Liquid handling device for performing assays

Abstract

The present invention relates to a device for measuring and / or processing a sample. The device comprises a rotatably arranged cylindrical sample holder, which is used to hold the sample at the side surface of the cylindrical sample holder. The cylindrical sample holder is configured to be at least partially covered by a liquid layer. The device further comprises: a liquid dispensing device, which is configured to add a liquid to the liquid layer; a reagent dispensing device, which is configured to add a reagent to the liquid layer; and a liquid contacting device, which is configured to distribute the liquid in the liquid layer. The liquid contacting device comprises a plate-like element, which is flexibly assembled relative to the cylindrical sample holder. The present invention relates to a method for measuring and / or processing a sample.

Description

Technical Field

[0001] The present invention relates to a device for measuring a sample and / or processing a sample; and a method for measuring a sample and / or processing a sample. Background Art

[0002] The progress in the field of "omics" (e.g., genomics, transcriptomics, proteomics, and metabolomics) over the past few decades has greatly promoted the understanding of human and non-human biology and diseases. The ability to perform biochemical assays in large-scale parallelism enables research groups to obtain unprecedented amounts of information. Using these data for statistical and big data analysis makes new discoveries possible and opens the way for a large number of applications in different fields (e.g., cancer research). Therefore, the pursuit of more biologically relevant data has promoted the development of high-throughput "omics" technology. In order to achieve wider adoption in research and pave the way for clinical groups to practice these technologies, it is highly desirable to increase the number of "omics" experiments that can be performed in assays and reduce the cost of each assay.

[0003] When performing large-scale parallel determinations (such as in next-generation DNA sequencing (NGS)), the chemicals and imaging steps in the process are usually repeated many times. A single cycle can, for example, involve rinsing the immobilized single-stranded DNA sample with a nucleotide solution (or reaction medium) equipped with a fluorophore, a second rinsing operation for removing excess nucleotides, and an imaging step for measuring fluorescence and determining which nucleotide (if any) is incorporated into the chain. Then, these operations or steps are repeated repeatedly until the sequence is established. Flux may be limited by the lead time of the rinsing step (adding reactants and / or removing excess reactants or other free-flowing reaction products) or by data acquisition time (i.e., the time required to collect data from all immobilized single-stranded DNA samples). Although there are NGS sequencing systems capable of rapid data acquisition, there is still a need in the art for improved liquid handling systems used in, for example, NGS applications. Summary of the invention

[0004] The following documents are incorporated by reference into the present disclosure as if fully set forth herein: WO 2016 / 030464 and WO 2017 / 144619.

[0005] It is an object of the present disclosure to alleviate at least some of the disadvantages associated with prior art techniques for performing biochemical assays.

[0006] In a first aspect of the present invention, there is provided a device for measuring a sample and / or processing a sample, the device comprising:

[0007] a cylindrical sample holder for holding a sample at a side surface of the cylindrical sample holder, wherein the cylindrical sample holder is rotatably arranged; wherein the cylindrical sample holder is configured to be at least partially covered by a liquid layer;

[0008] a liquid dispensing device configured to add liquid to the liquid layer;

[0009] a reagent dispensing device configured to add a reagent to the liquid layer;

[0010] A liquid contact device is configured to distribute liquid within the liquid layer, wherein the liquid contact device includes a plate-like element flexibly assembled relative to the cylindrical sample holder.

[0011] The device may further include a liquid removal device configured to remove liquid from the liquid layer. The liquid removal device may be at least partially contained in the liquid contact device. In other embodiments, the liquid removal device may be configured as a separate structure. Alternatively, the device does not include any liquid removal device. Alternatively, the liquid can be removed by allowing the liquid to leave the sample holder while the rotation stops or when the rotation speed of the sample holder decreases.

[0012] As mentioned above, the purpose of the present disclosure is to provide a kind of chemical composition and the device of characteristic that can quickly adjust the liquid layer.In other words, provide the device that can provide the specific reactant that will be present in the liquid layer at a given time and / or the position on the sample holder.Also comprise the ability of changing and controlling the concentration of the reactant, the local temperature of the liquid layer and the pH value of the liquid layer in the characteristic.It is recognized that the composition of the liquid layer acting as a reaction medium and the described rapid adjustment of characteristic need to involve the ability of adjusting the amount of liquid in the liquid layer by adding liquid to the sample holder from time to time, and displacing the liquid on the sample holder from time to time and removing liquid from the sample holder from time to time.

[0013] Herein, the term "liquid layer" refers to the liquid present on the side surface of the sample holder. The term "side surface" should be given its usual explanation, that is, it refers to the side of the cylindrical sample holder, excluding the bottom surface and the top surface. Therefore, the liquid will cover at least a portion of the surface of the sample holder, preferably substantially all of the surface of the sample holder. The liquid covering the sample holder will therefore form a liquid layer, wherein the liquid layer thickness is defined as the height of the layer at a specific position on the sample holder. During the rotation of the sample holder, a uniform liquid layer can be formed on the sample holder. The uniform liquid layer thickness is a substantially constant thickness around the circumference of the cylindrical sample holder. In this scenario, uniform thickness can refer to a thickness variation of less than 20% around the circumference, such as less than 15%, such as less than 10%, such as less than 5%. Preferably, the liquid layer should be thinner to reduce reagent consumption. The thickness of the liquid layer may be below 100 μm, such as below 30 μm, such as below 10 μm, such as in the range of 0.5 μm to 1.5 μm, such as in the order of 1 μm.

[0014] The liquid used in the device according to the first aspect may be any type of liquid used in microscopic imaging, such as a liquid suitable for NGS sequencing.The liquid may be water-based, ie contain a majority of water by weight.

[0015] The liquid dispensing device preferably includes a liquid channel capable of delivering liquid to the surface of the sample holder; and a liquid outlet connected to the liquid channel liquid, which is suitable for adding (dispensing) liquid from the liquid channel to the surface of the sample holder. The added liquid can have any preferred composition, concentration, pH value or temperature suitable for the purpose of the next step of the assay. For example, the liquid can have a reactant at or near zero concentration, and thus the increase in the thickness of the liquid layer causes dilution of any reagent that acts as a reaction medium that is already present in the liquid layer.

[0016] The liquid dispensing device preferably comprises a liquid outlet in liquid connection with a liquid reservoir via a liquid channel, the liquid dispensing device being adapted to add liquid from the liquid channel to the surface of the sample holder.The liquid channel in the present disclosure may comprise a flexible tube such as a flexible polymer tube.

[0017] Liquid can be distributed onto the rotating surface of the sample holder through the liquid outlet. The flow vector of the liquid can be directed at a small angle relative to the tangent of the surface. The speed of the liquid when leaving the channel is appropriately selected so that the speed is consistent with the peripheral speed of the surface of the rotating sample holder. In this way, the liquid deposited on the rotating surface stays at the position where it is deposited. Once the liquid is added to the sample layer, the liquid is usually unevenly distributed and needs to be redistributed or applied to cover each desired position of the sample layer. The liquid contact portion can be used to redistribute and / or change the thickness of the liquid layer, and control which areas of the sample layer are covered with liquid. The position and angle of attack of the liquid contact portion relative to the surface of the sample holder can be determined to adapt to the redistribution of the liquid on the surface or the adaptation of the thickness of the liquid on the surface.

[0018] The liquid outlet of the liquid dispensing device may preferably be in liquid connection with at least one liquid container suitable for containing liquid. The pressure in the liquid container may be regulated by a liquid regulating device, such as by a pump and / or a controllable valve, etc., and thus the flow of liquid from the liquid reservoir via the liquid channel to the outlet may be regulated. The pump and / or the controllable valve may be connected to a control device so that the amount of liquid added to the surface may be controlled by the user or preset according to a specific scheme. The liquid dispensing device may preferably be capable of delivering very small amounts of liquid with very high accuracy and precision.

[0019] In other examples, several liquid containers containing liquids with different chemical compositions (e.g., different substances, different concentrations, or different pH values) can be connected to the liquid dispensing device disclosed herein by liquid passages. In a preferred embodiment, the multiple liquid containers are connected to separate liquid dispensing devices. The advantage of this configuration is that, between different operations or steps of measuring, neither the liquid dispensing device nor the liquid removal device need to be rinsed, flushed, or cleaned. This can further reduce the single cycle time of measuring. However, the present invention also encompasses embodiments that distribute two or more different liquids by the same distribution outlet. In another example, the temperature of the liquid to be distributed can be controlled by controlling the temperature of the liquid in the liquid container, or by heating or cooling the liquid when arriving at the liquid dispensing device through the liquid passage.

[0020] The liquid contact portion may be adapted to mechanically contact the liquid layer present on the sample holder during rotation of the sample holder, thereby displacing the liquid in the liquid layer. It should be noted that mechanical contact here also includes the interaction between the liquid in the liquid layer and the liquid interacting with the liquid contact portion.

[0021] The liquid contact device includes a plate-like element configured to contact the surface of the liquid layer. The plate-like element is flexibly arranged relative to the cylindrical sample holder. This means that when no external force acts on the plate-like element, the plate-like element is arranged at a first predetermined distance from the cylindrical sample holder. This mode can be obtained, for example, when there is no liquid layer on the cylindrical sample holder. When the rotating liquid layer exerts a force on the plate-like element, the plate-like element will be further arranged at a larger second distance from the cylindrical sample holder. Therefore, the term "flexible arrangement" refers to arranging the plate-like element using a flexible portion or a similar arrangement that can shrink when a force is applied, thereby increasing the distance between the plate-like element and the sample container holder as long as a force is applied.

