WO2025210522A1

WO2025210522A1 - Automated sample preparation system implemented within a disposable cartridge

Info

Publication number: WO2025210522A1
Application number: PCT/IB2025/053438
Authority: WO
Inventors: Usama Ahmed Abbasi; Prakhar Jain
Original assignee: Microx Labs Inc
Current assignee: Microx Labs Inc
Priority date: 2024-04-02
Filing date: 2025-04-02
Publication date: 2025-10-09
Anticipated expiration: 2026-10-02

Abstract

According to this invention, there is provided an automated sample preparation system implemented within a disposable cartridge. The system utilizes finger prick blood or venous blood as an input sample and performs various sample processing steps, including fluidic metering, fluidic routing, and reagent storage with an integrated biosensor. The architecture, of this system, enables efficient and accurate testing of blood parameters, hematology parameters such as CBC, CD4-T cells count, but not limited to, Complete Blood Cell Count (CBC) and measurement of biomarkers such as Troponin, IL-6, Procalcitonin, but not limited to, CRP, Procalcitonin, and IL-6 through ELISA and is flexible to implement other electrochemistry techniques.

Description

AUTOMATED SAMPLE PREPARATION SYSTEM IMPLEMENTED

WITHIN A DISPOSABLE CARTRIDGE

FIELD OF THE INVENTION:

The present invention pertains to the field of biomedical engineering.

Particularly, this invention relates to the field of microfluidics and diagnostics.

Specifically, this invention relates to automated sample preparation system implemented within a disposable cartridge.

BACKGROUND OF THE INVENTION:

Microfluidics is both the science which studies the behaviour of fluids through microchannels, and the technology of manufacturing microminiaturized devices containing chambers and tunnels through which fluids flow or are confined.

Blood testing and sample preparation procedures often require sophisticated equipment, technical expertise, and specialized laboratory settings. This presents a challenge for home patients or at low resource settings who require regular monitoring of their health conditions. Furthermore, the existing sample preparation methods involve multiple manual steps and are prone to human error during operations, readability, usability, risks in transportation and contamination risks.

There is a need for a microfluidics cartridge, for use in diagnostics industry, which utilizes finger prick blood or venous blood as an input sample and performs various sample processing steps, including fluidic metering, fluidic routing, and reagent storage with an integrated biosensor.

PRIOR ART:

Prior art, US20190120840A1, discloses a microfluidic device that stores reagents off-chip, requiring bulky storage systems and mechanisms for dispensing and sample preparation. Another prior art, US8815537B2, describes leukocyte count using image analysis and semiautomatic sample preparation without the need for blisters and microfluidic valves. However, this system only performs leukocyte count and does not provide the count for red blood cells (RBCs) and platelets.

Another prior art, US7641856B2 describes sample preparation with all the reagents stored off the chip and number of microfluidic valves.

Additionally, another patent (WO 2019/167066 Al) describes sample preparation using microfluidic valves and on-chip reagent storage. However, it requires the use of multilayer pressure-sensitive adhesive (PSA) tapes and a special soft wall layer to create turbulence, which poses challenges for productization of the disposable cartridge.

Another prior art, JP2011227100A, describes automatic sample preparation using rotary valves and reagents stored on the cartridge. However, this mentioned art is limited to WBC sample preparation and does not include the counting of RBCs and platelets.

Furthermore, another patent (US9808802B2) also utilizes rotary valves with reagents stored in barrels. In this case, the biosensor is mounted off the chip, posing a potential risk of contamination at the fluidic interconnects due to the patient's blood, which could be problematic for end users when handling the system.

OBJECTS OF THE INVENTION:

An object of the invention is to provide an automated sample preparation system implemented within a disposable cartridge such that it does not require bulky storage systems.

Another object of the invention is to minimize the number of components requiring fabrication and assembly, resulting in a seamless manufacturing process and streamlined productization on an assembly line.

Yet another object of the invention is to provide an automated sample preparation system implemented within a disposable cartridge such that it provides leukocyte count as well as count for red blood cells (RBCs) and platelets. Still another object of the invention is to provide an automated sample preparation system implemented within a disposable cartridge such that it stores reagent on the chip itself.

An additional object of the invention is to provide an automated sample preparation system implemented within a disposable cartridge such that it overcomes currently known challenges for productization of the disposable cartridge.

Yet an additional object of the invention is to provide an automated sample preparation system implemented within a disposable cartridge such that it provides WBC count as well as count for red blood cells (RBCs) and platelets

Still an additional object of the invention is to provide an automated sample preparation system implemented within a disposable cartridge such that it does not provide any contamination risk.

SUMMARY OF THE INVENTION:

According to this invention, there is provided an automated sample preparation system implemented within a disposable cartridge.

The system utilizes finger prick blood or venous blood as an input sample and performs various sample processing steps, including fluidic metering, fluidic routing, and reagent storage with an integrated biosensor.

The architecture, of this system, enables efficient and accurate testing of blood parameters, hematology parameters such as CBC, CD4-T cells count, but not limited to, Complete Blood Cell Count (CBC) and measurement of biomarkers such as Troponin, IL-6, Procalcitonin, but not limited to, CRP, Procalcitonin, and IL-6 through ELISA and is flexible to implement other electrochemistry techniques.

Additionally, the invention encompasses application of this system for home monitoring of diseases, even by individuals lacking technical expertise e.g. patients themselves in haematology. The present invention addresses the challenges mentioned above, in prior arts, by introducing an innovative automatic sample preparation system integrated into a disposable cartridge. This system optimizes workflow of sample processing by incorporating liquid level sensing, multiple microfluidic valves, both active and passive, along with microchannels of various dimensions, a compartment for storing reagents (such as blister-packed reagents), pneumatic connectors, and motorized actuators. These components are seamlessly integrated within a compact instrument, ensuring efficient execution of unit operations. The cartridge's compact design, preferably measuring less than 100mm x 55mm, ensures its single -use functionality, ease of operation, simplified manufacturability, and enhanced convenience, while reducing the risks of contamination.

The disclosed invention presents a customizable microfluidic cartridge designed to accommodate the collection of various biological samples, including whole blood (from venous or capillary sources), platelet-rich plasma (PRP), spinal fluids, and more. This versatile cartridge performs multiple unit operations, such as metering, mixing, and incubation, to provide quantified outputs, such as the number of blood cells, their distribution, size, shape, and morphological analysis.

The cartridge incorporates various elements, including but not limited to microfluidic channels of different dimensions, both active and passive microfluidic valves, metering chambers, and chambers for conducting chemistries, such as reagent mixing, sample heating, and incubation. Additionally, blister compartments are included for convenient storage of reagents. The cartridge also features integrated biosensors that enable the detection and measurement of various biomarkers within the sample.

Among the various sensors that can be integrated, one notable type is a flow cell sensor. This sensor allows for identification, enumeration, and quantification of biological cells (or organisms like yeast and bacteria) using sensing principles such as impedance-based detection, optical detection, or fluorescence detection techniques

Further, the cartridge operates in conjunction with a portable instrument that drives the necessary unit operations. The instrument's functionality encompasses fluidic metering, fluidic routing, and blood dilution utilizing microfluidic valves and embedded actuators. By employing these elements, the cartridge system achieves precise and controlled sample processing.

Furthermore, the system ensures the usability of the invention for home patients, allowing them to monitor their health conditions independently. The simplified operation and integration of all essential components within the cartridge eliminate the need for technical expertise in haematology.

