System for analysis

Abstract

A device for analyzing a sample liquid is provided, the device including a microfluidic test card and a microfluidic chip for processing sample liquid presented from the microfluidic test card and returning the processed sample fluid to the microfluidic test card. The microfluidic test card includes a sample inlet configured to receive sample liquid, first and second pre-treatment test reagent channels having first and second test reagent outlets, respectively, for presenting test reagents to the microfluidic chip, a pre-treatment sample channel in fluid communication with the sample inlet for receiving sample liquid from the sample inlet and having a sample liquid outlet for presenting sample liquid to the microfluidic chip, first and second processed sample analysis channels for receiving processed sample liquid from the microfluidic chip, wherein the first and second processed sample analysis channels include first and second analysis zones, respectively, for analyzing the processed sample liquid, and a microfluidic chip contact region including the sample liquid outlet and the first and second test reagent outlets configured for connection and fluid communication with the microfluidic chip.

Description

Technical Field

[0001] The present invention relates to an apparatus for analyzing sample liquids, and a system including the apparatus. Background Technology

[0002] Accurate and precise diagnostic tests are a vital component of an effective and efficient healthcare system. Because achieving accuracy and precision typically requires laboratory-scale equipment, most diagnostic tests used in medical practice today are performed in centralized laboratory settings, negatively impacting the overall cost, time to result, and availability of diagnostic testing. Concepts have been proposed to enable on-demand diagnostic testing, but at the cost of reduced capacity to process clinical samples in the workflow while ensuring accuracy and precision.

[0003] Therefore, there is a need for low-cost, easy-to-use, and readily available diagnostic testing solutions. The World Health Organization (WHO) Sexually Transmitted Diseases Diagnostics Initiative has published the ASSURED benchmark to assess whether diagnostic solutions meet global needs. According to this benchmark, solutions need to be: affordable, sensitive, specific, user-friendly, robust, require no equipment, and deliverable to the end user.

[0004] Several point-of-care solutions have been proposed for other types of diagnostic tests, and some technologies have been successfully commercialized, but they still fall far short of the simplicity and convenience of a glucose test that allows monitoring glucose in a single drop of blood. Rapid diagnostic tests (RDTs) are among the simplest forms of point-of-care diagnostic tests, typically involving a nitrocellulose wick coated with reagent at a specific location. Fluid actuation is generated by capillary wicking of an aqueous liquid within the nitrocellulose strip, and the diagnosis is read by the human eye, common devices such as smartphones, or dedicated reader devices by detecting a colored band. Due to their simplicity, RDTs can be affordable and often require no equipment, but they typically fail to meet other requirements of the Assured standard. Sensitivity and specificity are often less than ideal because comprehensive quality control cannot be performed by humans as in a laboratory setting.

[0005] On a more complex level, on-the-spot solutions for molecular assays, including more sophisticated cartridges and instruments, are available. These systems achieve a more compact size than their centrally-laboratory counterparts by streamlining the workflow to a single-use cartridge actuated by the instrument in various ways. These systems are easier to use because reagent delivery is built into the single-use material, allowing users to simply apply the sample and run the appropriate procedure associated with the desired test cartridge. The need to provide mechanical, thermal, and optical interfaces between the instrument and the cartridge limits the extent to which the instrument can be miniaturized and implies prohibitive costs for many on-the-spot environments. Costs depend on the initial investment required for the instrument, consumable costs, maintenance, required infrastructure, operator time, and more. Summary of the Invention

[0006] The object of this invention is to mitigate, alleviate, or eliminate one or more of the aforementioned defects and disadvantages in the art, either alone or in any combination, and to solve at least one of the aforementioned problems. Another object of this invention is to provide an efficient or improved system for analyzing sample liquids (e.g., blood samples).

[0007] According to a first aspect of the present invention, an apparatus for analyzing sample liquids is provided. The apparatus includes a microfluidic test card and a microfluidic chip, the microfluidic chip being used to process sample liquids presented from the microfluidic test card and return the processed sample liquid fluid to the microfluidic test card. The microfluidic test card includes: a sample inlet configured to receive sample liquids; a first pretreatment reagent channel and a second pretreatment reagent channel, the first and second pretreatment reagent channels respectively having a first reagent outlet and a reagent outlet for presenting reagents to the microfluidic chip; a pretreatment sample channel fluidly connected to the sample inlet for receiving sample liquids from the sample inlet, and the pretreatment sample channel having a sample liquid outlet for presenting the sample liquids to the microfluidic chip; a first processed sample analysis channel and a second processed sample analysis channel. The first and second processed sample analysis channels are used to receive processed sample liquid from the microfluidic chip. Each channel includes a first analysis area and a second analysis area for analyzing the processed sample liquid, respectively. The microfluidic chip contact area includes a sample liquid outlet, a first test reagent outlet, and a second test reagent outlet. All outlets are configured for connection and fluid communication with the microfluidic chip. The microfluidic chip includes a sample liquid inlet. The sample liquid inlet is configured to be in fluid communication with and receive sample liquid from the sample liquid outlet of the test card; a first microfluidic channel system for processing the sample liquid, configured to be in fluid communication with the first test reagent outlet and thereby configured to receive the first test reagent from the first pretreatment test reagent channel, and further configured to be in fluid communication with the sample liquid inlet and thereby configured to receive sample liquid from the pretreatment sample liquid channel and allow contact between the sample liquid and the first test reagent within the first microfluidic channel system; and a second microfluidic channel system for processing The sample liquid is configured to be in fluid communication with the outlet of the second test reagent and thus configured to receive the second test reagent from the second pretreatment test reagent channel, and is further configured to be in fluid communication with the inlet of the sample liquid and thus configured to receive the sample liquid from the pretreatment sample liquid channel and allow the sample liquid and the second test reagent to contact within the second microfluidic channel system, wherein the first microfluidic channel system and the second microfluidic channel system respectively include a first outlet and a second outlet, the first outlet and the second outlet being configured to be in fluid connection with the first processed sample analysis channel and the second processed sample analysis channel of the microfluidic test card, respectively.

[0008] According to a second aspect of the invention, a system is provided comprising means for analyzing a sample liquid according to the first aspect, and a reader. The reader comprises a computational or lensless holographic microscope, preferably comprising a (partially or fully) spatially coherent light source and a complementary metal-oxide-semiconductor imager, wherein the reader is configured to receive the means for analysis and is further configured to allow the imager to image a first and a second detection area of ​​a test card, thereby allowing analysis of the sample liquid.

[0009] A reader can be a detection device.

[0010] According to another aspect of the present invention, a method for performing liquid sample processing and analysis on a microfluidic system is provided, the microfluidic system including a disposable microfluidic test card, the disposable microfluidic test card including a microfluidic sample processing area. The method includes: receiving a liquid sample into the microfluidic test card; transferring the received liquid sample to the microfluidic sample processing area via capillary action; and, as a timing event, performing the following in the microfluidic sample processing area: metering a predetermined volume of the transferred liquid sample; separating the predetermined volume of the transferred liquid sample from the remaining transferred liquid sample to provide a separated liquid sample having a predetermined volume; mixing or contacting the separated liquid sample with a test reagent; processing the separated liquid sample mixed or contacted with the test reagent to obtain a processed liquid sample; and performing analysis on the processed liquid sample on the microfluidic test card.