[0022] The term "plate-like element" as used herein refers to an element having a substantially flat surface suitable for contacting a liquid layer, such as a plate or other structure having a flat liquid contacting side, etc. The plate-like element may be attached to a support structure capable of holding the plate-like element at a distance from the surface of the sample holder.

[0023] By providing a plate-like element as described above, the thickness of the liquid layer can be controlled. By positioning the plate-like element so that it is in mechanical contact with the liquid layer, the thickness of the liquid layer can be reduced during rotation of the sample holder. During rotation, a predetermined force will be exerted on the liquid layer, thereby smearing the liquid in the liquid layer on the surface of the sample holder so that the thickness of the liquid layer is reduced. The flexibly arranged plate-like element allows the thickness of the liquid layer to be controlled by controlling the rotation speed of the cylindrical sample holder.

[0024] During operation, when the sample holder is rotated, the plate-like element slides against the liquid layer, with the flat surface against the curved surface of the liquid layer, thereby covering or immersing the sample or analyte fixed on the curved surface of the cylindrical sample holder. The plate-like element should preferably be positioned so that the line defined by the closest distance between the cylindrical surface and the substantially flat surface of the plate-like element (hereinafter referred to as the convergence line) is closer to the center of the plate-like element than to the edge of the plate-like element.

[0025] The plate-like element may be attached or mounted on a structure comprising a force control element, thereby obtaining a flexible assembly. The force control element is configured to control the amount of force applied by the plate-like element on the liquid layer. Thus, the thickness of the liquid layer will be automatically adjusted. Compared to a hypothetical assembly in which the plate-like element is fixedly mounted relative to the sample holder, the force control device is configured to allow a predetermined force from the plate-like element to be applied to the liquid layer, regardless of the amount of liquid in the liquid layer. Thus, a self-regulating function of the liquid layer can be achieved.

[0026] The arrangement of the force control element allows a substantially constant force to be applied to the liquid layer, regardless of the amount of liquid in the liquid layer, which in turn allows the liquid layer to have a substantially uniform thickness over the circumference of the sample holder. Consider the following example: In a first assay, a first cylindrical sample holder having a first circumference is provided. Automatic adjustment of the liquid layer by flexibly assembled plate-like elements provides a liquid layer having a first thickness over the circumference of the sample holder. In a second assay, the cylindrical sample holder is replaced by a second cylindrical sample holder having a second, larger circumference. Automatic adjustment of the liquid layer by flexibly assembled plate-like elements still provides a liquid layer having the same first thickness over the circumference of the sample holder.

[0027] The force control element may for example comprise a flexible portion such as a spring with a constant spring force. In other examples, the force control element may comprise a spring comprising an adjustment device for adjusting the spring force of the spring.

[0028] In other examples, the force control device may include an actuator adapted to adjust the force applied to the fluid layer. The force control device may also include a sensor or measuring device adapted to sense or measure the pressure applied by the plate-like element. Preferably, the sensor is configured to transmit its sensing or measurement to the actuator or a control device in communication with the actuator so that the applied force can be controlled based on the sensing or measurement.

[0029] In some embodiments, the force control element includes a flexible portion. The term "flexible portion" represents a portion of a force control element that exhibits elastic behavior when a force is applied thereto and subsequently withdrawn. When a force is applied to the fluid contact portion, the flexible portion will contract, thereby positioning the fluid contact portion farther from the surface of the sample holder. When the force is withdrawn, the flexible portion will expand, thereby positioning the fluid contact portion closer to the surface of the sample holder. In an example, the flexible portion is a spring. The fluid contact portion is then attached to the spring, which in turn is attached to a structure for holding the fluid contact portion. When a force is applied to the fluid contact portion, the structure should be substantially stationary relative to the sample holder.

[0030] Preferably, the flexibility of the flexible portion can be adjusted, for example by providing an adjustable spring. Therefore, the thickness of the fluid layer can be controlled by adjusting the flexibility of the flexible portion.

[0031] Preferably, the force applied by the force control device to the liquid contact portion should be small relative to the force applied by the liquid layer to the liquid contact portion, so that the distance between the liquid contact portion and the cylindrical sample holder can be determined by the balance between capillary action (i.e., surface tension) and lubricating forces. If the liquid contact portion provides a sufficiently small contact angle with the liquid in the liquid layer, the capillary force will attract the liquid contact portion to the sample holder. The lubricating layer formed between the rotating sample holder and the liquid contact portion will provide a force to resist the suction. Therefore, the distance between the liquid contact portion and the sample holder (or in other words, the thickness of the liquid layer) will self-adjust so that the forces balance each other.

[0032] In a simple model, one can consider the effects of surface tension and lubrication as pressures. The pressure due to surface tension depends roughly on the surface tension of the liquid, the contact angle of the liquid contact, and the thickness of the liquid layer. The lubrication pressure depends roughly on the viscosity of the liquid, the rotation rate, the radius of the sample holder, and the thickness of the liquid layer. Therefore, for a given rotation rate, the equilibrium thickness of the liquid layer can be calculated.

[0033] In some examples, the liquid contacting portion may include a polymer material. The liquid contacting portion may also include glass, metal, or any other suitable material.

[0034] The device preferably comprises a reagent dispensing device. Reagents are preferably provided to the liquid layer so that they can react with the sample. The reagent dispensing device can be arranged as a combined structure with a liquid dispensing device. In other words, the liquid dispensing device can be arranged to supply liquid and reagent to the side surface of the sample holder. However, the reagent dispensing device can also be arranged as a separate structure.

[0035] The reagent dispensing device can comprise the outlet for example formed by orifice, and this outlet is connected with the liquid channel liquid that is configured to reagent is transported to liquid outlet.Then, reagent can be distributed to liquid surface by outlet.The flow of reagent through passage can be controlled by pump.Dispensing device can accurately distribute very small amount of reagent liquid, such as in the scope of microliter, such as in the reagent liquid in the scope from 1 microliter to 500 microliters.

[0036] In some examples, the reagent dispensing device includes means for regulating flow through, preferably through at least one electrically controlled valve. In some examples, at least one electrically controlled valve may be used in conjunction with a pump for regulating the flow of the reagent liquid.

[0037] By rapidly alternating between dispensing or removing liquid, the local composition, concentration, pH and temperature of the reaction medium can be varied to meet the above objectives of the present invention.

[0038] The liquid contact portion can be arranged so that it can be positioned very close to the liquid layer or in mechanical (liquid) contact with the liquid layer, preferably by means of a translation platform and a rotation platform, preferably with micron precision. The liquid contact portion device is further preferably arranged to be stationary relative to the rotating sample. Therefore, when the liquid contact portion contacts the liquid layer, the relative movement of the liquid contact portion and the liquid layer will cause a mechanical force on the liquid layer. Depending on the shape and positioning of the liquid contact portion, the force will cause a local displacement of a portion of the liquid or a portion of the liquid to separate from the liquid layer.

[0039] The liquid contacting device, liquid dispensing device, liquid removal device, and liquid may be referred to as various "liquid manipulation devices". A common feature of all of the liquid manipulation devices disclosed herein is that they are compatible with, and in some cases take advantage of, the fact that manipulation can be performed while the sample holder is rotating. Due to the rotation of the sample holder, the liquid manipulation device can be arranged so that it is stationary or does not rotate with the sample holder during operation. This is advantageous because it provides much higher precision in its position relative to the rotating sample holder.

[0040] The "Next Generation Sequencing (NGS)" operation of the prior art generally involves the following steps: adding a reaction medium containing a set of reactants to a sample volume containing a set of immobilized target molecules; allowing time for the reaction to proceed; diluting the reaction medium and thereby preventing excess reactants that have not reacted with the target molecules from contaminating subsequent reaction medium; and detecting a signal indicating that a reaction has occurred in the sample volume. In this prior art operation, the step of diluting the excess reactants is performed by flushing the sample with a liquid having a zero concentration of reactants. Due to the laminar flow in this case, the time required to obtain a satisfactory low concentration of excess reactants in the sample volume depends on how fast the reactants will diffuse from the reaction medium liquid layer (covering the sample in the sample volume) to the zero concentration liquid layer flushed above the liquid layer covering the sample. The time required depends quadratically on the thickness of the liquid layer covering the sample volume, because the diffusion time increases quadratically with distance. In addition, the diffusion constant is generally reduced near the solid surface. In the prior art, this step is generally very time-consuming and may be rate-limiting for some applications.

[0041] In the present disclosure, a device is provided that is capable of rapidly adjusting the concentration of reactants or other substances in a liquid layer covering or submerging a sample on a rotating sample holder by providing a liquid manipulation device to manipulate the liquid on the sample holder (and reagents dissolved or immersed in the liquid), adding new liquid having a desired composition and concentration of substances and pH to the liquid layer, and dispersing the new liquid to the surface of the sample holder. It has been recognized that such rapid adjustment of the composition and properties of the liquid layer acting as a reaction medium requires the ability to adjust the amount of liquid in the liquid layer by occasionally adding liquid to the sample holder, and occasionally displacing the liquid on the sample holder and occasionally removing liquid from the sample holder.