According to this invention, there is provided a microfluidic cartridge-based blood collection and analysis system comprising: a cartridge having a linear actuator configured to be pressed to push down a cap plunger which drives blood filled inside a blood port, from a user, into a connected first metering chamber (WBC), via a microfluidic channel, and, parallelly, into a connected second metering chamber (RBC), via a microfluidic channel, in order to output metered blood, said cartridge’s bottom layer being a hydrophobic layer causing liquid meniscus surface to form a spherical shape upon contact in order to pronounce signal changes during sensing; plurality of fluid chambers including at least a first mixing chamber, a second mixing chamber, a third mixing chamber, a biosensor chamber, and plurality of blisters, said mixing chambers connected to the metering chambers via microfluidic channels, said blisters connected to metering chambers via fluidic channels, in that, o a second set of blisters configured to be pushed to dispense a diluent and metered blood into said first mixing chamber; o a third set of blisters containing diluent, pushed into said second mixing chamber after opening valves, facilitating mixing of diluent with the first diluted blood, by ferromagnetic beads, located inside the mixing chambers, to achieve a serial first dilution followed by a serial second dilution of 1:120 to reach cumulative dilution greater than 10,000 in order to ensure to ensure single cells pass through a subsequent optical detection system, located pursuant to the mixing chamber, - the mixing process preserving blood cells' size and shape due to a gentle micro mixing action enabled by ferromagnetic beads, eliminating the need for additional microfluidic valves for back-and-forth mixing as also transforming the shape of red blood cells (RBCs) to spherical, enhancing impedance measurement accuracy by reducing impedance variations caused by biconcave-shaped cells; o a fourth set of blisters being lysis buffer blisters, pushed into connected said third mixing chamber, via a microfluidic channel, after opening valves, enabling the lysis of red blood cells using a lysis buffer containing formic acid and saponin as surfactants; o a quench buffer, connected to said third mixing chamber, via a microfluidic channel, from a fifth set of blisters pushed into said third mixing chamber to quench the reaction; the quench solution preserving white blood cells (WBCs). ensuring their stability for subsequent analysis; an optical detection system disposed in a counting chamber, having optical detection windows which serve as a signal window for the measurement of liquid level detection at entrance of said counting chamber and comprising at least one laser diode and at least one PIN photodiode, the optical detection system being configured to detect a liquid level based on changes in light refraction such that when no liquid is present a flat-line electrical signal is generated and when the liquid enters the counting chamber a square pulse electrical signal is produced, o wherein the optical detection system further comprises an inclined optical window having a geometry of a right-angle prism filled with air, the inclined optical window compensating for refractive effects in a sensing region of a few microns to enhance signal strength; o wherein the optical detection system is further configured to measure a liquid meniscus formed at the optical window, the meniscus causing transient light reflection and refraction that produces a square pulse electrical signal for a short duration, the signal being used for sample volume measurement for both RBCs and white blood cells (WBCs); a controller configured to use the generated signals for counting red blood cells (RBCs) and for verifying sample metering.

In at least an embodiment, a linear actuator that, when pressed, drives the blood filled inside the blood port into metering chambers [first metering chamber, second metering chamber], while allowing excess blood to flow into a buffer zone. In at least an embodiment, said third mixing chamber comprising a magnetic flea configured to be rotated at RPM of 5,000 to completely lyse RBCs from the metered blood.

In at least an embodiment, a pressurized first vent, connected to an air solenoid valve, delivering positive pressure to drive blood from the first mixing chamber into a second RBC (third) metering chamber and waste chamber upon opening valves.

In at least an embodiment, a counting subsystem, configured to count RBCs and platelets, follows said optical detection system, in that, pressurized second vent, delivering positive pressure to drive diluted blood from the second mixing chamber into a biosensor (impedance measurement) upon opening a valve, said biosensor comprising: a fourth chamber in line with a counting chamber, configured to receive diluted blood, facilitating the counting of red blood cells (RBCs) as the diluted blood reaches said counting chamber, measurement beginning at an entry of said counting chamber and measurement ending at an exit of said fourth metering chamber; a. optical detection windows within the counting chamber, serving as signal windows for liquid level detection based on the principle of light refraction.

In at least an embodiment, said inclined optical window resembling a prism with angle of the prism between 30 and 60 degrees.

In at least an embodiment, valves enable opening of a passage for transfer of diluted blook from said third mixing chamber to said optical detection system upon the application of positive pressure on a third vent connected to said third mixing chamber.

In at least an embodiment, said biosensor featuring front and back side holes for fluid entry and exit, sealing of said biosensor on its front and back sides by a gasket with a wedge shape.

In at least an embodiment, said cartridge comprising: a capillary tube coated with Ethylenediaminetetraacetic acid (EDTA) for collecting blood; a designated blood port for transferring the collected blood; a cap configured to be pushed until it reaches a first stopper to prevent blood flow to a downward section; a capillary valve integrated within the cap to restrict movement of the collected blood based on capillary forces and geometry of the capillary valve structure, wherein the capillary forces and the capillary valve’s geometry act in tandem to prevent blood flow through the capillary valve, thereby functioning as a reliable stopper or barrier, said geometry being defined by a channel which is narrow in its initial section and expands in its further section to create a Laplace pressure barrier to stop flow unless extra pressure is applied externally.

In at least an embodiment, an actuator activates a microfluidic first valve, that stops blood (fluid) flow from flowing into other sections, of this system, except to a designated first metering section and to a designated second metering section; a linear actuator further pushes the cap to push the blood sample into two separate metering sections:

■ a first section is dedicated to counting of RBCs and platelets,

■ a second section is designed for WBCs count.

In at least an embodiment, said cartridge comprising: blister-packed reagents for convenient dispensing of liquids by pressing the blisters with actuators; a linear actuator that, when pressed against the blisters, increases the pressure inside the blisters, causing rupture of the bottom part of the blisters; a sharp conical pillar, positioned within the cartridge, facilitating controlled rupture of blisters by deflecting the bottom part of the blister, allowing efficient release of fluid into the microfluidic channel for subsequent analysis; a via located beneath a foil part of the cartridge, establishing a fluidic connection between the microfluidic channel and the metering section with the blister filled with reagents; an open microfluidic channel and said sharp conical pillars moulded directly on the cartridge; and a thin, soft, and flexible pressure-sensitive adhesive (PSA) layer of 50 to 200 microns thickness, with adhesive on one side, capable of closing the open microfluidic channel.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS:

This invention will now be described in relation to the accompanying drawings, in which:

Figure 1 illustrates, particularly, a blood port in the cartridge of this invention;

Figure 2 illustrates blood introduction inside the blood port of Figure 1 of the cartridge of this invention;

Figure 3 illustrates the port, of Figure 1, filled with blood sample and a capillary valve which stops blood movement into a downward section of fluid due to a capillary action and geometry of the valve;

Figure 4 illustrates microfluidic valves, except shown in black colour, are closed where movement of the plunger cap by linear actuator drives blood into a metering section;

Figure 5 illustrates a mechanism showing rupture of blister/s due to sharp conical pillar/s (14) placed beneath an aluminium foil, in the cartridge of this invention;

Figure 6 illustrates the position of cap during first hard stopper;

Figure 7 illustrates the disposable cartridge, of this invention, showing position of via beneath blisters;

Figure 8 illustrates the cartridge, of this invention, showing the position of the microfluidic valve, mixing chambers, waste chambers and bio-sensor;

Figure 9 illustrates this invention’s disposable cartridge showing metering sections and buffer zone filled with blood;

Figure 10 illustrates mixing and dilution of blood with diluent using the cartridge of this invention;

Figure 11 illustrates metering of the blood for second serial dilution using the cartridge of this invention;

Figure 12 illustrates blood mixing for achieving dilution ratio greater than 10000 and also Isysis of RBCs in chamber, using the cartridge of this invention;

Figure 13 illustrates final diluted blood pushed to an impedance sensor using the cartridge of this invention;

Figure 14 illustrates optical detection system to monitor liquid position inside the cartridge of this invention; Figure 14a shows the inclined surface when the chamber is filled with air (i.e. without any liquid) causing light to refract more;

Figure 14b shows a chamber which is filled with water which minimized the angle of refraction allowing more light to pass through the pin photodiode which is mounted in front of the laser diode;

Figure 14c illustrates a top view of the cartridge showing the liquid level sensing region of this invention;

Figure 15 illustrates signal from a PIN photodiode due to a meniscus of a liquid at an entry point using the cartridge of this invention;

Figure 16 illustrates leukocyte enumeration and haemoglobin detection using the cartridge of this invention;

Figure 17 illustrates a shape memory alloy used as a linear actuator for opening and closing of the valve for the cartridge of this invention;

Figure 18 illustrates sealing concept of an impedance sensor for the cartridge of this invention;

Figure 19 illustrates blister protector integrated with a gripper;

Figure 20 illustrates a disposable cartridge, of this invention, with blister protector and notch 90;

Figure 21 illustrates an isometric view of the disposable cartridge of this invention; and Figure 22 illustrates a shape memory alloy which is used as a linear actuator for opening and closing of the valve.

DETAILED DESCRIPTION OF THE ACCOMPANYING DRAWINGS:

Figure 4 illustrates microfluidic valves, except shown in black colour, are closed where movement of the plunger cap by linear actuator drives blood into a metering section; Figure 5 illustrates a mechanism showing rupture of blister/s due to sharp conical pillar/s (14) placed beneath an aluminium foil, in the cartridge of this invention;

Figure 6 illustrates the position of cap during first hard stopper;

Figure 14 illustrates optical detection system to monitor liquid position inside the cartridge of this invention;

Figure 14a shows the inclined surface when the chamber is filled with air (i.e. without any liquid) causing light to refract more;

Figure 18 illustrates sealing concept of an impedance sensor for the cartridge of this invention; Figure 19 illustrates blister protector integrated with a gripper;

In at least an embodiment, the cartridge can be broken into four sections:

• metering section,

• liquid / reagent storage section,

• section for running chemistry (mixing, incubation, lysis, dilution) in chambers, and

• detection section.