[0011] The further applicability of this disclosure will become apparent from the detailed description given below. However, it should be understood that while the detailed description and specific examples indicate preferred variations of the inventive concept, they are given by way of illustration only, as various changes and modifications within the scope of the inventive concept will become apparent to those skilled in the art based on this detailed description.

[0012] Therefore, it should be understood that the inventive concept is not limited to the specific steps of the described method or the components of the described system, as such methods and systems can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It must be noted that, unless the context clearly specifies otherwise, the articles “a,” “an,” “the,” and “said” are intended to mean one or more of the elements present, as used in this specification and the appended claims. Thus, for example, references to “unit” or “the unit” may include several means, etc. Furthermore, the words “comprising,” “including,” “containing,” and similar terms do not exclude other elements or steps. Attached Figure Description

[0013] The foregoing and other aspects of the inventive concept will now be described in more detail with reference to the accompanying drawings, which illustrate variations of the invention. The drawings should not be construed as limiting the invention to specific variations; rather, they are intended to explain and understand the inventive concept.

[0014] As shown in the accompanying drawings, the sizes of components, layers, and distances can be enlarged for illustrative purposes and are thus provided to demonstrate the overall structure for variations of the inventive concept. Throughout the text, the same reference numerals refer to the same elements.

[0015] Figure 1 One aspect of the inventive concept is illustrated schematically.

[0016] Figure 2 A microfluidic test card according to an embodiment is shown.

[0017] Figure 3 illustrates a microfluidic channel system according to an embodiment.

[0018] Figure 4 The fluid connection according to an embodiment is illustrated schematically.

[0019] Figure 5 The sample metering and / or channel system according to the embodiment is illustrated schematically.

[0020] Figure 6 A system based on a second concept and / or embodiment is shown.

[0021] Figure 7 The experimental results are presented. Detailed Implementation

[0022] This invention provides a technique for reducing and simplifying the entire sample liquid analysis workflow. A series of operations can be performed autonomously within a compact, disposable microfluidic test card, without the need for skilled personnel or laboratory equipment. This is achieved by precisely controlling capillary forces within the fluidic microchip structure, enabling the execution of a sequence of steps without further human intervention and / or additional instruments or actuations. Furthermore, for example, when combined with lensless computational microscopy and / or computer vision technologies, these autonomously driven microfluidic systems can be test card solutions for achieving desired point-of-care diagnostics.

[0023] Flow control operations can be achieved and controlled through capillary forces, which are used to propel liquids and control operations such as valve regulation, metering, temperature control, and conditional operations.

[0024] The inventive concept will now be described more fully below with reference to the accompanying drawings, in which presently preferred variations of the inventive concept are shown. However, the inventive concept can be practiced in many different forms and should not be construed as being limited to the variations set forth herein; rather, these variations are provided to achieve thoroughness and completeness and to fully communicate the scope of the inventive concept to those skilled in the art.

[0025] It should be understood that at least the channels of the device can be capillary channels. A capillary channel can be considered as a channel capable of providing a capillary-driven flow of liquid. It should also be understood that, according to specific embodiments of the invention, other channels of the system can be capillary channels and / or other types of channels.

[0026] In the following text, fluids are described as flowing through channels within a microfluidic system and arriving at specific locations at different times. The flow rates of these flows can be controlled in various ways to ensure that the fluid arrives at these locations at the described times. Capillary-driven fluid flow requires one or more contact surfaces that the fluid can wet. For example, surfaces comprising glass or silica can be used for capillary-driven aqueous liquid flows. Furthermore, suitable polymers, for example, possessing hydrophilic properties (either inherent to the polymer or acquired through modification, including, for example, chemical modification or coating), can promote or enhance capillary-driven flow.

[0027] For example, these flows can be controlled by adapting the length of the channel and / or by adapting the flow resistance of the channel. The flow resistance of the channel can be controlled by adapting the cross-sectional area of ​​the channel and / or the length of the channel. The flow resistance of the channel can further depend on the properties of the liquid, such as the dynamic viscosity of the liquid. Additionally or alternatively, the flow rate can be adapted by using a flow resistor.

[0028] To provide the desired capillary force, the size of the flow channel can be selected based on, for example, the properties of the liquid and / or material and / or the properties of the channel walls.

[0029] refer to Figure 1The first aspect of the inventive concept will now be discussed. An apparatus 1 for analyzing sample liquids is shown. The apparatus 1 includes a microfluidic test card 2 and a microfluidic chip 4, which processes sample liquids (not shown) presented from the microfluidic test card 2 and returns the processed sample liquids to the microfluidic test card 2.The microfluidic test card 2 includes: a sample inlet 6 configured to receive sample liquid; a first pretreatment reagent channel 8 and a second pretreatment reagent channel 10, each having a first reagent outlet 12 and a second reagent outlet 14 for delivering reagents to the microfluidic chip 4; a pretreatment sample channel 16 fluidly connected to the sample inlet 6 for receiving sample liquid from the sample inlet, and having a sample liquid outlet 18 for delivering the sample liquid to the microfluidic chip 4; a first processed sample analysis channel 20 and a second processed sample analysis channel 22. The first and second processed sample analysis channels are used to receive processed sample liquid from the microfluidic chip 4. The first and second processed sample analysis channels 20 and 22 respectively include a first analysis area 24 and a second analysis area 26 for analyzing the processed sample liquid; and a microfluidic chip contact area 28, which includes the sample liquid outlet 18, a first test reagent outlet 12, and a second test reagent outlet 14. These outlets are configured to connect and fluidly communicate with the microfluidic chip 4. The microfluidic chip 4 includes a sample liquid inlet 30, which is configured to connect with the sample liquid outlet of the test card 2. 18. Fluid communication and receiving of sample liquid from the sample liquid outlet; a first microfluidic channel system 32, configured to process the sample liquid, fluidly communicate with the first test reagent outlet 12 and thereby configured to receive the first test reagent from the first pretreatment test reagent channel 8, and further configured to fluidly communicate, for example via optional channel 36 or directly with the sample liquid inlet 30 and thereby configured to receive the sample liquid from the pretreatment sample liquid channel 16 and allow contact between the sample liquid and the first test reagent within the first microfluidic channel system 32; and a second microfluidic channel system 34, configured to process the sample liquid and fluidly communicate with the second test reagent from the first test reagent outlet 8; The reagent outlet 14 is fluidly connected and thus configured to receive the second test reagent from the second pretreatment test reagent channel 10, and is further configured to be fluidly connected, for example via optional channel 36 or directly to the sample liquid inlet 30, and thus configured to receive sample liquid from the pretreatment sample liquid channel 16 and allow contact between the sample liquid and the second test reagent within the second microfluidic channel system 34, wherein the first microfluidic channel system 32 and the second microfluidic channel system 34 respectively include a first outlet 40 and a second outlet 42, which are configured to be fluidly connected to the first processed sample analysis channel 20 and the second processed sample analysis channel 22 of the microfluidic test card 2, respectively.

[0030] The first and second test reagents can be independent, for example, test reagent liquids. The test reagent liquids can be, for example, buffer solutions or liquids containing reagents.

[0031] The first processed sample analysis channel and the second processed sample analysis channel may have a first processed sample inlet and a second processed sample inlet, respectively, and the first processed sample inlet and the second processed sample inlet are configured to be fluidly connected to a first outlet and a second outlet, respectively.