[0042] In order to meet the above objectives, it is recognized that a rotatable cylindrical sample holder, such as the sample holder defined in WO 2016 / 030464 and / or WO 2017 / 144619, forms a very suitable geometry and configuration to achieve accurate, rapid and repeatable control of the relevant parameters in order to successfully carry out the assay process. The rotation and axial translation of the sample holder allow rapid physical access to each point of the sample holder with sub-micron accuracy, and thus the parameters can be managed both locally (i.e. at the level of the individual sample) and globally (i.e. over the entire surface covered with the sample).

[0043] The sample holder can be made of a material selected from the group consisting of glass, metal (e.g., aluminum or steel) and plastic. Ideally, the sample holder is a hollow or solid circular symmetrical cylinder, i.e., a tube or a rod. In order to achieve smooth and accurate rotation, it is preferred that the sample holder is designed and manufactured so that it is symmetrical about the axis along its length, and the mass is evenly distributed in all radial directions from the axis. In order to ensure smooth and repeatable rotation around its axis, the sample holder can be held in place by a bearing, which is preferably an air bearing or an electric bearing to achieve non-contact suspension, and the bearing is assembled between the rotating sample holder and an internal or external fixed fitting.

[0044] The sample holder may be provided with a specified sample volume, i.e. a specific volume in which a sample may be contained or attached to a predefined location on the sample holder surface. The specified sample volume may also be provided on a sample container, for example wrapped around at least a portion of the sample holder.

[0045] The designated sample volume may be in the shape of a tiny ridge suitable for accommodating a sample, a well suitable for accommodating a sample, or in a dedicated groove or well, or between polymer film layers (eg, where the outermost polymer film layer is perforated).

[0046] The designated sample volumes may be separated from each other. In other examples, the designated sample volumes may be in contact with each other.

[0047] By functionalizing a portion of the surface of the above embodiments of the sample volume, the sample can also be fixed on the functionalized portion of the sample holder or sample container surface. In yet another embodiment, the entire sample holder surface is functionalized and the sample can be fixed at a random position on the sample holder surface.

[0048] The device may further comprise a housing in which the sample holder and the fluid handling device are housed. The fluid handling device may be attached to the housing so that they can remain stationary during rotation and / or translation of the sample holder.

[0049] In some embodiments, the plate-like element comprises at least one aperture configured to perform at least one of the following:

[0050] dispensing a liquid into the liquid layer; and

[0051] removing liquid from the liquid layer; and wherein the at least one orifice is in fluid communication with a fluid channel configured to perform at least one of:

[0052] delivering a fluid to the orifice, and

[0053] Fluid is delivered from the orifice.

[0054] The plate-like element may include at least one orifice serving as a liquid inlet, so that liquid can flow from the liquid layer to the liquid channel, thereby transporting the liquid away from the liquid layer. The plate-like element may further or alternatively include at least one orifice serving as an outlet, so that liquid is added to the liquid layer from the liquid channel. The device may also include a device for controlling the position of the plate-like element, such as making the corresponding orifice serving as the outlet and the inlet contact or not contact the liquid layer. The position control may include changing the relative position between the sample holder and the plate-like element along a direction substantially perpendicular to the direction defined by the axis of rotation of the sample holder.

[0055] In some examples, the liquid may be provided by means of a fluid communication or passage extending through the plate-like element. Thus, the plate-like element may include: a first surface, the first surface being configured to face the liquid layer; and a second surface, the second surface being opposite to the first surface; and a passage extending between these surfaces. The orifice may be arranged at the first surface, and the passage is arranged to provide fluid communication between the orifice and the liquid channel.

[0056] In the example where the liquid contact portion is provided with a liquid inlet for conveying liquid from the liquid layer, the liquid contact portion may be adapted to guide the liquid from the liquid layer to the orifice. By positioning the above-mentioned orifice acting as the inlet at the portion of the liquid contact portion where the liquid layer is compressed, i.e. before the liquid layer passes the confluence line, the positive pressure gradient caused by the shear force formed by the relative movement between the plate-like element and the cylindrical sample holder will push the liquid into the orifice, and the liquid can thus be removed from the liquid layer, and the thickness of the liquid layer is reduced. Therefore, the liquid removal device may be provided on the liquid contact portion.

[0057] Once the liquid reaches the inlet, the liquid may be subjected to negative pressure, ie, pressure below atmospheric pressure, to exert a suction force to transport the liquid to and through the fluid channel. The suction force may be accomplished, for example, by using a syringe pump or an impeller pump.

[0058] In the example where the liquid contact portion is provided with a liquid outlet for delivering liquid to the liquid layer, the orifice serving as the outlet should preferably be positioned after the confluence line so that liquid can be added through the outlet. The manner of adding liquid is similar to a Venturi jet pump. Therefore, the liquid dispensing device can be provided on the liquid contact portion.

[0059] In some embodiments, the liquid contact portion is provided with both a liquid inlet and a liquid outlet.

[0060] By controlling the flow rate of the liquid channels connected to the inlet and outlet respectively, the thickness of the liquid layer can be adjusted. The size of the plate-like element will vary according to the application, but the width of the contact portion of the element, i.e., the width of the element suitable for physical contact with the liquid layer, is preferably wider than the field of view (FOV) of the imaging system for studying the sample. In some examples, the plate-like element extends the length of the sample holder. The length should be at least long enough to leave room for at least one orifice on both sides of the confluence line. In some examples, the plate-like element extends the length of the sample holder. In order to operate the liquid contact device at different positions along the axis of rotation of the sample holder, this embodiment may include a device for realizing the relative translation of the liquid contact device and the cylindrical sample holder along the axis.

[0061] In certain embodiments, the liquid dispensing device includes a liquid outlet. The liquid dispensing device can add liquid to the surface of the sample holder through the liquid outlet. The liquid outlet is connected to the liquid channel so that the liquid dispensing device can receive liquid from the channel and add liquid to the surface of the liquid layer. The liquid outlet can be connected to the liquid contact portion, and the liquid contact portion can include a conduit and / or channel for delivering liquid to the surface of the sample holder. The liquid outlet can also be suitable for acting as a nozzle so that liquid can be ejected onto the surface of the sample holder.

[0062] In certain embodiments, the device further comprises a mixing tool, which is arranged to cause mixing in the liquid layer. In order to achieve a uniform concentration of reagent or other materials in the liquid layer, it is advantageous to cause mixing to help make the concentration uniform. The mixing tool may include a device for translating and / or rotating a platform to allow the mixing tool to move the liquid in the liquid layer so as to cause mixing. The mixing tool may include at least one of a brush, a plate-like element comprising at least one ridge and a plate-like element comprising at least one post. The plate-like element may be identical with the liquid contacting portion or may be a different second plate-like element.

[0063] In embodiments where the liquid contacting device is a plate-like element, the mixing means may be a ridge connected to the plate-like element on the side adapted to be in contact with the liquid layer.

[0064] In some embodiments, the device further comprises a temperature control device for raising or lowering the temperature of the liquid layer. The device may further comprise a device for regulating the temperature of the atmosphere surrounding the liquid layer and the temperature of the peripheral portion of the sample holder on which the surface sample or analyte being studied is placed.

[0065] Since the reaction rate depends largely on the temperature of the reaction medium, the ability to control and regulate the temperature not only in the liquid layer, but also in the surrounding environment of the layer is advantageous. Here, the surrounding environment refers to the side surface of the sample holder below the liquid layer and the atmosphere just above the liquid layer. Temperature regulation can be further divided into slow regulation, in which the main purpose is to maintain a stable temperature on each part of the liquid layer (reaction medium), and fast regulation, in which the temperature in the reaction medium in some determinations needs to be locally adjusted up and down in the order of seconds or even shorter.

[0066] In order to increase the temperature on the entire liquid layer, one embodiment includes a conductive lead attached to a cylindrical sample holder. Preferably, the lead is densely wound around the perimeter of the sample holder in a spiral manner in a single layer on all parts of the sample holder that require temperature regulation. The lead can also be provided with a thin electrically insulating coating to ensure that the resistance along the path of the wound lead is several orders of magnitude lower than any shorter path along the sample holder. In order to position the lead on the sample holder, the lead can be wound on the side surface of the sample holder in a spiral pattern. The lead is preferably ground and / or polished to about half of its thickness, thereby forming a flat surface of conductive material with a spiral shape covering the cylindrical shaft sample holder, and the sample can be directly placed on this flat surface. Alternatively, the sample holder can be provided with an additional layer therebetween. The additional layer can be equipped with a well or groove for accommodating a sample, etc. By passing a suitable current through the wire, ohmic losses will generate heat, which will be dissipated in the reaction medium of the sample layer. By controlling the current, the temperature of the liquid layer can be controlled with high precision.

[0067] Another way to adjust the temperature of the reaction medium is to add a fluid having a temperature higher than that of the liquid layer, and thereby gradually raise the temperature of the liquid layer, or, if desired, to add a fluid having a temperature lower than that of the liquid layer, and thereby gradually lower the temperature of the liquid layer. For acceleration, the change in temperature, the way to remove fluid from the liquid layer is crucial, because a fluid having an undesirable temperature can be replaced more quickly by a fluid having a desired temperature.

[0068] It becomes clear from the above that there are several ways to adjust the temperature of the reaction medium to a desired set point. It is also clarified that the need to simultaneously control the temperature of the liquid added to the liquid layer, the temperature of the atmosphere surrounding the liquid layer, and the temperature of the sample holder directly below the liquid layer.