In at least an embodiment, the system comprises a blood collection and analysis sub-system such that it addresses prior art’ s challenges by providing a streamlined process for blood collection and analysis.

In at least an embodiment, the invention discloses a blood collection and analysis system comprising a cartridge (1) having: a capillary tube coated with EDTA for collecting blood; a designated blood port (3) for transferring the collected blood; a cap (7) configured to be pushed until it reaches a first stopper (4, 8) to prevent blood flow to a downward section; a capillary valve (6) integrated within the cap (7) to restrict movement of the collected blood based on capillary forces and geometry of the capillary valve (6) structure, wherein the capillary forces and the capillary valve’s (6) geometry act in tandem to prevent blood flow through the capillary valve, thereby functioning as a reliable stopper or barrier; the specially designed cartridge (1) optimized for accurate blood component measurement.

This blood collection and analysis subsystem utilizes a capillary tube (200, Figure 2) coated with Ethylenediaminetetraacetic acid (EDTA) to collect blood, which is then transferred into a designated blood port (3, Figure 1). A cap (7, Figure 3) is configured to be gently pushed until it reaches a first stopper (manual hard stop 4, 8, Figure 1 and Figure 3), preventing blood from reaching a downward section using at least a capillary valve (6, Figure 3) which stops the blood flow due to capillary forces and due to geometry of the capillary valve (6, Figure 3) structure, which restricts movement of the fluid. The geometry is disclosed in figure 3 where the channel is narrow and then expanded to create a Laplace pressure barrier to stop the flow unless extra pressure is applied externally.

When the cap is pushed the blood first fills the WBC metering chamber then fills the RBC metering chambers and remaining blood flows into an overflow chamber 54. [see Figure 9]

Figures 6a and 6b illustrate the operational concept of the capillary stop valve within the microfluidic channel. The dimensions of the microfluidic channel connecting the blood port and the capillary valve are less than 100 microns, specifically less than 50 microns.

Consequently, the predominant force within these channels is surface tension, which significantly outweighs volumetric forces. The microfluidic side channels are constructed from hydrophilic materials such as acrylic or polycarbonate. As a result, these side channels act as a self-propelling force for liquid movement within the microchannel without necessitating external forces.

Although the bottom surface is hydrophobic, the overall effect leans towards hydrophilic dominance. The curvature of the liquid depicted in Figure 6a is concave on the right side, causing the pressure within the liquid to decrease by 2 * T * cos(0) / w, where '0' represents the contact angle of the liquid and 'w' denotes the width of the channel. This pressure decrease facilitates self-propelled movement of the liquid. Subsequently, the liquid advances to the corner point of the channel, where the contact angle changes due to wetting of the new surface with distinct geometry. This alteration in the contact angle results in an increase in pressure by 2 * T * cos(cp) / R, where ’cp’ signifies the new contact angle. Consequently, the liquid halts at the corner point depicted in Figure 6b.

Together, the capillary forces and the valve's (6) geometry work in tandem to prevent the fluid from flowing through the capillary valve (6); enabling it to act as a reliable stopper or barrier in various applications, such as microfluidics, lab-on-a-chip devices, and fluid control systems. The position of the cap (7) is shown in figure 6 during first hard stop pressed by a finger giving a user minimal space for further push. Subsequently, the cartridge (1), optimized for accurate sample metering, by a metering actuator (203, Figure 22) having a membrane wall (67) and a depression (13a) [first valve] inside the cartridge through which liquid flows - which serves as a valve sitting position, is inserted into the instrument. The isometric view of the disposable cartridge along with the cap (1) is shown in figure 21.

In at least an embodiment, the invention discloses a blood analysis system comprising a cartridge (1) having: an actuator configured to activate a microfluidic first set of valves (13) that selectively allows blood flow into a designated metering section while preventing flow into other sections; a linear actuator (10) designed to further push a cap (7) to transfer a blood sample (5) into two separate metering sections; a first metering section (12) dedicated to measuring red blood cells (RBCs) and platelets; a second metering section (11) specifically designed for white blood cell (WBC) count; a blood port (3) integrated within the cartridge (1), wherein the blood port (3) is designed to prevent excessive pushing of a stopper (4, 8), ensuring sample integrity and accurate analysis results; wherein the system enables precise and segregated measurement of RBCs, platelets, and WBCs within the metering sections for comprehensive blood analysis.

In at least an embodiment, the system comprising the blood collection and analysis subsystem further incorporates an actuator (203, Figure 22) that initially activates a microfluidic first valve (13, Figure 4), that stops blood (fluid) flow from flowing into other sections, of this system, except to a designated first metering section (11, Figure 4) and a designated second metering section (12, Figure 4). Subsequently, a linear actuator (10) further pushes the cap (7, Figure 3) to push the blood sample (5, Figure 2) into two separate metering sections: a first section (12, Figure 4) is dedicated to counting of RBCs and platelets (12, Figure 4), a second section (11, Figure 4) is designed for WBCs count (11, Figure 4). To ensure optimal performance and prevent sample contamination, the cartridge's blood port (3) is designed to prevent users from pushing the first stopper beyond a designated limit (4, 8, Figure 1, Figure 3), safeguarding the integrity of the sample (fluid) and maintaining accurate analysis results. The narrow steps 4, figure 1 restrict the motion of the cap.

Reference numeral 7 is cap.

Reference numeral 8a is step.

Cap (7) is such that it does not travel beyond step (8) in a notch used to receive the cap (7). Reference numeral 9 is cap guiding socket.

In at least an embodiment of the blood analysis system and cartridge, the microfluidic first valve (13) ensures controlled blood volume flow, allowing only a desired volume of blood to enter designated metering sections; thereby, facilitating accurate and reliable analysis of blood components.

In at least an embodiment of the blood analysis system and cartridge, a linear actuator (10) exerts controlled force/s on the cap (7); enabling precise transfer of the blood sample (5) into separate metering sections, ensuring accurate measurement of RBCs and platelets in one section and WBC count in another section.

In at least an embodiment of the blood analysis system and cartridge, the intelligently designed blood port (3) within the cartridge prevents users from pushing the stopper (4, 8) beyond a designated limit, safeguarding integrity of the blood sample and maintaining reliability of analysis results.

In at least an embodiment, the invention discloses a blood collection and analysis system comprising a cartridge (1) having: a cartridge with blister-packed reagents for convenient dispensing of liquids by pressing the blisters with actuators; a linear actuator that, when pressed against the blisters, increases the pressure inside the blisters, causing rupture of the bottom part of the blisters; a sharp conical pillar (14) with a diameter of 200 microns, positioned to deflect the bottom part of the blister and release the fluid upon rupture; a via (17) located beneath the aluminium foil part of the disposable cartridge, establishing a connection between the microfluidic channel and the metering section with the blister filled with reagents; an open microfluidic channel and sharp conical pillars moulded directly on the plastic cartridge; a thin, soft, and flexible pressure-sensitive adhesive (PSA) layer of 50 to 200 microns thickness, with adhesive on one side, capable of closing the open microfluidic channel; alternatively, direct ultrasonic welding of the thin plastic can be used to close the open microfluidic channel; furthermore, the open microfluidic channel can be thermally bonded with COC and Polystyrene, with a thickness ranging from 100 to 200 microns; in either case, the thin layer or thermal bonding serves to close the valve after pressing the bottom layer using a small linear actuator or shape memory alloy.

In at least an embodiment of the blood analysis system and cartridge, the cartridge with blister-packed reagents enables controlled and precise dispensing of fluids by pressing the blisters with actuators, ensuring accurate and reliable fluid release for performing desired analytical reactions.

In at least an embodiment of the blood analysis system and cartridge, the sharp conical pillar (14) positioned within the cartridge facilitates the controlled rupture of blisters by deflecting the bottom part of the blister, allowing efficient release of fluid into the microfluidic channel for subsequent analysis.

Blisters are located on the cartridge. Each blister has liquid inside it. The purpose of using the blister is to store the liquid and also to dispense the liquid inside the channel by applying pressure through linear actuators. Third blister’s purpose is to dilute the first serially diluted blood to achieve greater dilution ratio.

