the sequencing controls the flow of the deoxynucleotide triphosphate (dNTP) reagent sequence onto lanes of a multi-lane device.

[0097] While various examples of the present disclosure are shown and described herein, numerous variations, alterations, and substitutions will be conceivable to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the various examples described herein may be used when carrying out the present disclosure. The following claims define the scope of the present disclosure and are intended to encompass methods and structures within the scope of these claims, as well as their equivalents.

Claims

1. A method for preparing analytical sensor devices, The method involves introducing a loading solution onto the sensor device, wherein the sensor device includes a microwell array formed on a sensor array, and the loading solution contains a number of beads providing a ratio of at least two beads per microwell. Select incubation conditions suitable for loading the loading solution into approximately 65% ​​to approximately 95% of the microwells of the microwell array, A method comprising loading the sensor device by incubation of the loading solution with the sensor device using the selected incubation conditions.

2. The method according to claim 1, wherein the incubation of the sensor device with the loading solution is performed for about 10 minutes to about 1 hour.

3. The method according to claim 1 or 2, wherein the incubation of the sensor device with the loading solution is within a temperature range of about 4°C to about 60°C.

4. The method according to any one of claims 1 to 3, wherein the ratio of the bead diameter to the cross-sectional diameter of the microwell opening is about 60% to about 120%.

5. The method according to any one of claims 1 to 4, wherein the bead diameter is approximately 65% ​​or more of the microwell height and approximately 120% or less of the cross-sectional area of ​​the microwell opening.

6. The method according to any one of claims 1 to 6, further comprising performing a sequencing assay on the sensor device loaded with a sample of beads.

7. The method according to claim 6, wherein the bead sample includes a sample of hydrophilic polymer beads.

8. The method according to claim 7, wherein the hydrophilic polymer beads include hydrogel beads.

9. The method according to claim 6, wherein the sample of beads loaded onto the sensor device includes a sample of single-copy templated beads.

10. The method according to claim 6, wherein the sample of beads loaded into the sensor device includes a sample of amplification beads.

11. The method according to claim 6, wherein sequencing includes controllingly flowing a sequence of a deoxynucleotide triphosphate (dNTP) reagent onto the device.

12. The method according to claim 11, wherein the flow of the deoxynucleotide triphosphate (dNTP) reagent sequence onto the device is performed in a defined order.

13. The method according to claim 11, further comprising generating an output signal indicating the incorporation of a dNTP complementary to a target polynucleotide on a template bead.

14. The method according to any one of claims 1 to 13, wherein the sensor device comprises a chemisensitive field-effect transistor (chemFET) sensor device.

15. The method according to claim 14, wherein the chemFET sensor device is an ion-selective field-effect transistor (ISFET) sensor device.

16. The method according to claim 15, wherein the ISFET sensor device is selective for hydrogen ions.

17. The sensor device comprises at least 10 ７ ~10 ９ The method according to claim 16, comprising a number of sensors.

18. The device is a multi-lane sensor device, and the method is Introducing the loading solution onto the selected lane of the multi-lane device, The method according to any one of claims 1 to 17, comprising loading the multi-lane sensor device by incubation of the loading solution with the selected lane of the multi-lane device.

19. The method according to claim 18, further comprising performing a sequencing assay on the sensor device loaded with a sample of beads.

20. The method according to claim 19, wherein the bead sample includes a sample of a single copy mold bead.

21. The method according to claim 18, wherein sequencing includes controllingly flowing the sequence of a deoxynucleotide triphosphate (dNTP) reagent onto the lanes of the multi-lane device.

Images