[0069] The above embodiments for raising the temperature of the liquid layer allow for uniform and precise temperature regulation. However, in some assays, local and more instantaneous increases are desirable. In one embodiment, a radiation source for rapidly increasing the temperature is provided. The radiation source may include a radiation source in the mid-infrared (MIR) range, preferably a carbon dioxide (CO2) laser. The standard wavelength of a CO2 laser (i.e., 10,600 nanometers) is very effectively absorbed by a liquid layer containing water, and thus the energy absorbed from the source can be used simultaneously for heating and / or (if desired) evaporating the liquid layer. When the source is focused on a smaller point (e.g., 20×20 microns or less), a very small amount of radiation is required to raise the temperature of the irradiated volume. In order to accurately control the temporary increase in temperature, it is necessary to know or control the thickness of the liquid layer, and it is also necessary to know or control the amount of MIR radiation exposed to the target volume of the liquid layer. The control of the amount of radiation can be implemented in a variety of ways. Preferred embodiments include controlling the output power and duty cycle of a pulsed source, or using filters, beam splitters, shutters, and output power control for a continuous wave source. Preferred embodiments further comprise a temperature sensor monitoring the local temperature of the fluid at the irradiated volume.The radiation source may be positioned stationary, ie not co-rotating with the sample holder, so that the radiation is directed to a desired location on the rotating sample holder.

[0070] In yet another embodiment, the temperature control may be achieved by dielectric or capacitive heating between a conductive layer on the sample holder and an electrode in contact with the liquid layer. The temperature control means may also comprise a Peltier element.

[0071] In some embodiments, the device further comprises a device for measuring an amount indicating the concentration of the reagent in the liquid layer. Such a device may, for example, comprise a pH meter, but other amounts for measuring an amount indicating the concentration of a specific reagent are known to those skilled in the art.

[0072] In some embodiments, the device further comprises: an illumination device for illuminating the sample; an objective lens for collecting photons emitted or scattered from the sample; and a detection device for detecting the photons collected by the objective lens, wherein the optical contact between the objective lens and the sample is at least partially provided by the liquid layer. This type of embodiment may include a liquid contact device in the shape of a thin glass plate (e.g., a cover glass), which is arranged to contact the liquid layer and an immersion oil drop placed between the cover glass and the objective lens. In some embodiments, the excitation light (i.e., the illumination) and the emitted light (or photons) both pass through the same objective lens.

[0073] In some embodiments, the device further comprises

[0074] an irradiation device for simultaneously irradiating the sample with a line focus or an array of focuses; and

[0075] a detection device for simultaneously detecting photons emitted or scattered from the sample in the field of view array;

[0076] wherein an array of sub-observation volumes in the sample from which photons are emitted or scattered during imaging is defined by the volume in space where the array of line foci or focal spots of the illumination device overlaps with the corresponding array of fields of view of the detection device;

[0077] Therein, the sample holder is arranged such that by rotating the sample holder at least a portion of the sample can be transported through at least one of the sub-observation volumes.

[0078] Suitable illumination and detection arrangements are described in WO 2017 / 144619, where the definition of sub-observation volumes is also disclosed.

[0079] In some embodiments, the liquid contact portion is in the shape of a sickle, a cutting tool, a brush or a blade. The liquid contact portion should have sufficient mechanical stability so that the liquid contact portion can contact the liquid layer present on the sample holder and mechanically displace or remove the liquid. The liquid manipulation device may further include a device for adjusting the angle of attack of the liquid contact portion. By adjusting the angle of attack, the amount of liquid removed can be controlled. The liquid contact portion may further include a translation device, which is suitable for adjusting the distance from the surface of the sample holder where the liquid contact portion is positioned. Adjusting the distance from the surface is also a way to adjust the thickness of the liquid layer left on the surface after the liquid contact portion has contacted the liquid during the rotation of the sample holder.

[0080] In one embodiment, the liquid contact portion comprises at least one selected from a blade, a scraper or a brush. When the liquid contact portion contacts the liquid layer, the angle of attack can be positive, so that the liquid contact portion slides on the liquid surface, and the pressure from the liquid contact portion will therefore build up the droplet moving in front of the liquid contact portion. By selecting the correct angle of attack, the design with the correct rigidity (mechanical stability) of the liquid contact portion and selecting the position of the liquid contact portion relative to the rotating sample holder, the liquid droplet with reactant can be made to grow to the size that helps a part of liquid in the droplet to separate from the sample holder liquid layer. Alternatively, by gradually moving the liquid contact portion along the rotating sample holder, the droplet can be directed to the point where the droplet can be absorbed or directed to leave the sample holder surface.

[0081] In another embodiment, mechanical displacement of a portion of the liquid layer is achieved by arranging the liquid contact portion to have a negative angle of attack so that when the liquid contact portion contacts the liquid layer on the rotating axis, it cuts and separates the liquid on the surface in a lathe-like manner into a remaining portion that stays on the surface of the rotating axis and a detached portion that flows on the other side of the liquid contact portion, thereby enabling the detached portion of the liquid layer to be redirected away from the rotating axis. The detached portion can then be collected for reuse or controlled disposal through a liquid inlet positioned on a liquid removal device. For this purpose, the liquid contact portion in this embodiment includes a sharp leading edge in the direction of movement, which cuts and separates the liquid layer when the liquid contact portion is moved through the liquid layer.

[0082] In some embodiments, the liquid removal device of the present disclosure includes a liquid absorption device, which is suitable for removing liquid from the sample layer by conveying the liquid through the liquid absorption device to a liquid inlet that is arranged to be in fluid communication with the liquid removal device. The liquid removal device may include a liquid absorption device, which is suitable for being in liquid communication and contact with the liquid layer during rotation of the sample holder. The liquid absorption device preferably includes at least one conduit and / or channel arranged in the liquid contact portion. The liquid can be conveyed from the surface of the sample holder by means of capillary forces and / or other forces such as forces formed by a pump.

[0083] Thus, excess liquid can be removed from the surface in a fast and efficient manner.The thickness and shape of the liquid layer can be carefully controlled and adapted to the specific application.

[0084] In a second aspect of the present invention, a method for measuring and / or processing a sample in a device is provided, the device may be configured similarly to the device described above with reference to the previous embodiment. The method may include the following steps:

[0085] a) arranging a cylindrical sample holder in the device;

[0086] b) providing at least one sample to be investigated to the cylindrical sample holder;

[0087] c) providing a first type of liquid to the sample holder,

[0088] d) distributing the liquid to form a liquid layer on the sample holder;

[0089] e) providing a reagent to the liquid layer;

[0090] f) allowing at least one desired reaction to occur between the reactant and the at least one sample; and

[0091] g) removing liquid from the liquid layer.

[0092] The method disclosed in the second aspect of the present invention can alleviate at least some of the problems associated with the prior art. In particular, the liquid manipulation time required for removing excess reactant liquid from the surface of the sample holder can be greatly shortened. The method can preferably be performed in the device as described in the first aspect of the present disclosure.

[0093] In some embodiments, step bg) is performed when the sample holder is arranged in the device, preferably during rotation of the sample holder.

[0094] In some embodiments, step g) is performed by contacting the liquid layer rotating on the surface of the sample holder in such a way that excess reaction liquid is removed by separating the excess liquid from the liquid layer.

[0095] The liquid handling device described in relation to the first aspect of the present disclosure provides a liquid contacting portion and a liquid removing device adapted to remove liquid from a surface by physically contacting said liquid.

[0096] Step d) (wherein the liquid is distributed to form a liquid layer on the sample holder) may further comprise the step of controlling the thickness of the liquid layer by arranging a plate-like element flexibly assembled relative to the cylindrical sample holder as previously described in liquid contact with the liquid layer.

[0097] In some embodiments, step g) is further performed by a device as defined in conjunction with the first aspect for receiving liquid from the surface of the sample holder in the liquid inlet and transporting the liquid from the surface via the liquid channel. As disclosed in the first aspect of the present disclosure, the liquid handling device is capable of separating the liquid from the surface of the sample holder and transporting the liquid from the surface of the sample holder.

[0098] In some embodiments, the method further comprises a step h) of illuminating the sample and detecting photons emitted or scattered from the sample. This step may be performed while the sample holder is rotating. Rotation-based imaging solutions are further described in WO2016 / 030464 and WO2017 / 144619.

[0099] In some embodiments, the method further comprises a step g1) of diluting the concentration of the reagent in the liquid layer by adding the first type of liquid to the liquid layer.

[0100] In some embodiments, the method further comprises a step e1) of increasing the concentration of the reagent in the liquid layer by evaporating liquid from the liquid layer. This can be achieved, for example, by increasing the temperature or reducing or stopping the supply of liquid to the liquid layer.

[0101] In some embodiments, the method further comprises the step of inducing mixing in the liquid layer to homogenize the concentration of the reagent in the liquid layer.

[0102] In some embodiments, the method further comprises the step of adjusting the temperature of the liquid layer to a predetermined temperature. In further embodiments, the method may comprise adjusting the temperature to a first predetermined temperature when a reagent is added to the liquid layer, and adjusting the temperature of the liquid layer to a second predetermined temperature when the liquid is removed from the liquid layer.

[0103] In some embodiments, steps c)-h) are performed during rotation of the sample holder.