In at least an embodiment of the blood analysis system and cartridge, the via (17) located beneath the aluminium foil part of the cartridge provides a fluidic connection between the microfluidic channel and the metering section, enabling precise delivery of reagents to the desired analytical compartments. In at least an embodiment of the blood analysis system and cartridge, the open microfluidic channel, closed with a thin, soft, and flexible pressure-sensitive adhesive (PSA) layer or through direct ultrasonic welding or thermal bonding, effectively seals the fluidic pathway, maintaining fluid control and preventing unwanted leakage during the analysis process.

In at least an embodiment, the invention discloses a blood collection and analysis system comprising a cartridge (1) having: blister-packed reagents equipped with a blister protector, designed to prevent accidental blister rupture due to user pressure, thereby ensuring the system's integrity and reliability; first set of blisters (15) made of aluminium foil that can be directly bonded onto the disposable cartridge (1) using ultrasonic welding, enabling seamless and efficient production of cartridges on an assembly line; the integration of reagents within the disposable cartridge reduces reagent cost by minimizing fluid wastage associated with large-volume bottles used in automatic sample handling systems, resulting in a cost-effective and portable instrument suitable for remote resource settings; the system's design allows for self-tests to be performed by non-skilled individuals at home, utilizing finger-prick blood samples; the system's user-friendly nature is particularly beneficial for cancer patients, enabling them to monitor their blood cells during chemotherapy and facilitating personalized healthcare.

In at least an embodiment of the blood analysis system and cartridge, the blister protector incorporated within the blister-packed reagents mitigates the risk of mishandling, ensuring that the microfluidic device remains intact and maintains its reliability throughout the testing process.

In at least an embodiment of the blood analysis system and cartridge, the direct ultrasonic welding of the aluminium foil blisters onto the disposable cartridge enables efficient and rapid production, ensuring a secure and leak-proof attachment between the reagentcontaining blisters and the cartridge. In at least an embodiment of the blood analysis system and cartridge, the integration of reagents within the disposable cartridge reduces overall reagent cost by minimizing fluid wastage, enhancing the portability and affordability of the instrument, making it suitable for resource-limited settings and enabling convenient point-of-care testing.

In at least an embodiment of the blood analysis system and cartridge, the user-friendly design of the system enables non-skilled individuals to perform self-tests at home, utilizing fingerprick blood samples, thereby facilitating convenient and accessible monitoring of blood cells for various medical applications, including chemotherapy patients requiring regular blood cell analysis.

In at least an embodiment, the system comprises a reagent storage sub-system such that it comprises first set of blister-packed reagents (15, Figure 5) that can easily dispense fluid/s by pressing the blisters with actuators (10, Figure 5). Blister rupture occurs when the linear actuator is pressed against the blisters, resulting in an increase in pressure inside the first set of blisters (15, Figure 5). This elevated pressure causes a bottom part of the blister to deflect and press against a sharp conical pillar (14, Figure 5) which is, preferably, 200 microns in diameter; consequently, rupturing the bottom part of the blister and releasing its held fluid. A via (17, Figure 5) is provided just below a foil (preferably, Aluminium) part of the disposable cartridge (1) which connects a microfluidic channel (13) and the metering section/s (second valve 19 and third valve 21 Figure 8 connects the via 17 and the metering section 11 and 12, Figure 4) with the blister filled with reagents (16). The microfluidic open channel (13) and the sharp conical pillars (14, Figure 5) are moulded directly on the cartridges (preferably, made of plastic). The open microfluidic channel can be closed with a thin soft and flexible pressure sensitive adhesive (PSA) layer, preferably of thickness ranging from 50 micron to 200 micron, having glue on one side of the layer or can directly be ultrasonically welded using thin plastic. The open microfluidic channel can also be thermally bonded with the COC (Cyclic olefin copolymer) and Poly styrene of thickness 100 micron to 200 microns. In either of the case the thin layer can be used to close the first valve (13, Figure 4) after pressing a bottom layer being a membrane wall (67, Figure 22) using small linear actuator/s (203, Figure 22) which is made of shape memory alloy.

In at least an embodiment, the reagent storage sub-system comprises blister-packed reagents which are equipped with a blister protector (204, Figure 21), which serves to prevent blister rupture in the event of accidental user pressure. This protective mechanism minimizes risk of mishandling the microfluidic device, ensuring integrity and reliability of the system. The first set of blisters (15, Figure 5) are, typically, made of aluminium foil which can be directly bonded on the disposable cartridge (1) using ultrasonic welding enabling seamless and rapid production of cartridge on an assembly line. The advantage of storing the reagent in the disposable cartridge is that the reagent cost is minimized, as the bulky bottles required for automatic sample handling inside the instrument result in a significant wastage of fluid due to the larger volume needed. This makes the instrument portable and less expensive which can be used for remote resource settings. The above design offers additional advantages to non-skilled individuals, enabling them to perform self-tests at home using finger prick blood. This is especially useful for cancer patients to monitor their blood cells during the chemotherapy process.

In at least an embodiment, the invention discloses a blood collection and analysis system comprising a cartridge (1) having: a cartridge loaded inside the instrument, wherein microfluidic valves are selectively closed except for fourth valve (20) and fifth valve (22); a linear actuator that, when pressed, drives the blood filled inside the blood port into metering chambers (52, 53) [first metering chamber (52), second metering chamber (53), while allowing excess blood to flow into a buffer zone (54); an exposed first vent (48) connected to a rubber gasket, further connected to an air solenoid valve, which can be adjusted to atmospheric pressure or a higher positive pressure using a MOSFET or a relay switch; a sixth valve (30) opened to dispense diluent and metered blood into a first mixing chamber (33); ferromagnetic beads, shaped cylindrically with a diameter of 1mm and length of 5mm, driven by magnets mounted on motors with rapid polarity changes, facilitating efficient mixing of the blood and diluent in the mixing chamber; the mixing process preserving the cells' size and shape due to the gentle micro mixing action enabled by the ferromagnetic beads, eliminating the need for additional microfluidic valves for back-and-forth mixing; the dispensed liquid containing sphering reagents and fixating reagents that transform the shape of red blood cells (RBCs) to spherical, enhancing impedance measurement accuracy by reducing impedance variations caused by biconcave-shaped cells; a first dilution ratio of 1 : 120 for initial mixing of blood and diluent, ensuring sufficient dilution for RBC counting, followed by a second dilution ratio of 1:120 to achieve a dilution greater than 10,000, enabling precise measurement of single cells passing through the impedance chamber.

First metering chamber is to meter WBC blood.

Second metering chamber is to meter RBC blood.

In at least an embodiment of the blood analysis system and cartridge, the selective closing of microfluidic valves, activation of the linear actuator, and controlled dispensing of diluent and blood into the mixing chamber enable efficient and accurate mixing of blood and diluent, preserving the integrity of the cells and maintaining the desired dilution ratios for optimal analysis results.

In at least an embodiment of the blood analysis system and cartridge, the use of ferromagnetic beads and rapid polarity changes driven by magnets mounted on motors allows for rapid and effective mixing of blood and diluent, minimizing the need for additional microfluidic valves and reducing complexity in the mixing process.

These beads are located inside the mixing chambers. There are total three mixing chambers. One mixing chamber is to mix the blood and the diluent, the second is to mix the first diluted blood with diluent and third is to mix the blood and the lysis and quench. These beads are kept inside the mixing chambers and when the motor which has magnet mounted on it rotate the polarity of the north and south continuously changes which rotated the magnetic beads.

In at least an embodiment of the blood analysis system and cartridge, the inclusion of sphering reagents and fixating reagents in the dispensed liquid enhances the measurement accuracy of red blood cell volumes by transforming the cells to a spherical shape, thereby reducing impedance variations and ensuring precise assessment of cell characteristics.

In at least an embodiment of the blood analysis system and cartridge, the adjustable vent connected to a rubber gasket and controlled by an air solenoid valve, with options for atmospheric pressure or higher positive pressure, allows for regulated pressure conditions during the mixing process, ensuring optimal fluid dynamics and reliable analysis performance.