[0032] The first microfluidic channel system and the second microfluidic channel system may each have a first test reagent inlet and a second test reagent inlet, which are configured to be fluidly connected to the first test reagent outlet and the second test reagent outlet, respectively.

[0033] refer to Figure 2 The illustration shows a microfluidic test card 2 according to examples and embodiments. The microfluidic test card 2 includes: a sample inlet 6, which (located at the edge of the microfluidic test card 2 in this example, but it can alternatively be located at other locations) is configured to receive sample liquid; a first pretreatment reagent channel 8 and a second pretreatment reagent channel 10, the first and second pretreatment reagent channels having a first reagent outlet 12 and a second reagent outlet 14 for presenting test reagents to the microfluidic chip, respectively; and a pretreatment sample channel 16, which is in fluid communication with the sample inlet 6 for receiving sample liquid from the sample inlet, and the pretreatment sample channel has a function for... The sample liquid is delivered to the sample liquid outlet 18 of the microfluidic chip; a first processed sample analysis channel 20 and a second processed sample analysis channel 22 are used to receive processed sample liquid from the microfluidic chip, wherein the first processed sample analysis channel 20 and the second processed sample analysis channel 22 respectively include a first analysis area 24 and a second analysis area 26 for analyzing the processed sample liquid; and a microfluidic chip contact area 28, which includes the sample liquid outlet 18 and a first test reagent outlet 12 and a second test reagent outlet 14. Although the microfluidic test card 2 does not include the microfluidic chip 4, in Figure 2 In the image, the microfluidic chip is shown as a dark gray area in the chip contact area 28 located below the surface of the microfluidic test card 2 in an attempt to improve clarity.

[0034] Although a first pretreatment test reagent channel 8 and a second pretreatment test reagent channel 10 having a first test reagent outlet 12 and a second test reagent outlet 14, and a first processed sample analysis channel 20 and a second processed sample analysis channel 22 including a first analysis zone 24 and a second analysis zone 26 are shown, it should be understood that, alternatively, the apparatus and system may have only one of each of the presented channels, and the microfluidic chip and reader may be suitably adapted accordingly.

[0035] The microfluidic test card 2 allows for the introduction of reagents and samples, integration of additional components (such as capillary wicks and imaging areas), and provides a more convenient form factor for manual handling. The microfluidic test card 2 can consist of several patterned layers laminated onto each other, starting from, for example, an injection-molded substrate. To integrate the microfluidic chip 4 into / on the microfluidic test card 2, fluid needs to be transferred from the microfluidic test card 2 to the microfluidic chip 4 via capillary wicking / capillary force, and vice versa. This can be achieved by designing the outlet of the microfluidic chip 4 and by designing features of the foil stack that ensure rapid wicking to the surface of the microfluidic chip 4. The fluid transfer from the fluid channels in the microfluidic test card 2 to the capillary wick / channels, which act as waste reservoirs, can be designed to ensure a sufficiently low failure rate.

[0036] The microfluidic chip 4 can have precisely defined microfluidic channel geometry and surface properties. Fluid can be delivered via capillary wicking but can be stopped by a geometric feature known as a trigger valve and triggered to continue flowing across the valve again. For reliable operation, the microfluidic chip 4 can be constructed using a process that creates closed microfluidic channels by covering a first wafer (containing the chip) with a capping wafer. The process for the bottom wafer can achieve two etching depths, while the top wafer can have recesses in addition to fluid access holes, resulting in three levels of microfluidics that can be combined to achieve the desired component and system performance. The geometry and surface properties of the microfluidic channels can be controlled using silicon chip fabrication techniques. Both horizontal and vertical dimensions can be geometrically controlled using deep UV lithography and deep reactive ion etching (DRIE) techniques, respectively. Well-defined contact angles can be achieved using a single-layer coated silicon surface (including in the masked channels) covalently bonded from the vapor phase to the surface of the microfluidic chip 4. The manufacturing method of the microfluidic chip 4 can conform to silicon processing technology, which allows the manufacturing of the microfluidic chip 4 to be flexibly carried out at a selected manufacturing location.

[0037] The operation of the trigger valve can rely on three fluid levels to ensure reliable operation that maintains its leak-free capability. It can be reliably triggered and can operate without forming unwanted bubbles (which could otherwise hinder system operation). The ability to program complex fluid manipulation sequences allows for the integration of complete sample workflows into autonomously operating silicon microfluidic chips, such as microfluidic chip 4.

[0038] Combining a trigger valve with a high-resistance fluid channel can produce programmable or predeterminable delayed functions. For example, by adjusting the channel fluid resistance and relying on the coordination of several competing menisci, the fluid can be actuated in complex sequence of steps, including reversal of fluid motion, which is generally considered impossible in capillary-driven systems. By adjusting the channel size, specific operations can be performed conditionally, as discussed below. The microfluidic chip 4 is designed to accept three fluids: for example, blood samples, aqueous dilution test reagent solutions, and aqueous erythrocyte lysis test reagent solutions. According to the example microfluidic chip 4, a sequence of operations can be performed on the sample, some of which can be gated by reagents. First, the sample can arrive at the sample inlet of the microfluidic chip 4 and be converted into three simultaneous streams. Two of these streams can be designed to meter specific volumes of sample, for example, 100 nL-1000 nL (e.g., 600 nL) and 5 nL-50 nL (e.g., 10 nL), respectively, while the third stream can remove excess sample. In subsequent steps, by using... Figure 2 and Figure 4 The similar designs shown or Figure 2 and Figure 4 The design of the type shown, which replaces the upstream portion of the fluid plug with a test reagent solution, can isolate the metered volume from the trailing sample liquid plug. Subsequently, for example, a metered 10 nL volume of sample can be diluted, for example, by 400-fold with the dilution test reagent, while for example, a metered 600 nL volume can be mixed with the lysis test reagent at a ratio of, for example, 1:5.

[0039] The microfluidic chip 4 and the microfluidic test card 2 can be arranged such that their respective channels are oriented in different planes (e.g., parallel planes), for example, the microfluidic chip 4 and the microfluidic test card 2 can be arranged vertically relative to each other. It should be understood that, for example, liquid communication between the first pretreatment reagent channel and the second pretreatment reagent channel, and between the channels of the first microfluidic channel system and the channels of the second microfluidic channel system, can be achieved through channels or openings, for example, whose direction or flow direction is orthogonal to the planes of the microfluidic chip 4 and the microfluidic test card 2.