[0104] In some embodiments, step g) of removing liquid is performed while the sample holder is stationary. BRIEF DESCRIPTION OF THE DRAWINGS

[0105] Hereinafter, the present invention will be described in detail with reference to exemplary embodiments as shown in the following drawings, in which

[0106] In the figure:

[0107] Figure 1 A schematic diagram of a liquid manipulation device according to some embodiments of the present invention is shown.

[0108] Figure 2 A schematic illustration of the arrangement of a liquid manipulation device and a specified sample volume according to some embodiments of the present invention is shown.

[0109] Figure 3 A schematic illustration of the arrangement of a liquid manipulation device and a specified sample volume according to some embodiments of the present invention is shown.

[0110] Figure 4 A schematic illustration of a device comprising several liquid manipulation devices according to an embodiment of the invention is shown.

[0111] Figure 5 Schematic illustration showing how the thickness of the liquid layer can be varied in embodiments of the invention.

[0112] Figure 6 The various steps in a determination cycle according to an embodiment of the present invention are schematically shown.

[0113] Figure 7 A schematic illustration of a device comprising several liquid manipulation devices according to an embodiment of the invention is shown.

[0114] Figure 8 A schematic illustration of a liquid handling device according to an embodiment of the present invention is shown.

[0115] Fig. 9 A schematic illustration of a liquid handling device according to an embodiment of the present invention is shown.

[0116] Fig.10 A schematic illustration of a liquid handling device according to an embodiment of the present invention is shown.

[0117] Fig.11 A schematic illustration of a liquid handling device according to an embodiment of the present invention is shown. DETAILED DESCRIPTION

[0118] The present invention is described more fully below with reference to the accompanying drawings, in which currently preferred embodiments of the present invention are shown. However, the present invention may be implemented in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided for thoroughness and completeness and to illustrate various aspects of the present invention to the skilled person. As shown, the size of layers and regions may be exaggerated for illustrative purposes, and are therefore provided to show the overall structure of embodiments of the present invention. Identical elements are numbered the same.

[0119] Figure 1 Embodiments of the invention are schematically depicted. Figure 1 A liquid handling device 100 according to the present invention is shown. The liquid handling device 100 comprises a liquid channel 101, a liquid orifice serving as a combined liquid inlet 104 and liquid outlet 103, and a liquid contact portion 105. In this document, the liquid contact portion 105 is represented by a brush comprising a plurality of bristles adapted to contact a liquid layer 107 on a sample layer 109 present on a cylindrical sample holder 111. However, the liquid contact portion 105 may also be in the shape of, for example, a blade, a plate-like element, or have a scraper-like shape. The sample layer 109 is the outer surface of the sample holder 111. In Figure 1In the embodiment shown in , the liquid channel 101 is constituted by a flexible tube, but other channels are also conceivable. The liquid manipulation device 100 further comprises a translation device 113, which is suitable for adjusting the position of the liquid manipulation device 100 relative to the sample layer 109. The translation device may include an electric motor, which is suitable for adjusting the position of the liquid manipulation device 100 along the sample layer 109 and is capable of adjusting the distance between the outer surface of the sample holder 111 and the liquid contact portion 105. The liquid manipulation device 100 may further include an angle adjustment device 115 suitable for adjusting the angle α. The angle α is defined here as the angle between the tangent line of the surface of the sample holder 111 and the liquid contact portion 105 when the liquid contact portion 105 is not yet in mechanical contact with the liquid layer 107 on the surface of the sample holder 111 or the surface itself. Once in contact, the liquid contact portion can bend at a smaller angle and slide against the surface according to its mechanical properties, the applied force against the surface and the friction force. The angle adjustment device 115 may also include an electric motor.

[0120] Preferably, at least one of the translation device 113 and the angle adjustment device 115 may include a force control element adapted to control the force with which the fluid contact portion 105 contacts the fluid layer 107. The force control element may include a flexible portion that contracts when a large force is applied and expands when the force is released.

[0121] In the illustrated embodiment, the liquid inlet 104 and the liquid outlet 103 are in liquid connection with the liquid channel 101. Therefore, the liquid channel 101 can be used to supply liquid to the liquid outlet 103 so that the liquid layer 107 can be disposed on the sample layer 109.

[0122] The liquid manipulation device 101 further includes a pressure regulator 117, which is suitable for adjusting the pressure at which the liquid contact portion 105 contacts the liquid layer 107. The pressure regulator 117 may further include a pressure sensor, which is suitable for sensing the pressure at which the liquid contact portion 105 contacts the liquid layer 107. In other examples, the pressure regulator 117 may include a flexible portion such as a spring, or be composed of a flexible portion such as a spring.

[0123] In another embodiment, the liquid contact portion 103 is slightly inclined so that when the liquid wavelet in front of the liquid contact portion 105 reaches a certain amplitude, the accumulated liquid leaks through the liquid contact portion 105 on only one side (similar to the way a snow plow is used to push snow off the road). By moving the inclined liquid contact portion 105 in the axial direction of the rotating cylindrical sample holder 111, the liquid is gradually moved toward the end of the sample holder 111 as the relative movement of the inclined liquid contact layer forms a spiral on the sample holder 111.

[0124] In an embodiment where the liquid contact portion 105 is a brush, a plurality of bristles can be positioned in an array spaced apart from one another so that the spaces between the bristles achieve a capillary action on the liquid in contact with the brush, and thus the liquid can be absorbed by the brush itself and subsequently transported to the liquid channel 101 via the liquid inlet 104, where the liquid can be transported to a container suitable for accommodating excess liquid. Such a brush can combine the mechanical removal of liquid disclosed above with the absorption of excess liquid. Negative pressure in the liquid channel 101 (e.g., negative pressure formed by a pump) can transport the liquid away through the liquid channel 101.

[0125] In a preferred embodiment, the bristles of the brush are made of fibers which, depending on the fiber material, have a diameter thin enough to bend by the friction caused by the sample layer 109 or a thin lubricating layer formed under the fibers, thereby sliding lightly on the surface of the rotating sample holder 111 or the sample layer 109, but are hard enough to do mechanical work on the liquid layer 107 and deform the surface of the liquid.

[0126] Preferably, the liquid handling device 100 shown above is arranged in a device further comprising a reagent dispensing device suitable for dispensing reagents into the liquid layer. The reagent dispensing device can have a configuration similar to the liquid handling device 100. The reagent dispensing device does not need to include a liquid contact portion or a liquid inlet 103. The liquid outlet of the reagent handling device is preferably arranged to be communicated with an outlet valve fluid, and the outlet valve is configured to dispense reagents into the liquid layer 107.

[0127] Figure 2 An embodiment is shown in which a sample layer 209 is disposed on the surface of a sample holder 211. The sample layer 209 includes designated sample volumes 210, which are shown herein as having functionalized surfaces 212 that enable the sample to be immobilized in the designated sample volumes 210. Below the sample layer 209, a conductive layer 214 is disposed. The conductive layer 214 is preferably made of a thin layer of a noble metal deposited on the sample holder 211. The noble metal is preferably gold.

[0128] Functionalized surfaces can be provided in several ways.When, for example, cylindrical sample holder 211 has the outer layer of fluorinated polymer film, sample layer 209 can be functionalized by being used to remove the first preparation step of adhesion / functionalization by oxidation surface and by dry etching such as plasma or corona etching or wet etching such as sodium ammonia etching.When the adhesion characteristics on the surface have increased, the subsequent optional step can be applied, wherein the surface is coated with a functionalized compound layer to introduce more specific functionalization. Functionalized compounds can be monomers that introduce functionality, such as (3-aminopropyl) triethoxysilane or such as polyethyleneimine, including oligomers / polymers of specific functional groups such as carboxylic acids, amines, thiols, hydroxyls, oligonucleotides.In one example, in addition to the functional group, the functionalized compound can also include a cleavable group so that the sample can be separated.

[0129] The cylindrical sample holder 211 can be produced from glass using a lathe. After additional grinding and polishing to minimize surface roughness and deviation from the ideal cylindrical shape, the surface of the cylinder is covered with a thin layer of precious metal such as gold. Gold is functionalized with thiols, which are used as intermediate self-assembly layers and have functional groups that can fix target molecules, cells or tissue samples. The advantage of this method is that after completing the determination of the sample, the immobilized sample on the thiolated surface can be made active, and therefore the sample holder 211 can be reused in subsequent determinations.

[0130] In another embodiment, the sample holder 211, which preferably has a gold layer surface, is selectively functionalized using a patterning technique such as photolithography to produce an array of functionalized sites. For example, if the sample consists of target molecules such as single-stranded DNA molecules, each molecule can be fixed to a specified point or a specified well in this way. In a preferred embodiment, patterning for creating the array of functionalized sites for the immobilized sample is accomplished according to the following steps: i) rotating the sample holder; ii) adding photoresist to the surface of the sample holder 111 using the liquid manipulation device 100' disclosed herein; iii) distributing the photoresist in a uniform layer having a desired thickness on a desired surface area of ​​the sample holder 207 using a liquid manipulation device 200 according to the present invention; iv) illuminating the surface of the sample holder 207 using a modulated light source through an imaging optical device (not shown); v) similar to steps i) and ii), adding and distributing a developer, i.e., a liquid that removes the irradiated portion of the photoresist or the unirradiated portion of the photoresist, onto the surface; vi) rinsing and removing the developed portion (in the case of using a positive photoresist) or the undeveloped portion (in the case of using a negative photoresist); and finally vii) functionalizing the exposed surface of the sample holder 207.