In at least an embodiment, the system comprises a process flow and fluidic routing subsystem such that once the cartridge is loaded inside the instrument (for readings) and, thereafter, all the microfluidic valves are closed except a fourth valve (20, Figure 8) and a fifth valve (22, Figure 8) and, subsequently, the linear actuator is pressed to push down a cap plunger (7, Figure 6) which drives the blood filled inside the blood port (3) into a first metering chamber (52, Figure 9) and into a second metering chamber (53, Figure 9). Excess blood collected, inside the port, flows into a buffer zone (54, Figure 9). A first vent (48, Figure 7) is exposed to atmospheric pressure. A sixth valve (30, Figure 8) is opened and the second set of blisters (45, Figure 7) is pushed to dispense the diluent and the metered blood into a first mixing chamber (33, Figure 8). The blood and the diluent are mixed using ferromagnetic beads (205, Figure 23) driven by a magnet mounted on motors placed inside the instrument. The shape of the ferromagnetic bead is cylindrical; typically, with diameter 1mm and length 5mm. The magnets mounted on the motor rapidly changes polarity from north to south which drives motion of the magnetic beads. The amount of mixing is dependent on geometry of the beads, speed of the motor expressed in RPM, and the size of magnets mounted on the motor. The diluent and the blood require only gentle micro mixing to preserve all cells without changing size and shape of the cells. The advantage of mixing, using ferromagnetic beads, is that mixing is rapid and requires no extra microfluidic valves as in the case of back and forth mixing requiring multiple microfluidic valves actuation. The dispensed liquid contains the sphering reagents and the fixating reagents also which makes the RBCs spherical in shape. This is essential since the impedance magnitude of the cells depends on the geometry of the cells, making them spherical from biconcave shape reduces the impedance variation and gives better measurement of the volume of the cells. The first vent (48, Figure 7) is either exposed to atmospheric pressure or a higher positive pressure. The first vent (48, Figure 7) is connected to a rubber gasket, which is further connected to an air solenoid valve. The ends of the air solenoid valve can be connected to a positive pressure or atmospheric pressure using a MOSFET or a relay switch. The blood and the diluent are mixed in 1:120 in first dilution. Since the dilution required for counting the RBCs is more than 10000 to ensure single cell is passed through the impedance chamber. Therefore, further second dilution of 1:120 is required to reach the dilution greater than 10,000. In terms of working, higher dilution greater than 10,000 is not possible, in single step, as this requires 30 mL of diluent for 3 microliters of blood for achieving dilution of 10,000.

The first mixing chamber (33, figure 11) is RBC mixing chamber for mixing the blood and the diluent for 1st dilution and this is connected to chamber 55 (Figure 11) for metering the new mixed first serially diluted blood. When chamber 33 is presusurized the blood flow from chmber 33 to chamber 55 and extra blood flow to chamber 57. Blister connected to valve 31, figure 8 is opened and liquid is dispensed from this blister to sweep the liquid present in chamber 55. valve 28 and 26 is also opened at the same time to drag the first diluted metered blood into chamber 34, figure 8.

In at least an embodiment, the invention discloses a blood collection and analysis system comprising a cartridge (1) having: a pressurized first vent (48), delivering positive pressure to drive blood from the first mixing chamber (33) into the waste chamber (57) upon opening valves (28, 27) [eighth valve (28), seventh valve (27)] (Figure 11); a waste chamber (57) designed to receive excess blood from the mixing chamber; a third set of blisters (47) containing diluent, pushed into the second mixing chamber (34) (Figure 7) after opening valves (26, 28) [ninth valve (26), eighth valve (28)] (Figure 8), facilitating the mixing of diluent with the first diluted blood to achieve a serial dilution greater than 10,000; a fourth set of blisters (44), pushed into the third mixing chamber (35) after opening valves (19, 18) [i.e. tenth valve (18) and second valve (19)], enabling the lysis of red blood cells using a lysis buffer containing formic acid and saponin as surfactants; a magnetic flea rotated at 5000 RPM to facilitate complete lysis of red blood cells upon the introduction of the metered blood and lysis buffer into the chamber; a quench buffer from fifth set of blisters (46) pushed into the third mixing chamber (35) to quench the reaction, the quenching reagent composed of sodium chloride, sodium bicarbonate, and sodium phosphate; the quench solution preserving white blood cells (WBCs) for a minimum of fifteen minutes, ensuring their stability for subsequent analysis. In at least an embodiment of the blood analysis system and cartridge, the pressurized vent delivering positive pressure, in conjunction with the opening of specific valves, facilitates the controlled flow of blood from the mixing chamber to the waste chamber, ensuring efficient removal of excess blood and enabling subsequent steps in the analysis process.

In at least an embodiment of the blood analysis system and cartridge, the introduction of diluent from third set of blisters (47) into the second mixing chamber (34), followed by the lysis of red blood cells using a lysis buffer from fourth set of blisters (44), ensures the serial dilution and complete lysis of red blood cells, preparing the sample for subsequent analysis steps.

Fourth set of blisters (44) is the lysis buffer blister which Is connected to mixing chamber 35. these blisters are connected to the mixing chamber through a microfluidic channel and can be isolated when valve is closed.

In at least an embodiment of the blood analysis system and cartridge, the rotation of the magnetic flea at 5000 RPM within the third mixing chamber (35) enables thorough lysis of red blood cells, facilitating accurate analysis of white blood cells and ensuring reliable measurement results.

In at least an embodiment of the blood analysis system and cartridge, the introduction of a quench buffer from fifth set of blisters (46) into the third mixing chamber (35) after lysis ensures the efficient quenching of the reaction, preserving the stability of white blood cells for a minimum of fifteen minutes, allowing sufficient time for subsequent analysis steps to be performed accurately. Reference numeral 29 is a microfluidic valve for venting air and pressurizing the chamber 35. Quench buffer is connected to mixing chamber 35 through a microfluidic channel and this can also be isolated using valve 29. the small port close to 29 is the via which connects the quench blister to the mixing chamber.

In at least an embodiment, the first vent (48) is pressurized with positive pressure to drive the blood from the first mixing chamber (33) into the second RBC (third) metering chamber (55, figure 11) and waste chamber (57, Figure 11) after opening the eighth valve (28, Figure 8) and seventh valve (27, Figure 8) shown in figure 11. The excess of the blood from the first mixing chamber (33) flows into a waste chamber (57). Thereafter, third set of blisters (47, Figure 7) containing diluent is pushed after opening the valves (26, 28; Figure 8) [ninth valve (26), eighth valve (28)] into the second mixing chamber (34, Figure 7) sweeping the metered blood inside the third metered chamber (55, Figure 11). The diluent and the first diluted blood are mixed to achieve serial dilution of greater than 10,000. Reference numeral 31 is a microfluidic valve for second serial dilution. Thereafter, fourth set of blisters (44, Figure 7) is pushed after opening the valves (19, 18) [tenth valve (19), second valve (18)] into the third mixing chamber (35) for the lysis of the red blood cells. The Lysis contains formic acid and saponin as surfactant to lyse RBCs. Once the metered blood is pushed into the third mixing chamber (35) along with a lysis buffer, the magnetic flea is rotated at RPM of 5,000 to completely lyse the RBCs and, subsequently, a quench buffer from fifth set of blisters (46, Figure 7) is pushed into the third mixing chamber (35) to quench the reaction. The composition of the quenching reagent is sodium chloride, sodium bicarbonate and sodium phosphate. The quench solution preserves the WBCs for longer period of time at least for fifteen minutes.

Reference numeral 32 is a buffer chamber for extra blood flow.

Reference numeral 37 is a waste chamber as well as measurement chamber for volume dispensed for RBC count.

Reference numeral 38 is a second waste chamber for wash buffer volume measurement.

Reference numeral 39 is a third waste chamber as well as measurement chamber for volume dispensed for WBC count.

Reference numeral 40 is a second waste chamber for washing. Reference numeral 41 is a buffer chamber to prevent overflow. Reference numeral 42 is a chamber for Hemoglobin measurement. Reference numeral 43 is a connection port for wash buffer blister. Reference numeral 51 is a WBC metered volume.

Reference numeral 56 is a buffer overflow chamber.

In at least an embodiment, the invention discloses a blood collection and analysis system comprising a cartridge (1) having: a pressurized second vent (49) (Figure 7), delivering positive pressure to drive diluted blood from the second mixing chamber (34) into a biosensor (Impedance measurement) (36) upon opening eleventh valve (25); fourth chamber (59) and counting chamber (58) (Figure 13) within the biosensor, facilitating the counting of red blood cells (RBCs) as the diluted blood reaches chamber (58); optical detection windows within the counting chamber (58), serving as signal windows for liquid level detection based on the principle of light refraction; an optical detection system comprising laser diodes and PIN photodiodes at each entry point of the chamber, detecting the position of the liquid through the refractive index change; a flat-line signal from the photodiodes when no liquid is present, transforming into a square pulse electrical signal as the liquid enters the entry point of the chamber, indicating the arrival of the liquid; laser diode (60) and PIN photodiode (61) (Figure 14) integrated within the system for accurate signal measurement; a liquid meniscus with curvature at the optical detection windows, causing light reflection and refraction and resulting in a square pulse electrical signal for a short duration (Figure 15); the same principle applied for white blood cell (WBC) sample volume measurement, enabling precise analysis using the optical detection system.