[0040] For example, you can refer to Figure 4 Further descriptions are available upon request. Figure 1 and Figure 2 The described embodiments. Figure 4 A microfluidic arrangement is shown for capillary-driven fluid connections between capillary flow channels (e.g., between pretreatment sample channels 8, 10, 2014 or pretreatment test reagent channels 16, 2014 and their connected / corresponding microfluidic channel systems 32, 34, 2016). The microfluidic arrangement includes: a first microfluidic system or microfluidic test card 2, comprising a first surface and a first capillary flow channel 208, wherein the first capillary flow channel 208 has an extension in a first plane, and the first surface includes an outlet opening 209 (e.g., a sample / test reagent outlet) in a plane other than the first plane, the outlet opening defining an outlet region in the first surface and adapted to allow fluid communication with the first capillary flow channel, thereby forming a flow outlet of the first capillary flow channel; and a second microfluidic system, comprising a second surface and a second capillary flow channel, wherein the second capillary flow channel is parallel to the first surface... The second plane has an extension, and a portion of the second surface includes an inlet opening in a plane different from the second plane. This inlet opening defines an inlet region in the second surface and is adapted to allow fluid communication with a second capillary flow channel, thereby forming a flow inlet for the second capillary flow channel. The first and second microfluidic systems are arranged such that the first and second surfaces are in contact such that the flow outlet and flow inlet interfaces are connected, thereby allowing capillary-driven fluid communication between the first and second capillary flow channels. At least a portion of the outlet region overlaps with the inlet region, and the overlapping portion of the inlet region is smaller than the outlet region. Similarly, fluid communication between the outlet of a microfluidic chip and the outlet of a processed sample analysis channel can be a fluid connection, as referenced in [reference missing]. Figure 4 As described.

[0041] The first microfluidic channel system 32 and the second microfluidic channel system 34 may each include a first sample metering capillary channel and a second sample metering capillary channel for providing a predetermined sample volume. Therefore, the device 1 allows for the processing of two predetermined sample volumes of liquid samples in parallel or sequentially.

[0042] This predetermined sample volume can be referenced. Figure 5 The arrangement shown is provided. Although reference is made in the discussion to the first microfluidic channel system 32, Figure 5 The arrangement can be a first microfluidic channel system 32 or a second microfluidic channel system 34, or a part thereof. Figure 5 This demonstrates a sample liquid for providing a predetermined sample volume. Figure 5A first microfluidic channel system 32 (not shown) is configured to receive sample liquid via a sample liquid inlet 30 and a sample liquid outlet 18. The first microfluidic channel system 32 further includes a first sample processing channel 120 connected to the sample liquid inlet 30. The first sample processing channel 120 branches into a second sample processing channel 122 terminating at a first valve 130 and into a third sample processing channel 124. The third sample processing channel 124 branches into a fourth sample processing channel 126 terminating at a second valve 132 and into a fifth sample processing channel 128 terminating at a third valve 134, wherein the fifth sample processing channel 128 has a predetermined volume. The first valve 130, the second valve 132, and / or the third valve 134 may be trigger valves. A trigger valve can stop the main liquid flow in its closed state and allow the main liquid flow through the trigger valve in its open state. The trigger valve can be opened by a secondary flow (i.e., changed to its open state) and can allow a combined flow of the main and secondary flows through the output of the trigger valve. Such a trigger valve is known in the art as a capillary trigger valve. The first microfluidic channel system 32 shown is further configured to be in fluid communication with the first test reagent outlet 12 via a test reagent inlet 13 arranged for receiving the first test reagent. The first test reagent inlet 13 can therefore be arranged for receiving the test reagent.

[0043] The microfluidic channel system 32 further includes a first trigger channel 150 arranged to connect the first test reagent inlet 13 to the second valve 132. The microfluidic channel system 32 further includes a second trigger channel 152 connecting the second valve 132 and the first valve 130.

[0044] The microfluidic channel system 32 further includes an outlet channel 154 having a first end 1542 and a second end 1544. The first end 1542 is connected to a first valve 130. A first sample processing channel 120 is arranged to draw sample liquid from the sample inlet 30 by capillary action to fill the first sample processing channel 120, the second sample processing channel 122, the third sample processing channel 124, the fourth sample processing channel 126, and the fifth sample processing channel 128. The flow of sample liquid is stopped by these valves when the first valve 130, the second valve 132, and the third valve 134 are in their closed state.

[0045] The first trigger channel 150 is arranged to draw test reagent from the first test reagent inlet 13 via a liquid path including the second trigger channel 152 to the outlet channel 154 by capillary action, and to open the second valve 132 and the first valve 130, thereby opening another liquid path including the fourth processed sample channel 126, the third processed sample channel 124, and the second processed sample channel 122. This opened liquid path allows samples present in the fourth processed sample channel 126, the third processed sample channel 124, and the second processed sample channel 122 to be replaced by test reagent from the first trigger channel 150 and flow into the outlet channel 154 together with test reagent from the second trigger channel 152, thereby isolating the sample liquid present in the fifth processed sample channel 128 from the adjacent sample liquid. The first sample processing channel 120 and / or the fifth sample processing channel 128 can be adapted, for example, by adapting their respective geometries (e.g., cross-sectional dimensions and / or shapes), such that capillary forces (or capillary pressures) prevent sample liquid present in the first sample processing channel 120 and / or the fifth sample processing channel 128 from flowing toward the discharge channel 154. The second sample processing channel 122, the third sample processing channel 124, the fourth sample processing channel 126, the first trigger channel 150, the second trigger channel 152, and / or the discharge channel 154 can be adapted, for example, by adapting their respective geometries (e.g., cross-sectional dimensions and / or shapes), such that sample liquid present in the second sample processing channel 122, the third sample processing channel 124, and the fourth sample processing channel 126 can be replaced by test reagent from the first trigger channel 150 and flow into the discharge channel 154 together with test reagent from the second trigger channel 152.

[0046] The volume of the isolated sample liquid corresponds to the volume of the fifth sample processing channel 128, thereby providing a sample liquid with a predetermined sample volume.

[0047] Therefore, the microfluidic channel system 32 of the present invention can provide a sample liquid with a predetermined volume. The sample liquid with the predetermined sample volume is isolated from adjacent sample liquids in the microfluidic channel system 32 without actively controlling the flow within the microfluidic channel system 32.

[0048] like Figure 5As illustrated in the example, the microfluidic channel system 32 may further include a timing channel 160 connecting the test reagent inlet 13 and the third valve 134. The timing channel 160 may be arranged to draw test reagent from the first test reagent inlet and thus from the first pre-treated test reagent channel 8 through capillary action to the output 1342 of the third valve 134 (which may be the second outlet 40) and open the third valve 134, thereby allowing isolated sample liquid present in the fifth channel to flow through the output 1342 of the third valve 134 along with the test reagent from the timing channel 160. The output 1342 of the third valve 134 may be the outlet of the microfluidic channel system 32, configured for direct fluid communication with the first treated sample channel 20, or for fluid communication with the first treated sample channel via a channel for treating sample liquid. The test reagent may be, for example, a lysis test reagent for lysing, for example, red blood cells, or a dilution test reagent for diluting the sample liquid.

[0049] The first processing channel system 32 may further include a channel 190 connected to the valve 138.

[0050] Therefore, isolated sample liquid can be extracted from the microfluidic channel system 32. The isolated sample liquid can, for example, be provided to a microfluidic test card for analysis and / or further processing. For analysis, precise metering of the sample liquid to be analyzed can be advantageous, which is allowed by the microfluidic channel system 32 of the present invention. The timing channel 160 can be configured to open the third valve 134 after the sample liquid present in the fifth processing sample channel 128 is isolated from the adjacent sample liquid. The timing channel 160 can also be configured to open the third valve 134 after the sample liquid and test reagent reach the second end 1544 of the discharge channel 154. Figure 5 As shown in the example, timing channel 160 may include a first flow resistor 162. The flow resistance of the first flow resistor 162 can be selected to control the flow rate from the test reagent inlet 13 to the third valve 134, such that the third valve 134 can be opened after the sample liquid in the fifth sample processing channel 128 is isolated from the adjacent sample liquid. Alternatively, the flow resistance of the first flow resistor 162 can be selected to control the flow rate from the test reagent reservoir to the third valve 134, such that the third valve 134 can be opened after the sample liquid and test reagent reach the second end 1544 of the discharge channel 154.