[0131] The advantage of this embodiment is that the position and size of the active sites can be very carefully controlled. The feature size using this embodiment is limited by the optics used in the irradiation step and in principle by diffraction. Another important advantage of the embodiment is that more or less the same device can be used both for the preparation of sample immobilization (functionalization) and for the analysis of the sample.

[0132] exist Figure 2 In the embodiment, the samples are arranged in designated sample volumes 210, which may have the shape of wells separated by walls of photoresist. The designated sample volumes are suitable for being covered with a reaction liquid suitable for reacting with the samples that may be immobilized in the designated sample volumes 210.

[0133] Figure 2 It also shows something like Figure 1 The liquid handling device 200 of the liquid handling device shown in FIG. Figure 2 In FIG. 2 , the liquid contact portion 205 is shown as a blade. The blade is preferably provided with a conductive layer.

[0134] The conductive layer 211 acts as an electrode and can be connected to a stationary voltage source 218 during rotation via a slip ring 216. Since the liquid manipulation device 200 also includes a conductive layer in contact with or close to the liquid layer 207, the liquid manipulation device 200 also acts as an electrode, which can be connected to the potential of the conductive layer relative to the sample holder via a wire 220. This arrangement makes it possible to maintain an electric field on the sample layer, or to flow a current through the sample layer, or to measure the capacitance between the conductive fiber and the conductive layer.

[0135] The measurement of the applied electric field or capacitance can be used for many different purposes, such as controlling the distance of the device to the sample holder 207, pulling charged liquid from the surface of the sample holder 207, and the orientation of dipoles or molecules in the sample.

[0136] In many assays, the chemical species involve charged particles such as ions, dipoles, etc. Therefore, the liquid manipulation device 200 can be used to generate a gradient of electric potential (electric field) across the sample layer, or to allow a flow of charge to or from the sample layer.

[0137] Figure 3 Shown with Figure 2 A similar arrangement, except that the liquid contacting portion 305 is a brush rather than a blade. At least some of the bristles of the brush may include conductive fibers. The conductive fibers may be carbon fibers such as graphite fibers. The remaining reference numerals end with the same two digits and are referred to as the beginning Figure 2 The corresponding structure is shown in .

[0138] Figure 4A schematic illustration of the inventive concept according to some embodiments is disclosed. In this schematic illustration, a rotating sample holder 411 has a sample layer 407 extending around the circumference of the sample holder 411. An illumination device 422 and a detection device 424 are arranged so that a sample in a sample layer arranged on the surface of the sample holder 411 can be investigated. An imaging optics 426 is further shown. The imaging optics may include a beam splitter, a reflector, a microlens array, a tubular lens, a dichroic mirror, etc. Preferably, the illumination device and the detection device are positioned in parallel, but other non-parallel positions are also conceivable.

[0139] Provides such as Figures 1 to 3 and Figures 7 to 10 100, 200, 300, 700, 800, 900, 1000, etc. are shown in the figure. In a preferred embodiment, the liquid manipulation devices 400a-f are capable of adding liquid to the sample layer 407, manipulating the liquid present on the sample layer 407, and / or removing liquid from the sample layer. Each liquid manipulation device 400a-f is connected to a liquid container 428a-f, which is suitable for containing liquid to be supplied to the sample layer, or receiving liquid removed from the liquid layer 407 by a liquid removal device.

[0140] The liquid supplied from liquid containers 428a-f can be a composition such as a reagent, a buffer solution and / or a cleaning solution. Each liquid container can include a liquid different from the other containers or a liquid of different concentrations. By arranging several different containers that are each connected to the liquid handling device liquid, the steps of rinsing, flushing and / or cleaning the liquid handling device before adding different liquids can be omitted. On the contrary, new liquids can be added from different liquid handling devices. This further reduces the cycle time of the assay.

[0141] In order to deliver liquid to and / or from the liquid containers 428a-f, the liquid handling device preferably comprises at least one pump and / or a controllable valve such as an electrically controllable valve (not shown). Thus, a higher precision regarding the amount of liquid provided to the liquid layer 40 can be provided.

[0142] At least one of the liquid manipulation devices 400a-f is a reagent dispensing device adapted to provide a reagent to the liquid layer.

[0143] At least one of the liquid manipulation devices 400a-f is a liquid contacting device adapted to manipulate a liquid layer.

[0144] At least one of the liquid manipulation devices 400a - f is a liquid dispensing device adapted to add liquid to the liquid layer 407 .

[0145] At least one of the liquid manipulation devices 400a - f is a liquid removal device adapted to remove liquid from the liquid layer 407 .

[0146] Figure 5 Shown is a schematic diagram of how the thickness of the reaction medium (i.e., the liquid layer covering the sample) in a specific sample volume increases and decreases with different moments. Initially, before Example A, the liquid layer has a high concentration of excess reactant. At point A, liquid is partially displaced or partially removed from a specific sample by a liquid contact device or a liquid removal device, which means that the quantity of the reactant in the sample volume is reduced because the reactant is removed or displaced to other positions on the sample. At point B, liquid with zero concentration is added to the liquid layer by a liquid dispensing device, and the remaining reactant begins to diffuse into the added liquid. After a period of time (if the thickness of the remaining liquid after step A is on the order of several microns, then it may be on the order of 10 milliseconds), the larger part of the remaining reactant has diffused into the main body of the thicker liquid layer now. At point C, liquid is partially displaced by the liquid contact device again or is partially removed by the liquid removal device from a specific sample volume, and the quantity of the reactant present in the specific sample volume has now significantly reduced. Steps B and step C can be repeated thereafter until the concentration of the remaining reactant is sufficiently low. In step D, liquids with different reactants are added via the reagent dispensing device to effect the next reaction in the assay.

[0147] Figure 6Schematically illustrate the various steps in the determination cycle. Initially, i.e., before step A), the liquid layer actually has a reactant of zero concentration. In step A), the liquid handling device liquid suitable for adding liquid will have a desired pH value and temperature, a liquid containing a desired composition and concentration of reactants is distributed to the liquid layer. In optional step B), the liquid layer is subjected to a liquid handling device, which distributes (smears) the liquid added in step A). ​​The step B) is intended to ensure that each sample volume obtains the same concentration and the same liquid layer thickness. If there is a correct target molecule or analyte in the sample volume, the reaction between the reactant and the analyte (target molecule) can occur in the sample volume, as represented by F in the figure. The purpose of the following step C) is to reduce the concentration of the excess reactant (at d) that does not participate in any reaction but is still present in the sample volume) to a sufficiently low level. Step C) is completed by using a liquid handling device suitable for partially displacing or partially removing a portion of liquid from the sample layer. In step D), the liquid handling device distributes the liquid of the reactant close to zero concentration. By repeating steps C) and D) and ending with step C), the concentration is gradually reduced to the desired level. Similarly, step B) can be used to make the surface of the liquid layer uniform, which is advantageous from an optical point of view in the subsequent step E). In step E), the sample volume is irradiated and the light emitted from the sample volume due to the irradiation is detected. For example, if a reaction has occurred between the reactant and the analyte and the fluorescent group attached to the reactant is therefore still present in the sample volume, light emission from the sample volume may occur. This completes the cycle of whether a reaction has been detected or not.

[0148] Figure 7Different examples of liquid manipulation devices 700a-c are schematically depicted. In this example, the liquid manipulation devices 700a-c are stationary relative to the rotating sample holder 711. The liquid layer 707 on the sample holder rotates together with the sample holder 711, and therefore has a speed of the peripheral speed of the sample holder 711 when passing through the liquid manipulation devices 700a-c. All liquid manipulation devices 700a-c are equipped with translation platforms 713a-c and rotation platforms 715a-c so that the liquid manipulation devices can be accurately positioned near or in contact with the liquid layer 707. Such translation platforms 713a-c and rotation platforms 150a-c may include actuators, gears, etc. The translation and / or rotation platforms 713a-c, 715a-c are preferably controlled by a control unit (not shown) suitable for controlling the position of the liquid manipulation device. The rotation platform is used to control the angle of attack of the liquid manipulation device interacting with the liquid layer. The control unit can communicate with sensors or measuring devices to accurately provide feedback about various parameters of the rotation and / or translation platform, such as feedback related to the position of the liquid manipulation device, the angle between the liquid contact portion and the liquid layer, or the pressure applied by the liquid manipulation device to the liquid layer.

[0149] Depicted on the left side of the figure is a liquid manipulation device 700a, which is suitable for removing a portion of liquid S from a liquid layer. The liquid manipulation device includes a liquid contact portion 705a (shown herein as a flexible blade with a sharp shedding edge) that, when immersed in a liquid layer 707, separates the liquid into a shedding portion S and a remaining portion R, which is redirected away from the liquid layer and which rests on a rotating sample holder 711, due to the relative movement of the liquid in the liquid layer and the liquid contact portion 705a. The redirected liquid is captured in an inlet 703a or orifice that is in liquid communication with a liquid channel 701a.