Meniscus is formed inside the waste chamber. The hydrophobic nature of the surface makes the surface of liquid concave towards the flow direction also the optical detection region of the measurement volume is inclined at 45 degrees. The hydrophobic nature as well as this 45- degree tilt scatters more light which acts as better sensor for liquid level detection.

In at least an embodiment of the blood analysis system and cartridge, the pressurized vent delivering positive pressure, in conjunction with the opening of eleventh valve (25), ensures controlled flow of diluted blood from the second mixing chamber (34) to the biosensor (36), allowing for accurate counting of red blood cells (RBCs) within the designated counting chamber (58).

In at least an embodiment of the blood analysis system and cartridge, the optical detection windows within the counting chamber (58), based on the principle of light refraction and reflection, provide reliable liquid level detection for precise analysis of red blood cells (RBCs), with signal measurement facilitated by laser diodes and PIN photodiodes at each entry point of the chamber.

In at least an embodiment of the blood analysis system and cartridge, the integration of laser diode (60) and PIN photodiode (61) within the system enables accurate measurement of the electrical signal, transforming light refraction into a square pulse signal as the liquid enters the entry point of the chamber, facilitating reliable detection of the liquid level for precise analysis of blood components.

In at least an embodiment of the blood analysis system and cartridge, the utilization of the optical detection system, employing laser diodes (60) and PIN photodiodes (61), enables efficient and accurate measurement of liquid levels within the counting chamber, ensuring precise analysis of red blood cells (RBCs) and white blood cells (WBCs) based on their respective counting principles.

The optical detection system is placed in front of the measurement chambers. More specifically they are located near the inclined surface which is inclined at 45 degrees. The optical detection system comprises of laser diode which is placed in front of these chambers and the sensor PIN photodiode is placed opposite to it.

Figure 14a illustrates the angle of refraction is more when the detection region is filled with air.

Figure 14b illustrates the angle of refraction is minimized when the detection region is filled with liquid.

In at least an embodiment, the system comprises a counting subsystem which is configured to count RBCs and platelets; using the system and cartridge of this invention. For counting of RBCs and platelets, the second vent (49, Figure 7) is pressurized to push the diluted blood from the second mixing chamber (34) into a biosensor (Impedance measurement) (36, Figure 8) after opening the eleventh valve (25, Figure 8). The blood is pushed to the chambers (58, 59) [counting chamber (58), fourth chamber (59)] sequentially (Figure 13). The measurement begins at the entry of counting chamber (58) and ends at the exit of fourth metering chamber (59). The counting of the RBCs starts when the diluted blood reaches counting chamber (58). The counting chamber (58) has optical detection windows (207, Figure 14a and 14b) at entrance of the chamber which serve as a signal window for the measurement of liquid level detection. The principle of liquid level detection is based on refraction of light. When there is no liquid, the laser beam falling on the optical window goes straight but in the presence of liquid due to change in the refractive index the light refracts. This refraction can easily be detected using PIN Photodiode. The measurement is stopped once the liquid reaches the exit point of fourth metering chamber (59). The optical detection system can detect entry of liquid level in counting chamber (58) and at the exit of fourth chamber (59). The optical photodiode and laser are present at each entry and exit point of the chamber to detect the position of the liquid. The signal from the photodiode is a flat line when there is no liquid but as the liquid enters inside the entry point of the chamber the laser beam refracts and gives a square pulse as electrical signal indicating liquid has reached. Figure 14 shows a laser diode (60) and PIN photodiode (61) for measurement of the signal. The liquid shown in black colour forms a meniscus with curvature which bounces most of the light from the laser diode and gives square pulse for short duration of time shown in Figure 15. The same principle is adopted for WBC volume measurement.

X-axis: time

Y-axis: voltage

Figure 14a illustrates the angle of refraction is more when the detection region is without any liquid.

In accordance with this invention, the liquid level detection system is based on law of refraction. The cartridge has an inclined plane (300) with right angle prism shape, at a small section (Figure 14a and Figure 14b where laser light falls) where a light of laser diode is incident. The incline (300) is provided to compensate for refraction caused by the liquid in the sensing region. This sensing region is in a few microns; which is why there is a need to improve signal strength. The diameter of the laser diode, preferably, is 1mm which falls on the inclined surface of the cartridge. In the absence of water (or any fluid), the light is refracted at a relatively higher angle. However, when liquid water with refractive index 1.33 or more is filled (Figure 14b) there is a significant change in the refraction angle of the laser (from the laser diode). This liquid level detection is used to, precisely, monitor the metering accuracy of the sample and the reagents and serves as one of the checkpoints in accurate sample preparation. Since the volume of the waste chambers (57) are fixed, therefore, the liquid level detection system allows to precisely monitor liquid volume. Figure 14a shows the optical window with inclined surface having geometry of right-angle prism filled with air (i.e. without any liquid) causing light to refract more and Figure 14b shows a chamber which is filled with water which minimized the angle of refraction allowing more light to pass through the pin photodiode which is mounted in front of the laser diode. The volume of the region where most of the laser falls has volume less than 100 nanolitre therefore measurement inaccuracy in volume is less than few hundred nanolitre.

Figure 14c illustrates a top view of the cartridge showing the liquid level sensing region of this invention.

In the realm of liquid sensing technology, optimizing the cartridge design is crucial for accurate and efficient detection. One innovative aspect of this invention revolves around a bottom layer of the cartridge, particularly focusing on its material composition and surface characteristics. This aspect enhances the sensing mechanism by leveraging hydrophobic materials to induce specific surface properties, ultimately influencing the behaviour of light upon interaction with the liquid surface.

Hydrophobic Surface:

The surface of the bottom layer of the cartridge is intentionally crafted from hydrophobic materials. This choice ensures that when liquid comes into contact with the sensing region, it exhibits distinct surface properties. Unlike conventional surfaces, the hydrophobic nature of the material causes the liquid meniscus surface to form a spherical shape (5, figure 24) upon contact. This spherical surface configuration plays a pivotal role in scattering a greater amount of light, thereby facilitating a more pronounced signal change during sensing processes.

Inclined Liquid Sensing Region:

Within the cartridge design, the liquid sensing region is strategically inclined at an angle, as illustrated in Figures 14a and 14b. This inclination serves a dual purpose, resembling a prism with angle of the prism between 30 and 60 degrees. The angle of inclination is carefully selected to be greater than 30 degrees yet less than 60 degrees, optimizing its prism-like behaviour. Prism-Like Behaviour:

The inclined liquid sensing region functions akin to a prism, refracting light as it passes through. The angle of refraction experienced by the light depends on several factors, including the wavelength of the light, the angle of the prism (in this case, the inclination of the sensing region), and the refractive index of the liquid being sensed. This prism-like behaviour enhances the interaction between light and liquid, contributing to the accuracy and sensitivity of the sensing mechanism.

The prism-like behaviour of the sensing region guarantees the accurate metering of both liquid and samples, while also enabling the monitoring of fluid routing in the desired direction. These checkpoints are essential for ensuring precise sample preparation, and any deviations in metering accuracy or blister rupture are rigorously monitored by the liquid level sensor.

The measurement process for cell counting commences only when the liquid has reached the bottom sensing region of the measurement chamber, as depicted in Figure 14c. The measurement concludes when the liquid reaches the top measurement sensing region. This sequential approach ensures that the counting of cells occurs precisely within the designated measurement zones, facilitating accurate and reliable results.

The incorporation of hydrophobic materials and the strategic inclination of the liquid sensing region within the cartridge design significantly enhances the sensing mechanism's performance. By leveraging these features, the cartridge facilitates a more efficient interaction between light and liquid, resulting in improved signal detection and overall sensing accuracy. This innovative approach represents a significant advancement in liquid sensing technology, with potential applications across various fields, including medical diagnostics, environmental monitoring, and industrial quality control.

In at least an embodiment, the invention discloses a blood collection and analysis system comprising a cartridge (1) having: twelfth valve (23) and thirteenth valve (24), enabling the opening of a passage for sample transfer from the third mixing chamber (35) to the impedance sensor upon the application of positive pressure on third vent (50); a leukocyte counting process initiated as the liquid reaches the entry point of chamber (64) and terminated at the entry point of chamber (65); a haemoglobin detection chamber (66), featuring an optical path length of 3mm for accurate absorbance measurement using a green laser or collimated green LED; the utilization of a laser diode or collimated green LED to measure the absorbance and determine the concentration of haemoglobin in the blood sample; an incubation period following the mixing of the quench into the mixing chamber (65), with the duration ranging from 2 minutes to 5 minutes based on the concentration of saponin in the lysis buffer.