[0051] Therefore, the length of the timing channel 160 can be reduced while still allowing the third valve 134 to be opened after the sample liquid in the fifth sample processing channel 128 is isolated from the adjacent sample liquid.

[0052] like Figure 5As illustrated in the example, the microfluidic channel system 32 may further include a capillary pump 174 arranged to empty the sample liquid inlet 30 and / or a sample reservoir connected thereto. The capillary pump 174 may be arranged to empty the sample liquid inlet 30 after the first sample processing channel 120, the second sample processing channel 122, the third sample processing channel 124, the fourth sample processing channel 126, and the fifth sample processing channel 128 have been filled with sample liquid. The capillary pump 174 may be a paper pump and / or a microfluidic channel structure configured to draw liquid from the sample liquid inlet 30. During the emptying of the sample liquid inlet 30 by the capillary pump 174, capillary pressures or capillary forces in the second sample processing channel 122, the fourth sample processing channel 126, and the fifth sample processing channel 128 may resist the drawing of sample liquid from the first sample processing channel 120, the second sample processing channel 122, the third sample processing channel 124, the fourth sample processing channel 126, and the fifth sample processing channel 128 in the direction toward the sample liquid inlet 30. The capillary pressure or capillary force in the second sample processing channel 122, the fourth sample processing channel 126, and the fifth sample processing channel 128 can be higher than the capillary pressure or capillary force generated by the capillary pump 174, thereby avoiding the emptying of the second sample processing channel 122, the fourth sample processing channel 126, and the fifth sample processing channel 128.

[0053] Therefore, the sample liquid inlet 30 can receive sample liquid with a volume greater than the combined volume of the first sample processing channel 120, the second sample processing channel 122, the third sample processing channel 124, the fourth sample processing channel 126, and the fifth sample processing channel 128, thereby reducing the need to limit the volume of sample liquid received by the sample liquid inlet 30. When sample liquid is present in the sample liquid inlet 30 after filling the first sample processing channel 120, the second sample processing channel 122, the third sample processing channel 124, the fourth sample processing channel 126, and the fifth sample processing channel 128, additional sample liquid can be drawn from the sample liquid inlet 30 by capillary action when the first valve 130, the second valve 132, and / or the third valve 134 are opened. After filling the first sample processing channel 120, the second sample processing channel 122, the third sample processing channel 124, the fourth sample processing channel 126, and the fifth sample processing channel 128, the liquid in the sample liquid inlet 30 is emptied, allowing capillary pressure or capillary force at the interface between the sample liquid in the first sample processing channel 120 and the sample liquid inlet 30 to resist the aspiration of sample liquid from the first sample processing channel 120 in the direction from the sample liquid inlet 30.

[0054] Capillary pump 174 can be connected to sample liquid inlet 30 via second flow resistor 172. The flow resistance of second flow resistor 172 can be selected to control the flow rate from sample liquid inlet 30 to capillary pump 174, such that sample liquid inlet 30 can be emptied after the first sample processing channel 120, second sample processing channel 122, third sample processing channel 124, fourth sample processing channel 126, and fifth sample processing channel 128 have been filled with sample liquid. Capillary pump 174 can be connected to sample reservoir via pump capillary channel 170, and pump capillary channel 170 may include second flow resistor 172.

[0055] The microfluidic channel system 32 may further include a shut-off valve 136 connected to a second end 1544 of the discharge channel 154.

[0056] The microfluidic channel system 32 may further include a vent 180 connected to the shut-off valve 136. The vent 180 may be arranged to allow gas communication between the shut-off valve 136 and the surrounding environment of the microfluidic channel system 32, allowing gas present in the discharge channel 154 to escape. Gas present in one or more of the first sample processing channel 120, the second sample processing channel 122, the third sample processing channel 124, the fourth sample processing channel 126, the first trigger channel 150, and the second trigger channel 152 may escape through the vent 180 via the discharge channel 154. Additionally, gas present in one or more of the first sample processing channel 120, the second sample processing channel 122, the third sample processing channel 124, the fourth sample processing channel 126, the fifth sample processing channel 128, the first trigger channel 150, and the second trigger channel 152 may escape through the output terminal 1342 of the third valve 134. Gas present in these channels may cause gas pressure to form in these channels, which may react against the liquid flow caused by capillary action in the channels. This formation can be avoided by allowing the gas to escape, thereby allowing for improved flow of the sample liquid and / or test reagents.

[0057] Referring now to Figure 3(a), the microfluidic chip 4 of the device 1 for analyzing sample liquids according to an embodiment will be discussed. The illustrated embodiment includes references to... Figure 5The microfluidic channel systems 32 and 34 are discussed and illustrated. The device 1 according to the example may include two microfluidic channel systems, namely a first microfluidic channel system 32 and a second microfluidic channel system 34, as shown in Figure 3(a). For improved clarity, Figure 3(b) schematically shows a microfluidic chip 4 similar to the microfluidic chip discussed with reference to Figure 3(a), wherein a single microfluidic channel system 32 is shown to enhance clarity. Microfluidic chip 4 includes: a sample liquid inlet 30 configured to be in fluid communication with and receive sample liquid from a sample liquid outlet of a test card (not shown); a first microfluidic channel system 32 configured to process the sample liquid, be in fluid communication with a first test reagent outlet and thereby configured to receive a first test reagent from a first pretreatment test reagent channel, and be further configured to be in fluid communication with the sample liquid inlet 30 and thereby configured to receive sample liquid from the pretreatment sample liquid channel and allow contact between the sample liquid and the first test reagent within the first microfluidic channel system 32; and a second microfluidic channel system 34 (not shown in FIG. 3(b)) configured to process the sample liquid and be... For fluid communication with the second test reagent outlet 14 and thus configured to receive the second test reagent from the second pretreatment test reagent channel, and further configured for fluid communication with the sample liquid inlet 30 and thus configured to receive sample liquid from the pretreatment sample liquid channel and allow contact between the sample liquid and the second test reagent within the second microfluidic channel system 34, wherein the first microfluidic channel system 32 and the second microfluidic channel system 34 (only the first microfluidic channel system is shown in FIG. 3(b)) respectively include a first outlet 40 and a second outlet 42 (only the first outlet is shown in FIG. 3(b)), which are configured to be fluidly connected to the first processed sample analysis channel 20 and the second processed sample analysis channel 22 of the microfluidic test card 2, respectively.

[0058] It should be understood that the first microfluidic channel system 32 and the second microfluidic channel system 34 may have one or more shared channels and / or components, but typically each microfluidic channel system has a single microfluidic channel.