[0150] In the center of the figure, a liquid handling device 700b is depicted, which is suitable for distributing (adding) a portion of liquid to the liquid layer 707 of the sample holder. The liquid is fed to the outlet 704b via the liquid channel 701b. Preferably, the flow of the liquid is regulated so that when the liquid leaves the outlet, the velocity vector of the liquid in the peripheral direction is consistent with the peripheral velocity of the rotating sample holder. In this way, the added liquid stays at the position where it is distributed on the circumference of the sample holder. The amount of liquid distributed can be determined by a liquid control device such as a controllable valve.

[0151] Described on the right side of the figure is a liquid handling device 700c, which is suitable for liquid being displaced or distributed to another position from a position on the sample holder. The liquid handling device comprises a liquid contact portion 705c, which slides on the liquid layer 707 and forms a droplet in front of the liquid contact portion 705c. In this example, the liquid contact portion comprises a brush 705c with bristles 706c. Brush is in contact with liquid channels 701c liquids. The purpose of liquid channels is to feed the liquid of the reactant of zero concentration to the brush through the capillary action of bristles 706c, so that only the outer end of the brush absorbs liquid from the liquid layer. Like this, only the outer end of the brush is polluted by the reactant, and therefore can be quickly rinsed between uses.

[0152] Figure 8 A liquid handling device suitable for removing a portion of liquid from a liquid layer is shown in more detail. A liquid contact portion 805 having a curved blade with an edge 806 is immersed in a liquid layer 807, and the relative movement of the liquid in the liquid layer 807 and the liquid contact portion separates the liquid into a falling portion S and a remaining portion R, which is redirected away from the liquid layer by the blade, and the remaining portion stays on a rotating sample holder. The function of the liquid contact portion 805 is similar to that of a cutting tool in a lathe. The shape of the liquid contact portion 805 is selected so that the falling liquid S is guided to an orifice or inlet 803 that is fluidly connected to a liquid channel 801 with minimal interruption. This is like the way to select a front angle in a traditional cutting tool. The shape of the side of the liquid contact portion 805 facing the rotating sample holder 811 is selected to have a circular shape to avoid sudden pressure changes in the liquid remaining on the sample holder. This is to avoid cavitation in the liquid remaining on the sample holder. Otherwise cavitation may damage the sample. By creating a negative pressure in the liquid channel 801, for example by means of a pump connected to the liquid channel, the detached liquid S trapped in the orifice can be further transported to a container for subsequent recovery or disposal. The figure also summarizes a sample holder 811a provided with a metal layer 814. On top of the metal layer, the sample volume 810 has been defined by a pattern of photoresist, and the sample in the formed recess has been fixed in the recess by previous functionalization of the surface of the exposed part of the metal layer.

[0153] Also in this example, the liquid manipulation device includes a translation device 813 and a rotation device 815, such as those described above.

[0154] Fig. 9A liquid handling device 900 suitable for removing liquid is schematically depicted from another angle. The liquid contact portion 905 has an edge 906 having a certain width in a direction roughly consistent with the direction of the axis of rotation of the rotating sample holder. In this way, liquid can be partially removed from the entire section of the sample holder. This width can be as small as a few tenths of a micron, but can also be on the order of centimeters. The shape of the side of the liquid contact portion 905 facing outward from the rotating sample holder is designed to guide the detached liquid toward the inlet 904 connected to the liquid channel 901.

[0155] Fig.10 Schematically a schematic diagram of a device 1000 according to an embodiment of the invention is shown. In the schematic diagram a liquid layer 1007 is provided on a sample holder 1011. In liquid contact with the liquid layer a liquid contact portion 1005 is provided which is depicted herein as a plate-like element.

[0156] The first part of the plate-like element, i.e. the proximal part 1005a, is provided with a liquid inlet 1004 in liquid connection with the liquid channel 1001a. The liquid inlet is preferably provided with a negative pressure so that liquid from the liquid layer 1007 can be guided to the liquid inlet from the liquid layer 1007 so that the liquid can be removed from the liquid layer 1007. The negative pressure can be provided by a pump (not shown) connected to the liquid channel 1001a. In the depicted embodiment, the liquid inlet 1004 and the liquid channel 1001a form a part of a fluid removal device 1013 capable of removing liquid from the liquid layer.

[0157] A liquid outlet 1003 in liquid connection with the liquid channel 1001b is provided at the second distal end 1005b of the plate-like element 1005. The liquid outlet is configured to dispense liquid to the rotating sample holder so that the amount of liquid and / or the thickness of the liquid layer 1007 can be increased. The liquid outlet 1003 may be provided with at least one valve configured to control the amount of liquid dispensed to the liquid layer.

[0158] The plate-like element 1005 is attached to a supporting structure 1025, which includes a flexible portion 1022 acting as a force control element, which is described herein as being in the form of two pistons 1022a movably attached in a housing 1022b. The force control element is configured to contract and retract when applying and releasing force, respectively. It is also conceivable that the flexible portion 1022 may include at least one spring. The structure 1025 is attached to the housing of the device via the flexible portion 1022. In order not to complicate the accompanying drawings, this connection is not shown, but can be realized by a manner known to those skilled in the art. When the force from the liquid layer 1007 is applied to the plate-like element, depending on the force, the flexible portion will contract a predetermined amount so that the plate-like element 1005 is moved a certain distance from the surface of the sample holder 1011. In the example where the flexible portion includes a spring, the spring force will determine how much the spring will compress under the given applied force from the liquid layer 1007. The force exerted by the fluidic layer depends on the rotation speed of the sample holder 1011 and the amount of liquid present in the liquid layer 1007. The flexible portion 1022 and the connection between the liquid channel and the support structure may be protected by sheaths 1030a-d.

[0159] The provision of the flexible portion 1022 allows the formation of an automatically adjusted thickness of the liquid layer. For example, if the first sample holder 1111 is replaced by a second sample holder having a larger diameter, for example to replace the sample, the flexible portion 1022 allows the formation of the same thick liquid layer 1007 as formed using the first sample holder.

[0160] During operation, when sample holder 1011 rotates, the plate-like element 1005 slides against the liquid layer when the flat surface is against the liquid layer. The liquid contact portion 1005 should preferably be positioned so that the convergence line L defined by the closest distance between the cylindrical surface and the substantially flat surface of the plate-like element is closer to the center of the plate-like element than to the edge of the plate-like element. By placing the liquid inlet 1004 in the place where the liquid layer 1007 is compressed by the liquid contact portion 1005, that is, before the liquid layer passes through the convergence line L, the positive pressure gradient caused by the shear force formed by the relative movement between the liquid contact portion and the cylindrical sample holder will push the liquid into the inlet 1004, and the liquid can therefore be removed from the liquid layer 1007, and the thickness of the liquid layer 1007 is reduced. At the same time, if the outlet 1003 is placed after the convergence line, liquid can be added in a manner similar to a venturi jet pump through the inlet. By controlling the flow of the liquid channel connected to the inlet and outlet respectively, the thickness of the liquid layer can be adjusted. The size of the plate-like element will vary depending on the application, but the width of the contact portion of the element, i.e. the width of the element in physical contact with the liquid layer, is preferably wider than the field of view (FOV) of the imaging system. The length should be at least long enough to leave space for at least one orifice on both sides of the confluence line. In order to move the liquid contact portion 1005 to different positions along the rotation axis of the sample holder 1011, the support structure 1025 may include a device for realizing relative translation of the liquid manipulation device. By translating the liquid contact portion 1005 in a direction substantially perpendicular to the direction defined by the rotation axis of the sample holder 1011, the inlet 1004 can be moved out of contact with the liquid layer 1007 or in contact with the liquid layer. When the inlet does not contact the liquid layer, the liquid contact portion can be used for liquid distribution, i.e. for redistributing and / or changing the thickness of the liquid layer without removing liquid from the liquid layer. The flexible connection of the liquid contact portion 1005 to the support structure 1025 can provide self-regulation of the thickness of the liquid layer. A liquid flow adapted to compensate for the loss of liquid of the liquid layer (eg by evaporation) may be supplied via the liquid outlet 1003. The added liquid will be distributed by the liquid contact portion such that the resulting thickness balances the force exerted by the flexible connector.

[0161] As shown in this figure, the plate-like element 1005 may include a first surface arranged to face the liquid layer, a second surface opposite to the first surface, and an orifice arranged at the first surface. In this example, the orifice is represented by an outlet 1003 and an inlet 1004. Further, a passage may be provided between the first surface and the second surface, thereby allowing a fluid to be communicated between a liquid channel such as the first liquid channel 1001a or the second liquid channel 1001b and the orifice.

[0162] The outlet 1003 and the liquid channel 1001b are included in a liquid dispensing device 1015, which is capable of adding liquid to the surface of the sample holder 1011. The liquid outlet 1003 is connected to the liquid channel 1001b via a passage through the plate-like element, so that the liquid outlet 1003 can receive liquid from the channel and add the liquid to the surface of the liquid layer. The liquid outlet 1003 can also be adapted to function as a nozzle so that the liquid can be ejected onto the surface of the sample holder. Preferably, the liquid dispensing device 1015 includes at least one valve, which is adapted to control the amount of liquid provided to the sample holder 1007.

[0163] Liquid can be distributed to the rotating surface through the liquid outlet 1003, and its flow vector is directed at a small angle relative to the tangent of the surface. The speed of the liquid when leaving the liquid outlet 1003 is appropriately selected to make the speed consistent with the peripheral speed of the surface of the rotating sample holder 1011. In this way, the liquid deposited on the rotating surface stays at the position where it is deposited. Once the liquid is added to the sample holder, the liquid is usually unevenly distributed and needs to be redistributed or smeared to cover each desired position of the sample layer. The liquid contact portion 1005 of the liquid manipulation device can be used to redistribute and / or change the thickness of the liquid layer and control which areas of the sample layer are covered with liquid.