In at least an embodiment of the blood analysis system and cartridge, the opening of twelfth valve (23) and thirteenth valve (24), coupled with the application of positive pressure on third vent (50), ensures controlled sample transfer from the third mixing chamber (35) to the impedance sensor, facilitating accurate leukocyte counting within the designated chambers.

In at least an embodiment of the blood analysis system and cartridge, the haemoglobin detection chamber (66), featuring an optical path length of 3mm, allows for precise absorbance measurement using a green laser or collimated green LED, enabling accurate determination of haemoglobin concentration in the blood sample.

In at least an embodiment of the blood analysis system and cartridge, the utilization of a laser diode or collimated green LED for absorbance measurement ensures accurate determination of haemoglobin concentration, enhancing the system's ability to provide reliable and precise measurement results.

In at least an embodiment of the blood analysis system and cartridge, the incubation time following the mixing of the quench into the mixing chamber 65, with variations ranging from 2 minutes to 5 minutes, ensures optimal reaction and stabilization based on the saponin concentration in the lysis buffer, leading to accurate haemoglobin concentration measurement and three -part Leukocyte Count.

In at least an embodiment, the system comprises a counting subsystem which is configured to count WBCs; using the system and cartridge of this invention. For counting of WBC’s, valves (23 and 24) [twelfth valve (23), thirteenth valve (24)] are opened and, subsequently, positive pressure is applied on the third vent (50) to push the sample from the third mixing chamber (35) into the impedance sensor. The counting of the leukocyte starts when the liquid reaches the entry point of the chamber (64, Figure 24) and stop at entry point of chamber (65, Figure 24). The haemoglobin detection chamber (66) has an optical path length of 3mm for measurement of the absorbance using green laser. The laser diode or collimated green LED can be used for the measurement of the absorbance and measurement of the concentration of the haemoglobin. The incubation time required after the quench is mixed into the mixing chamber (65) can vary, typically, from 2 min to 5 min depending on the concentration of the saponin inside the lysis buffer.

In at least an embodiment, the invention discloses a blood collection and analysis system comprising a cartridge (1) having: a valve mechanism utilizing a working principle for opening and closing the valve, as depicted in Figure 17; the utilization of COC material as a membrane to provide valve closure; a shape memory alloy actuated by the application of 12V, resulting in length expansion and direct pressure on the COC membrane (67) to effect valve closure; the linear actuator returning to its initial position upon voltage drop to 0; a compact footprint of the shape memory alloy actuator, measuring only 3mm, enabling the placement of multiple valves within a small footprint.

In at least an embodiment of the blood analysis system and cartridge, the working principle for the valve's opening and closing, as illustrated in Figure 17, ensures efficient and reliable valve operation, allowing for precise control of fluid flow within the system.

In at least an embodiment of the blood analysis system and cartridge, the utilization of COC material as a membrane for valve closure provides a reliable seal, preventing unintended fluid leakage and ensuring the integrity of the system during operation.

In at least an embodiment of the blood analysis system and cartridge, the actuation of a shape memory alloy by the application of 12V, resulting in length expansion and direct pressure on the COC membrane (67), allows for reliable and responsive valve closure, enhancing the system's performance and fluid control capabilities. In at least an embodiment of the blood analysis system and cartridge, the compact footprint of the shape memory alloy actuator, measuring only 3mm, facilitates the incorporation of multiple valves within a limited space, optimizing the system's design and enabling efficient utilization of available resources.

Another aspect of the invention is the working principle for opening and closing of the valves. The working principle for the valve's opening and closing is shown in Figure 22. The COC material (67, Figure 22) is used as a membrane to close the valve. The shape memory alloy (203, Figure22) is actuated by applying 12V, resulting in an increase in length and directly pressing the COC membrane (67) to close the valve. When the voltage is dropped to 0, the linear actuator returns to its initial position. The footprint of the shape memory alloy actuator is only 3mm, allowing multiple valves to be placed in a small footprint. It should be noted that apart from shape memory alloy valves, various other kinds of valves such as motorized motors, pneumatic valves can be used.

In at least an embodiment, the invention discloses a blood collection and analysis system comprising a cartridge (1) having: the integration of an impedance sensor within a disposable cartridge, providing a compact and self-contained measurement solution; an impedance sensor design featuring side holes for fluid entry and exit, as illustrated in Figure 18 (36), offering improved fluid flow dynamics and optimized sensor performance; the sealing of the impedance sensor on the sides accomplished by a rubber gasket with a wedge shape (labelled as 63 in Figure 18); the placement of the rubber gasket, equipped with inlet holes, inside the slot (70) on each entry and exit side, followed by the positioning of the impedance sensor on top; the top cover (62) securely pressed and ultrasonically welded, ensuring a reliable and leak-proof flow through the system; the application of pressure during top cover placement causing the gasket to expand in all directions, effectively sealing the side holes and enhancing fluid control; the top cover featuring three holes on each side for the electrical connection of the sensor, facilitating the integration of spring connector pogo pins for reliable electrical contact; the provision of sufficient space within the three holes to accommodate exposed pads with a diameter greater than 1mm, ensuring optimal electrical connection for accurate sensor measurement.

In at least an embodiment of the blood analysis system and cartridge, the integration of the impedance sensor within a disposable cartridge ensures a compact and user-friendly measurement solution, allowing for convenient and efficient blood analysis.

In at least an embodiment of the blood analysis system and cartridge, the impedance sensor design with side holes for fluid entry and exit offers improved fluid flow dynamics, enhancing the accuracy and reliability of measurement results.

In at least an embodiment of the blood analysis system and cartridge, the secure sealing of the impedance sensor on the sides using a rubber gasket with a wedge shape ensures a robust and leak-proof flow, preventing fluid leakage and maintaining the integrity of the system during operation.

In at least an embodiment of the blood analysis system and cartridge, the ultrasonic welding of the top cover, pressed in conjunction with the rubber gasket, guarantees a reliable and durable seal, enabling consistent and accurate fluid flow through the system.

In at least an embodiment of the blood analysis system and cartridge, the integration of spring connector pogo pins for electrical connection facilitates reliable and efficient communication with the impedance sensor, ensuring precise and accurate measurement of blood samples.

Another aspect of the invention is the integration of the impedance sensor (36, Figure 18) within the disposable cartridge. Unlike traditional top and bottom holes, the impedance sensor features side openings (36, Figure 18) for the entry and exit of the fluid, as depicted in the figure 18 (36). The sealing of the sensor on the sides is achieved using a rubber gasket with a wedge shape (63 in Figure 18). The gasket, which includes inlet holes, is placed inside the slot (70) on each entry and exit side, followed by the placement of the impedance sensor on top of it. Subsequently, the top cover (62) is positioned and pressed for ultrasonic welding to ensure a leak-proof flow. This is possible because when the pressure is applied while pressing the top cover the gasket expands in all the direction sealing the side holes. The top cover features three holes on each side for the electrical connection of the sensor, which can be achieved using spring connector pogo pins. The dimension of the exposed pads inside the three holes are greater than 1mm diameter providing sufficient space for the electrical connection. However, it should be noted that the biosensor is not restricted solely to impedance measurement, but it can also function as an impedance sensor, optical sensor, or both simultaneously.

In at least an embodiment, a gripper (91, Figure 19) is provided which is a part of the disposable cartridge having QR code or RFID code which stores the following information:

(a) Production date of the microfluidic disposable cartridge

(b) Batch number of the production

(c) Lot number

(d) Calibration data for the biosensor

(e) Storage condition of the disposable cartridge such as temperature and humidity.

The cartridge is designed in such a way that it can only be inserted into the instrument in one direction. The 'L'-shaped notch (90), shown in Figure 20, ensures that the cartridge can be inserted inside the instrument in only one direction.

TECHNICAL ADVANTAGES:

In this invention, the inventors present the inventive steps taken in the development of a disposable cartridge, designed for single-time use. The cartridge incorporates on-board reagents, integrated sensor(s), chambers for mixing, microchannels for routing fluids (liquid and air), and a system for aligning the cartridge. It also includes a valve system that enables tight control over flow steps for fully automatic sample preparation. Moreover, the design aims to minimize the number of components requiring fabrication and assembly, resulting in a seamless manufacturing process and streamlined productization on an assembly line. The present invention provides several notable advantages over traditional sample preparation methods. The integration of multiple microfluidic valves, blister packed reagents, and actuators within the disposable cartridge streamlines the sample processing workflow, minimizing manual steps and reducing the risk of errors and contamination. The portable instrument facilitates the driving of necessary unit operations, eliminating the reliance on complex laboratory setups. Consequently, the invention enables accurate and efficient blood testing, making it accessible to home patients without specialized knowledge in haematology.