[0059] Figures 3(a, b) further illustrate the first sample processing channel 120 connected to the sample liquid inlet 30. These illustrations in the figures can be seen from the perspective of... Figure 5Furthermore, this can be further understood with reference to the discussion of the first microfluidic channel system, but analogously or similarly, with reference to the discussion of the second microfluidic channel system. The first sample processing channel 120 branches into a second sample processing channel 122 terminating at the first valve 130, and into a third sample processing channel 124. The third sample processing channel 124 branches into a fourth sample processing channel 126 terminating at the second valve 132, and into a fifth sample processing channel 128 terminating at the third valve 134, wherein the fifth sample processing channel 128 has a predetermined volume. The first valve 130, the second valve 132, and / or the third valve 134 can be trigger valves. A trigger valve can stop the main fluid flow in its closed state and allow the main fluid flow through the trigger valve in its open state. The trigger valve can be opened by a secondary flow (i.e., changed to its open state), and can allow a combined flow of the main and secondary flows through the output of the trigger valve. Such a trigger valve can be known in the art as a capillary trigger valve. The first microfluidic channel system 32 shown is further configured to be in fluid communication with the first test reagent outlet 12 via a test reagent inlet 13 arranged for receiving the first test reagent. The first test reagent inlet 13 can therefore be arranged for receiving the test reagent.

[0060] The microfluidic channel system 32 further includes a first trigger channel 150 arranged to connect the first test reagent outlet 12 to the second valve 132. The microfluidic channel system 32 further includes a second trigger channel 152 connecting the second valve 132 and the first valve 130.

[0061] The first processing channel system 32 further includes a discharge channel 154 having a first end 1542 and a second end 1544. The first end 1542 is connected to a first valve 130, and the second end is connected to a shut-off valve 136, which is gas-connected to a vent 180 arranged to allow communication with an ambient gaseous medium (e.g., air). The first processed sample channel 120 is arranged to draw sample liquid from the sample inlet 30 by capillary action to fill the first processed sample channel 120, the second processed sample channel 122, the third processed sample channel 124, the fourth processed sample channel 126, and the fifth processed sample channel 128. The flow of these sample liquids is stopped by the first valve 130, the second valve 132, and the third valve 134 when they are in their closed state. One, more, or all of these valves may be capillary-triggered valves.

[0062] The first trigger channel 150 is arranged to draw test reagent from the first test reagent inlet 13 via a liquid path including the second trigger channel 152 to the outlet channel 154 by capillary action, and to open the second valve 132 and the first valve 130, thereby opening another liquid path including the fourth processed sample channel 126, the third processed sample channel 124, and the second processed sample channel 122. This opened liquid path allows samples present in the fourth processed sample channel 126, the third processed sample channel 124, and the second processed sample channel 122 to be replaced by test reagent from the first trigger channel 150 and flow into the outlet channel 154 together with test reagent from the second trigger channel 152, thereby isolating the sample liquid present in the fifth processed sample channel 128 from the adjacent sample liquid. The first sample processing channel 120 and / or the fifth sample processing channel 128 can be adapted, for example, by adapting their respective geometries (e.g., cross-sectional dimensions and / or shapes), such that capillary forces (or capillary pressures) prevent sample liquid present in the first sample processing channel 120 and / or the fifth sample processing channel 128 from flowing toward the discharge channel 154. The second sample processing channel 122, the third sample processing channel 124, the fourth sample processing channel 126, the first trigger channel 150, the second trigger channel 152, and / or the discharge channel 154 can be adapted, for example, by adapting their respective geometries (e.g., cross-sectional dimensions and / or shapes), such that sample liquid present in the second sample processing channel 122, the third sample processing channel 124, and the fourth sample processing channel 126 can be replaced by test reagent from the first trigger channel 150 and flow into the discharge channel 154 together with test reagent from the second trigger channel 152.

[0063] The volume of the isolated sample liquid corresponds to the volume of the fifth sample processing channel 128, thereby providing a sample liquid with a predetermined sample volume, such as 600 nl or 10 nl, to name just a few examples.

[0064] Therefore, the microfluidic channel system 32 of the present invention can provide a sample liquid with a predetermined volume. The sample liquid with the predetermined sample volume is isolated from adjacent sample liquids in the microfluidic channel system 32 without actively controlling the flow within the microfluidic channel system 32.

[0065] like Figure 5As illustrated in the example, the microfluidic channel system 32 may further include a timing channel 160 connecting the test reagent inlet 12 and the third valve 134. The timing channel 160 may be arranged to draw the test reagent from the first test reagent inlet and thus from the first pre-treatment test reagent channel 8 via capillary action to the output 1342 of the third valve 134 (which leads to the second outlet 40) and open the third valve 134, thereby allowing isolated sample liquid present in the fifth channel to flow through the output 1342 of the third valve 134 along with the test reagent from the timing channel 160. The output 1342 of the third valve 134 may be an outlet for direct fluid communication with the first treated sample channel 20, or, as in the illustrated example, an outlet for fluid communication with the first treated sample channel via a channel 35 for treating sample liquid (e.g., lysis or mixing). The test reagent may be, for example, a lysis test reagent for lysing, e.g., red blood cells, or a dilution test reagent for diluting the sample liquid. The system may be designed to appropriately dilute the sample with the test reagent when the sample is actuated from the sample channel 128. In embodiments having two or more microfluidic channel systems 32, 34, the microfluidic channel systems can be designed and operated similarly to those discussed above, and can alternatively be designed for metering different sample volumes, dilution processing times, etc.

[0066] The capillary pump 174 can be exemplarily arranged to empty the sample inlet / reservoir / entry port 30, for example, after the first sample processing channel 120, the second sample processing channel 122, the third sample processing channel 124, the fourth sample processing channel 126, and the fifth sample processing channel 128 have been filled with sample liquid. A vent 180 is further shown, which is arranged to allow communication with the surrounding gaseous medium (e.g., air).

[0067] The microfluidic chip can contact the microfluidic chip contact area of ​​the microfluidic test card. Preferably, the microfluidic chip and the microfluidic test card are integrated.

[0068] The device can be configured to provide a capillary-driven liquid flow through a channel. For example, the channel may have capillary dimensions and / or the flow may be assisted by a capillary pump or a paper pump (e.g., a pump driven by capillary or wicking effects, such as a paper pump). Pressure-assisted capillary-driven flow may be used in embodiments.

[0069] The device may include, in a suitable location, capillary valves (e.g., capillary trigger valves) that are connected to the fluid passage of the device, and these capillary valves are used to manipulate or control the flow of the device.

[0070] The first pretreatment test reagent channel and the second pretreatment test reagent channel may further have a first test reagent inlet and a second test reagent inlet respectively, which are fluidly connected to the first test reagent reservoir and the second test reagent reservoir (preferably blister type reservoir).

[0071] The test card can be further configured to contact an analyzer and / or detector used for detecting and analyzing the sample liquid and / or the components of the sample liquid.

[0072] The sample liquid can be blood or blood-derived, the first test reagent can be a lysis test reagent for lysing red blood cells, and the second test reagent can be a dilution test reagent for diluting the blood sample.

[0073] The sample liquid can be blood or a liquid derived from blood. The first test reagent can be a lysis buffer for lysing red blood cells present in the first microfluidic channel system, and the second test reagent can be a dilution buffer for diluting blood samples present in the second microfluidic channel system.