[0164] Fig.11 A schematic diagram of an apparatus 1100 according to an embodiment of the present invention is shown. In the schematic diagram, a liquid layer 1107 is disposed on a cylindrical sample holder 1111, which is rotatably arranged in the apparatus around its longitudinal axis. In liquid contact with the liquid layer, a liquid contact portion is provided, which is depicted herein as a plate-like element 1105. The plate-like element 1105 is flexibly assembled in the device housing. A flexible assembly is realized, in which the plate-like element 1105 is attached to a flexible portion 1122, which in turn is attached to the device housing. The flexible portion 1122 can be, for example, a spring.

[0165] The device is further provided with a liquid dispensing device 1115 which is capable of adding liquid to the surface of the sample holder 1111. The liquid dispensing device may also be used to provide reagents to the liquid layer, in which case the liquid dispensing device doubles as a reagent dispensing device.

[0166] The liquid dispensing device 1115 is arranged to add liquid to the surface of the sample holder 1111. Preferably, the liquid dispensing device 1115 includes at least one valve, which is suitable for controlling the amount of liquid 1140 provided to the sample holder 1111. The liquid can be distributed to the rotating surface by the liquid dispensing device 1115, and its flow vector is directed at a small angle relative to the tangent of the surface. The speed of the liquid when leaving the liquid dispensing device 1115 is appropriately selected so that it is consistent with the peripheral speed of the surface of the rotating sample holder 1111. In this way, the liquid deposited on the rotating surface stays at the position where it is deposited. Once the liquid is added to the sample holder, the liquid is usually unevenly distributed and needs to be redistributed or applied to cover each desired position of the sample layer. The plate-like element 1105 of the liquid manipulation device can be used to redistribute and / or change the thickness of the liquid layer, and control which areas of the sample layer are covered with liquid. The plate-like element 1105 may also be arranged so that liquid droplets dispensed from the liquid dispensing device 1115 that do not adhere to the sample holder but instead bounce off are redirected back toward the sample holder.

[0167] When a force from the liquid layer 1107 is applied to the plate-like element, the flexible portion will contract, the amount of which depends on the thickness of the liquid layer and the rotation of the sample holder, so that the plate-like element 1105 is moved a certain distance from the surface of the sample holder 1111. In the example where the flexible portion comprises a spring, the spring force will determine how much the spring will compress under a given applied force from the liquid layer 1107. The force applied by the liquid layer depends on the rotation speed of the sample holder 1111 and the amount of liquid present in the liquid layer 1107. Preferably, the spring force is small compared to the capillary force acting on the plate-like element 1105, so that the thickness of the liquid layer 1107 is mainly determined by the balance between the surface tension and the lubrication pressure, while the spring force mainly serves to ensure that there is contact between the liquid layer 1107 and the plate-like element 1105.

[0168] The provision of the flexible portion 1122 allows the formation of a liquid layer of automatically adjusted thickness. For example, if the first sample holder 1111 is replaced by a second sample holder having a larger diameter, for example in order to replace the sample, the flexible portion 1122 allows the formation of a liquid layer 1107 of the same thickness as that formed with the first sample holder. Obtaining a liquid layer of the same thickness with a fixedly assembled liquid contact portion would place extreme demands on the manufacturing tolerances of the sample holder, especially when the desired thickness of the liquid layer is in the range of several μm.

[0169] The device 1100 may further be provided with a liquid removal device, such as Figures 1 to 10 As described in any one of .

[0170] Although the present invention has been described with reference to various exemplary embodiments, it will be appreciated by those skilled in the art that various changes may be made and elements thereof may be replaced with equivalents without departing from the scope of the present invention. In addition, many modifications may be made to adapt specific circumstances or materials to the teachings of the present invention without departing from the essential scope of the present invention. Therefore, it is intended that the present invention is not limited to the specific embodiments disclosed as the best mode for carrying out the present invention, but that the present invention will include all embodiments falling within the scope of the appended claims.

[0171] Furthermore, variations of the disclosed embodiments are understood and accomplished by the skilled person in practicing the claimed invention by studying the drawings, the disclosure, and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.

Claims

1. A device for measuring and / or processing a sample, the device comprising: a cylindrical sample holder for holding a sample at a side surface of the cylindrical sample holder, wherein the cylindrical sample holder is rotatably arranged; wherein the cylindrical sample holder is configured to be at least partially covered by a liquid layer; a liquid dispensing device configured to add liquid to the liquid layer, wherein the liquid dispensing device is connected to a first container for containing the liquid; a reagent dispensing device configured to add a reagent to the liquid layer, wherein the reagent dispensing device is connected to a second container for containing the reagent; A liquid contact device configured to distribute liquid within the liquid layer, wherein the liquid contact device includes a plate-like element having a substantially flat surface suitable for contacting the liquid layer, the plate-like element being flexibly assembled relative to the cylindrical sample holder so that the plate-like element can be arranged at a first predetermined distance from the cylindrical sample holder when no external force acts on the plate-like element, and is arranged at a second, greater distance from the cylindrical sample holder when the liquid layer exerts a force on the plate-like element when the cylindrical sample holder is rotated, wherein the plate-like element is attached to a force control element configured to control the amount of force exerted by the plate-like element on the liquid layer, wherein the force control element includes a flexible portion configured to contract when a force is applied from the liquid layer when the cylindrical sample holder is rotated, thereby increasing the distance between the plate-like element and the sample holder as long as the force is applied.

2. The device according to claim 1, wherein: The plate-like element comprises at least one aperture configured to perform at least one of the following: dispensing a liquid into the liquid layer; and removing liquid from the liquid layer; and wherein, The at least one orifice is in fluid communication with a liquid channel configured to at least one of: delivering a fluid to the orifice, and Fluid is delivered from the orifice.

3. The device according to claim 2, wherein: The plate-like element comprises: a first surface configured to face the liquid layer; a second surface, the second surface being opposite to the first surface; and a passage extending between the first surface and the second surface; wherein the at least one orifice is arranged at the first surface; and Therein, the passageway is arranged to provide fluid communication between the orifice and the liquid channel.

4. A device according to any one of the preceding claims, wherein: The device further comprises mixing means arranged to induce mixing in said liquid layer.

5. The device according to claim 4, wherein: The mixing tool includes at least one of the following: brush; a plate-like element comprising at least one ridge; A plate-like element comprising at least one post.

6. The device according to any one of claims 1 to 3, further comprising: an irradiation device, the irradiation device being used to irradiate the sample; an objective lens for collecting photons emitted or scattered from the sample; as well as a detection device for detecting photons collected by the objective lens; Therein, the optical contact between the objective lens and the sample is at least partially provided by the liquid layer.

7. The device according to any one of claims 1 to 3, further comprising: Means for measuring an amount indicative of the concentration of said reagent in said liquid layer.

8. A method carried out in a device for measuring and / or processing a sample, the method comprising the following steps: a) arranging a cylindrical sample holder in the device; b) providing at least one sample to be investigated on the cylindrical sample holder; c) providing a first type of liquid to the sample holder, d) distributing the liquid to form a liquid layer having a uniform thickness on at least one circumference on the cylindrical sample holder by arranging a plate-like element and rotating the cylindrical sample holder about a longitudinal axis, the plate-like element being flexibly assembled relative to the cylindrical sample holder so that the plate-like element can be arranged at a first predetermined distance from the cylindrical sample holder when no external force acts on the plate-like element, and at a second, greater distance from the cylindrical sample holder when the liquid layer exerts a force on the plate-like element when the cylindrical sample holder is rotated, and wherein the plate-like element is attached to a force control element configured to contract when a force is exerted from the liquid layer when the cylindrical sample holder is rotated, thereby increasing the distance between the plate-like element and the sample holder as long as a force is applied; e) providing a reagent to the liquid layer; f) allowing at least one desired reaction to occur between the reagent and the at least one sample; g) Removing liquid from the liquid layer in a cylindrical sample holder arranged in the device.

9. The method according to claim 8, further comprising the step h) of illuminating the sample and detecting photons emitted or scattered from the sample.

10. The method according to claim 8 or 9, wherein the method further comprises a step g1) of diluting the concentration of the reagent in the liquid layer by adding the first type of liquid to the liquid layer.

11. The method according to claim 8 or 9, further comprising the step e1) of increasing the concentration of the reagent in the liquid layer by evaporating liquid from the liquid layer.

12. The method according to claim 8 or 9, further comprising the step of inducing mixing in the liquid layer to make the concentration of the reagent in the liquid layer uniform.

13. The method according to claim 8 or 9, further comprising the step of adjusting the temperature of the liquid layer to a first predetermined temperature.

14. The method according to claim 8 or 9, wherein: The steps following steps a) and b) are performed during rotation of the sample holder.

15. The method according to claim 8 or 9, wherein: The step of removing liquid is performed while the sample holder is stationary.

16. The method according to claim 8 or 9, wherein: Step d) further comprises controlling the thickness of the liquid layer by arranging a plate-like element flexibly assembled with respect to the cylindrical sample holder in liquid contact with the liquid layer.

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