The TECHNICAL ADVANCEMENT of this invention lies in the following:

1] how to improve signal strength in liquid level sensing;

2] how to determine location of liquid in a sensing region;

3] how to collect blood sample.

The cartridge, of this invention, has 4 sections: metering, liquid storage, section for running chemistry (mixing, incubation, lysis, dilution) in chambers and finally detection. The entire flow is controlled with valves with feedback from liquid level sensors.

While this detailed description has disclosed certain specific embodiments for illustrative purposes, various modifications will be apparent to those skilled in the art which do not constitute departures from the spirit and scope of the invention as defined in the following claims, and it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the invention and not as a limitation.

Claims

CLAIMS,

1. A microfluidic cartridge-based (1) blood collection and analysis system comprising: a cartridge having a linear actuator configured to be pressed to push down a cap plunger (7, Figure 6) which drives blood filled inside a blood port (3), from a user, into a connected first metering chamber (WBC) (52, Figure 9), via a microfluidic channel, and, parallelly, into a connected second metering chamber (RBC) (53, Figure 9), via a microfluidic channel, in order to output metered blood, said cartridge’s bottom layer being a hydrophobic layer causing liquid meniscus surface to form a spherical shape upon contact in order to pronounce signal changes during sensing; plurality of fluid chambers including at least a first mixing chamber (33), a second mixing chamber (34), a third mixing chamber (35), a biosensor chamber (36), and plurality of blisters (45, 47, 44), said mixing chambers connected to the metering chambers via microfluidic channels, said blisters connected to metering chambers via fluidic channels, in that, o a second set of blisters (45, Figure 7) configured to be pushed to dispense a diluent and metered blood into said first mixing chamber (33, Figure 8); o a third set of blisters (47) containing diluent, pushed into said second mixing chamber (34) (Figure 7) after opening valves (26, 28), facilitating mixing of diluent with the first diluted blood, by ferromagnetic beads (205, Figure 23), located inside the mixing chambers, to achieve a serial first dilution followed by a serial second dilution of 1 : 120 to reach cumulative dilution greater than 10,000 in order to ensure to ensure single cells pass through a subsequent optical detection system, located pursuant to the mixing chamber, - the mixing process preserving blood cells' size and shape due to a gentle micro mixing action enabled by ferromagnetic beads, eliminating the need for additional microfluidic valves for back-and-forth mixing as also transforming the shape of red blood cells (RBCs) to spherical, enhancing impedance measurement accuracy by reducing impedance variations caused by biconcave-shaped cells; o a fourth set of blisters (44) being lysis buffer blisters, pushed into connected said third mixing chamber (35), via a microfluidic channel, after opening valves (19, 18), enabling the lysis of red blood cells using a lysis buffer containing formic acid and saponin as surfactants; o a quench buffer, connected to said third mixing chamber (35), via a microfluidic channel, from a fifth set of blisters (46) pushed into said third mixing chamber (35) to quench the reaction; the quench solution preserving white blood cells (WBCs), ensuring their stability for subsequent analysis; an optical detection system disposed in a counting chamber (58), having optical detection windows (207, Figure 14a, 14b) which serve as a signal window for the measurement of liquid level detection at entrance of said counting chamber (58) and comprising at least one laser diode (60) and at least one PIN photodiode (61), the optical detection system being configured to detect a liquid level based on changes in light refraction such that when no liquid is present a flat-line electrical signal is generated and when the liquid enters the counting chamber a square pulse electrical signal is produced, o wherein the optical detection system further comprises an inclined optical window (300) having a geometry of a right-angle prism filled with air, the inclined optical window compensating for refractive effects in a sensing region of a few microns to enhance signal strength; o wherein the optical detection system is further configured to measure a liquid meniscus formed at the optical window, the meniscus causing transient light reflection and refraction that produces a square pulse electrical signal for a short duration, the signal being used for sample volume measurement for both RBCs and white blood cells (WBCs); a controller configured to use the generated signals for counting red blood cells (RBCs) and for verifying sample metering.

2. The system as claimed in claim 1 wherein, a linear actuator that, when pressed, drives the blood filled inside the blood port into metering chambers (52, 53) [first metering chamber (52), second metering chamber (53)], while allowing excess blood to flow into a buffer zone (54).

3. The system as claimed in claim 1 wherein, said third mixing chamber (35) comprising a magnetic flea configured to be rotated at RPM of 5,000 to completely lyse RBCs from the metered blood.

4. The system as claimed in claim 1 wherein, a pressurized first vent (48), connected to an air solenoid valve, delivering positive pressure to drive blood from the first mixing chamber (33) into a second RBC (third) metering chamber (55, figure 11) and waste chamber (57, Figure 11) upon opening valves (28, 27).

5. The system as claimed in claim 1 wherein, a counting subsystem, configured to count RBCs and platelets, follows said optical detection system, in that, pressurized second vent (49) (Figure 7), delivering positive pressure to drive diluted blood from the second mixing chamber (34) into a biosensor (impedance measurement) (36) upon opening a valve (25), said biosensor comprising: a fourth chamber (59) in line with a counting chamber (58), configured to receive diluted blood, facilitating the counting of red blood cells (RBCs) as the diluted blood reaches said counting chamber (58), measurement beginning at an entry of said counting chamber (58) and measurement ending at an exit of said fourth metering chamber (59); a. optical detection windows within the counting chamber (58), serving as signal windows for liquid level detection based on the principle of light refraction.

6. The system as claimed in claim 1 wherein, said inclined optical window resembling a prism with angle of the prism between 30 and 60 degrees.

7. The system as claimed in claim 1 wherein, valves (23, 24) enable opening of a passage for transfer of diluted blook from said third mixing chamber (35) to said optical detection system upon the application of positive pressure on a third vent (50) connected to said third mixing chamber (35).

8. The system as claimed in claim 1 wherein, said biosensor featuring front and back side holes for fluid entry and exit, sealing of said biosensor on its front and back sides by a gasket with a wedge shape.

9. The system as claimed in claim 1 wherein, said cartridge comprising: a capillary tube (200) coated with Ethylenediaminetetraacetic acid (EDTA) for collecting blood; a designated blood port (3) for transferring the collected blood; a cap (7) configured to be pushed until it reaches a first stopper (4, 8) to prevent blood flow to a downward section; a capillary valve (6) integrated within the cap (7) to restrict movement of the collected blood based on capillary forces and geometry of the capillary valve (6) structure, wherein the capillary forces and the capillary valve’s (6) geometry act in tandem to prevent blood flow through the capillary valve, thereby functioning as a reliable stopper or barrier, said geometry being defined by a channel which is narrow in its initial section and expands in its further section to create a Laplace pressure barrier to stop flow unless extra pressure is applied externally.

10. The system as claimed in claim 1 wherein, an actuator (203, Figure 22) activates a microfluidic first valve (13, Figure 4), that stops blood (fluid) flow from flowing into other sections, of this system, except to a designated first metering section (11, Figure 4) and to a designated second metering section (12, Figure 4); a linear actuator (10) further pushes the cap (7, Figure 3) to push the blood sample (5, Figure 2) into two separate metering sections:

■ a first section (12, Figure 4) is dedicated to counting of RBCs and platelets (12, Figure 4),

■ a second section (11, Figure 4) is designed for WBCs count (11, Figure 4).

11. The system as claimed in claim 1 wherein, said cartridge comprising: blister-packed reagents (15) for convenient dispensing of liquids by pressing the blisters with actuators (10); a linear actuator that, when pressed against the blisters, increases the pressure inside the blisters, causing rupture of the bottom part of the blisters; a sharp conical pillar (14), positioned within the cartridge, facilitating controlled rupture of blisters by deflecting the bottom part of the blister, allowing efficient release of fluid into the microfluidic channel for subsequent analysis; a via (17) located beneath a foil part of the cartridge, establishing a fluidic connection between the microfluidic channel (13) and the metering section (11, 12) with the blister filled with reagents; an open microfluidic channel and said sharp conical pillars moulded directly on the cartridge; and a thin, soft, and flexible pressure-sensitive adhesive (PSA) layer of 50 to 200 microns thickness, with adhesive on one side, capable of closing the open microfluidic channel.