[0074] According to a second aspect of the invention, a system is provided, which will now be referred to... Figure 6 The system is discussed below. The system includes a device for analyzing a sample liquid according to the first aspect, and a reader. The reader includes a computational or lensless holographic microscope, preferably comprising a laser diode and a complementary metal-oxide-semiconductor imager, and wherein the reader is configured to receive the device for analysis and is further configured to allow the imager to image a first and a second detection area of ​​a test card, thereby analyzing the sample liquid.

[0075] It should be understood that the apparatus and system, and embodiments thereof, can be used for blood analysis as discussed herein, but can alternatively be used for other analytical or lab-on-a-chip applications, such as PCT reactions. Consider any suitable application in which liquids and / or reagents, etc., are manipulated as implemented by the apparatus and / or system of the present invention.

[0076] Device development and testing

[0077] The following will discuss the apparatus (e.g., already referenced). Figure 3a , Figure 3b and Figure 5 The development and testing of embodiments of the device discussed. This device can be suitably used in embodiments of the system.

[0078] A microfluidic chip is integrated into a plastic microfluidic test card, which allows for the introduction of reagents / test reagents and samples, the integration of additional components such as capillary wicks and imaging areas, and provides a more convenient form factor for manual handling. As described herein, the microfluidic test card consists of several patterned layers laminated onto each other starting from an injection-molded substrate. By integrating the microfluidic chip into the microfluidic test card, fluid can be transferred from the microfluidic test card to the microfluidic chip via capillary wicking, and vice versa. This is achieved by designing the microfluidic chip outlet and inlet / outlet and by designing features of the foil stack that ensure rapid wicking to the microfluidic chip surface. Similarly, the transfer from the fluid channels in the microfluidic test card to the capillary wick, which acts as a waste reservoir, is tailored to ensure a sufficiently low failure rate.

[0079] Development and testing of holographic microscopes

[0080] The apparatus discussed above, along with the system according to an embodiment of the second aspect, presents a sample to a computational or lensless holographic microscope comprising a laser diode and a complementary metal-oxide-semiconductor (MOS) imager having a pixel size of 1.1 µm and an array size without additional optical components. A microfluidic test card is positioned directly above the image sensor, the flow chamber is positioned above the sensor surface, and the laser diode is positioned above the image sensor to ensure uniform illumination. The laser diode operates in a stroboscopic mode (i.e., spontaneous emission mode) with 2 µs pulses below the laser threshold to ensure a sufficiently broad spectrum to prevent unwanted interference fringes due to unintended thickness variations in the microfluidic test card stack. The hologram captured by the imager is the result of interference between the partially coherent beam emitted by the laser diode and the light scattered by the flow chamber and other particles within the chamber, at a frame rate of 21 frames per second (synchronized with the laser pulses).

[0081] The hologram was then reconstructed into a microscopic image.

[0082] Evaluation using clinical samples

[0083] Performance of the device and system:

[0084] The experiment is performed using the apparatus described above and the system including such apparatus and reader according to the embodiment of the second aspect.

[0085] The performance of the apparatus and system described in this paper was evaluated using residual blood samples obtained and tested on the same day from KU Leuven Hospital. To train the WBC CNN, pure cellular fractions of neutrophils, eosinophils, monocytes, and lymphocytes were prepared via magnetic bead-based separation. The samples were aliquoted and run on a Sysmex XN-350, which served as a reference apparatus.

[0086] against Figure 7 a and Figure 7 The series of samples in b shows the results of the obtained RBC and total WBC counts as a function of the counts obtained using the reference instrument.

[0087] The results demonstrate how this system, through autonomous liquid sample handling and a lensless in-flow microscopy system, can combine these results to achieve a point-of-care diagnostic solution for complete blood cell counts in a form factor and cost that would otherwise be unimaginable. The microfluidic chip enables autonomous handling of sample and liquid reagent inputs without electrical, optical, or mechanical input from the instrument. The use of computational in-flow microscopy technology avoids the need for optical systems and their associated volume, weight, complexity, and cost.

[0088] Manufacturing of microfluidic test cards

[0089] The channels of the microfluidic test card are constructed by cutting channels through a 42 μm thick double-sided pressure-sensitive adhesive (PSA). This PSA is sandwiched between two 100 µm thick hydrophilic optically clear PET foils. The foils have a SiO2 coating, achieving a contact angle of <20° with deionized water. This arrangement results in the top and bottom of the fluid channels being PET foils with hydrophilic surfaces exposed to the channels, and the sidewalls being edges cut from the PSA. Typical channel widths range from 500 µm to 1 mm. The foil arrangement is supported by a substrate that acts as a structural support for the laminated foils and a housing for the microfluidic chip and capillary wick located in a recess in the substrate. The capillary wick is blotting paper from Ahlstrom. The capillary microfluidic structure is created using a stack of hydrophilic biocompatible foils. This foil assembly is attached to a backbone component, which also contains the microfluidic chip-cell and the fluid drainage medium.

[0090] Most components are manufactured on-site. The PMMA baseplate is molded in a rapid prototyping facility (prototype mold). The channel cutouts of the double-sided PSA and the fluid access holes in other layers are manufactured using high-precision laser cutting in a dedicated laser processing workshop. The microfluidic chip is manufactured as described above.

[0091] Assembly was performed under a flow hood to avoid particulate contamination that could be detrimental to fluid flow or LFI imaging.

[0092] The different components are positioned one above the other using assembly jigs. These jigs are made by laser-cutting acrylic sheets into approximately 10 cm x 10 cm plates and cutting holes at specific locations for inserting metal pins. These metal pins have matching positions on the different layers. The bottom PSA, bottom hydrophilic foil, middle PSA (with channels cut out), and top hydrophilic foil are aligned one above the other using these metal pins. The release liner on the PSA is removed before placing the additional layers on top. This arrangement is gently pressed to adhere the different layers together. All layers are handled with tweezers, and only at the very edges. This is to avoid excessive contact that could be detrimental to the hydrophilic layer or LFI imaging.

[0093] Use tweezers to insert the microfluidic chip into the recessed area on the substrate. The operator needs to pay attention to the orientation, as the microfluidic chip is square (not designed to prevent incorrect insertion), and the fluid channels need to be connected to the correct fluid paths on the microfluidic test card.

[0094] The paper core is cut to a specific size using laser cutting. Like a microfluidic chip, the paper core is inserted into the substrate using tweezers.

[0095] Then place the base plate in the same fixture as before, and place the four layers (see below) on top (remove the last liner from the bottom PSA). The base plate has the same alignment as the foil. Gently press the assembly again to ensure it adheres.

[0096] The component then passes through a roller laminator. The laminator has a degree of plasticity thanks to the rollers covered with silicone. The microfluidic test card passes through the roller laminator once. Lamination is used to fix the layer and the base plate (which contains the microfluidic chip and paper core). After this lamination, the test card is ready for use.

[0097] Holographic microscopy computational methods

[0098] Evaluation using clinical samples

[0099] The CBC parameters of interest are total white blood cell count (WBC), counts of different WBC cell populations (i.e., WBC differentiation), and red blood cell count (RBC).

[0100] The accuracy of a clinical testing paradigm for white blood cells (WBCs) was evaluated in venous whole blood samples covering a broad range of hematocrit (HCT) values. These samples were anonymized and exceeded the requirement of being drawn from blood obtained on the same day from UZ Leuven Gasthuisberg Hospital. The normal range for HCT in healthy individuals is 35% to 50%, between 35% and 45% for women and between 40% and 50% for men. HCT values ​​were subdivided into five ranges: 1) HCT up to 34% (low), 2) HCT from 35% to 39% (normal for women, low for men), 3) HCT from 40% to 44% (normal), 4) HCT from 45% to 50% (normal for men, high for women), and 5) HCT above 50% (high). Samples from two different donors in each category were tested five times (N = 5 per sample, N = 10 per category, N = 50 in total).

[0101] Because different blood donors exhibit wide variations in their blood composition and fluid properties, it is of interest to separate HCT parameters from other blood properties to determine the impact of HCT on the accuracy and precision of clinical testing. To this end, processed blood samples with three very different HCT values ​​were prepared from whole blood from the same donor by centrifuging two equal aliquots of blood and transferring the plasma from one component to another, resulting in lower HCT (between 20% and 24%) and higher HCT (between 50% and 54%) from the original normal HCT (35% to 45%) sample. This process was performed on two different donors, with each sample tested four times (N = 4 per sample, N = 8 per category, N = 24 in total).

[0102] Extensive replicate testing was performed on three randomly selected whole blood samples to assess the reproducibility of WBC results using the imprecisely measured coefficient of variance (CV%). For the purposes of this study, it was concluded that whole blood stability can be achieved for at least 3 days when plasma is replaced with Alsever's solution and samples are stored in a refrigerator (2°C–8°C). This extended shelf life is necessary to allow for replicate testing over multiple days to obtain approximately 20–60 replicates of the same sample, as the goal is to obtain ≥10 successful tests per sample to assess accuracy. Whole blood stability has been shown to have no adverse effect on test performance (data not shown).

[0103] The accuracy and precision of RBC counting were evaluated in whole blood samples diluted with phosphate-buffered saline (PBS) in a microfluidic chip at dilution ratios from 200 to 800 times to assess the effect of dilution ratio on the results.

[0104] Each blood sample was measured before and after testing on the Sysmex XN350 to obtain a reference CBC value and confirm sample integrity. For samples undergoing long-term testing (i.e., samples used for accuracy and repeatability testing), additional intermediate Sysmex measurements were required.

[0105] After introducing the initial sample into the inlet port on the silicon microfluidic chip, the first observation of the test was performed under an infrared (IR) microscope to evaluate the performance of the microfluidic chip. Whole blood (6 µL) was dispensed into the blood inlet of the microfluidic test card using a pipette, and the internal microfluidic chip volumetrics were visualized by IR imaging. Excess whole blood was removed by directing it to the "waste" channel (integrated into the microfluidic test card and directly connected to the card-mounted paper pump integrated into the microfluidic test card). After precise cell volumetrics on the microfluidic chip, 30 µL of lysis test reagent was dispensed into the corresponding inlet of the microfluidic chip, and IR imaging was performed again after dilution, mixing, and lysis.

[0106] Once the sample is visualized at the outlet of the microfluidic chip, the microfluidic test card is removed from the IR microscope and inserted into the LFI reader, where holographic / LFI images are generated and collected. LFI data is collected at a high frame rate (21 frames per second (fps)).

[0107] Holographic / LFI images are uploaded to a cloud-based storage solution for processing.

Claims

1. An apparatus for analyzing sample liquids, the apparatus comprising: Microfluidic test cards, and A microfluidic chip is used to process the sample liquid presented from the microfluidic test card and return the processed sample liquid to the microfluidic test card. The microfluidic test card includes: The sample inlet is configured to receive sample liquid. A first pretreatment test reagent channel and a second pretreatment test reagent channel, the first pretreatment test reagent channel and the second pretreatment test reagent channel respectively having a first test reagent outlet and a second test reagent outlet for presenting test reagents to the microfluidic chip. A pretreatment sample channel, which is in fluid communication with the sample inlet, is used to receive sample liquid from the sample inlet, and the pretreatment sample channel has a sample liquid outlet for presenting the sample liquid to the microfluidic chip. A first processed sample analysis channel and a second processed sample analysis channel are used to receive processed sample liquid from the microfluidic chip. The first processed sample analysis channel and the second processed sample analysis channel respectively include a first analysis area and a second analysis area for analyzing the processed sample liquid. The microfluidic chip contact area includes the sample liquid outlet, a first test reagent outlet, and a second test reagent outlet. The sample liquid outlet, the first test reagent outlet, and the second test reagent outlet are all configured for connection and fluid communication with the microfluidic chip. The microfluidic chip includes: A sample liquid inlet is configured to be in fluid communication with and receive sample liquid from the sample liquid outlet of the test card. A first microfluidic channel system, configured to process sample liquid, fluidly communicate with a first reagent outlet and thereby configured to receive a first test reagent from a first pretreatment test reagent channel, and further configured to fluidly communicate with a sample liquid inlet and thereby configured to receive sample liquid from the pretreatment sample liquid channel and allow contact between the sample liquid and the first test reagent within the first microfluidic channel system. A second microfluidic channel system, configured to process sample liquid, fluidly communicate with the outlet of the second test reagent and thereby receive the second test reagent from the second pretreatment test reagent channel, and further configured to fluidly communicate with the sample liquid inlet and thereby receive sample liquid from the pretreatment sample liquid channel, allowing contact between the sample liquid and the second test reagent within the second microfluidic channel system. The first microfluidic channel system and the second microfluidic channel system each include a first outlet and a second outlet, which are configured to be fluidly connected to the first and second processed sample analysis channels of the microfluidic test card, respectively. The first microfluidic channel system and the second microfluidic channel system respectively include a first sample metering channel and a second sample metering channel for providing a predetermined first sample volume and a predetermined second sample volume, and the sample liquid having the predetermined first sample volume is isolated from the adjacent sample liquid in the first microfluidic channel system, and the sample liquid having the predetermined second sample volume is isolated from the adjacent sample liquid in the second microfluidic channel system.

2. The apparatus according to claim 1, wherein, The microfluidic chip makes contact with the microfluidic chip contact area of ​​the microfluidic test card.

3. The apparatus according to claim 2, wherein, The microfluidic chip is integrated with the microfluidic test card.

4. The apparatus according to any one of claims 1-3, wherein, The device is configured to provide a capillary-driven liquid flow through a channel.

5. The apparatus according to any one of claims 1-3, wherein, The first pretreatment test reagent channel and the second pretreatment test reagent channel further have a first test reagent liquid inlet and a second test reagent liquid inlet, respectively, which are fluidly connected to the first test reagent reservoir and the second test reagent reservoir.

6. The apparatus according to claim 5, wherein, The first and second test reagent reservoirs are blister-type reservoirs.

7. The apparatus according to any one of claims 1-3, wherein, The sample liquid is blood or a liquid derived from blood. The first test reagent is a lysis buffer for lysing red blood cells present in the first microfluidic channel system, and the second test reagent is a dilution buffer for diluting blood samples present in the second microfluidic channel system.

8. A system comprising means for analyzing a sample liquid according to any one of the preceding claims, and a reader. in, The reader includes a computational or lensless holographic microscope comprising a laser diode and a complementary metal-oxide-semiconductor imager, and The reader is configured to receive the device for analyzing the sample liquid and is further configured to allow the imager to image the first and second analysis areas of the test card, thereby allowing the analysis of the sample liquid.

Images