Mucosal-adhering polymer drug delivery composition and method

Abstract

A card reading system in which electrical contact between an assay card and a card reader is made through vertical movement of the card interface relative to the assay card. In one embodiment, the assay card reading system includes: (a) a card reader including at least one card interface having a plurality of card interface contacts; (b) an assay card having card contacts; and (c) a vertical movement mechanism causing relative vertical movement between the assay card and the card interface to bring at least a portion of the card interface contacts into contact with the card contacts.

Description

Cross - reference to related applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 527,659, filed on July 19, 2023, and U.S. Provisional Patent Application No. 63 / 599,854, filed on November 16, 2023. The entire disclosure of each of these is incorporated herein by reference.

Technical Field

[0002] The present disclosure generally relates to a test reader and a disposable microfluidic test cartridge / card, as well as a multiplexed microfluidic - based assay system including individual components, methods of using the same for performing diagnostic assays, and related test kits.

Background Art

[0003] Clinical laboratory testing plays a vital role in patient diagnosis, monitoring, and treatment by analyzing various types of biological specimens and providing actionable clinical results for physicians and nurses on the front lines of healthcare. Clinical laboratory testing in hospitals and other healthcare facilities is generally conducted in central laboratories, utilizing large, high-throughput equipment to batch process large numbers of clinical samples in parallel, in order to improve efficiency. However, traditional central laboratories have significant drawbacks, making it difficult to improve the quality, speed, and cost-effectiveness of clinical testing over the past few decades. To ensure the reliability of tests, clinical laboratories require the employment of highly trained staff on a 24-hour basis to implement rigorous, labor-intensive quality control and calibration procedures daily. The shortage of skilled personnel and the associated labor costs are significant burdens, potentially greatly increasing the cost of providing testing services, especially in low-capacity centers. Except for annual preventive testing performed after primary care visits or less common specialized tests performed in central satellite laboratories, most tests are conducted for acute patient care in hospitals, emergency departments, emergency care centers, and physicians' clinics. Timeliness of test results is crucial for patient care. However, centralized clinical laboratories typically experience long turnaround times, which can delay treatment decisions that impact patient outcomes and lead to delayed discharges. A balance exists between providing rapid test results and batching tests to manage costs. Turnaround times can vary depending on how quickly test samples can be transported to the appropriate central facility and the rules implemented to allow a certain number of samples to arrive before being processed as a batch. In hospital laboratories, high-throughput equipment designed to process hundreds or thousands of samples at once is often used to process only a small number of samples to avoid delays. When used in this manner, the efficiency and cost savings of using large equipment are completely lost. Finally, outpatient facilities such as physicians' clinics, surgical centers, and long-term care facilities lack access to testing unless they are located near a central laboratory.

[0004] Physicians have long spoken of revolutionizing healthcare with “near-patient” or point-of-care testing. Point-of-care testing platforms are portable in vitro diagnostic systems designed to be simple and easy to use (“low complexity”), controlling all sources of error so that anyone can achieve reliable results without extensive knowledge or training. Point-of-care testing can drastically reduce the turnaround time for test results and offers portability, allowing tests to be performed virtually anywhere, including outpatient facilities that would normally lack access to testing. Despite its many advantages, point-of-care testing has been used sparingly and has not evolved as rapidly as expected. This is due to several challenges. Disposable test cartridges / cards for point-of-care platforms often cost 10 to 30 times more than reagent packs for tests performed with conventional high-throughput testing equipment. Despite performing only one test at a time, point-of-care test readers still require a relatively large footprint and are expensive relative to their throughput. For example, operating multiple point-of-care devices to meet daily demand as an alternative to a central laboratory in facilities that perform a large number of tests daily is neither practical nor cost-effective. Finally, point-of-care testing platforms often offer only a very limited menu of tests with any given device. In such cases, dozens of different devices would be needed to replace the versatility of a central laboratory.

[0005] To overcome the limitations of existing platforms, point-of-care testing devices need to continue evolving to become more versatile laboratory alternatives. This need includes incorporating multiplexing capabilities that enable the performance of numerous tests using diverse testing modalities and configuring various test panels to be operational. Furthermore, this capability should be provided within a highly integrated, compact test reader and disposable test cartridge that utilize a small footprint and can both be manufactured easily and at low cost, making the platform scalable and cost-effective compared to central laboratory testing. At the heart of these requirements is the ability to precisely control small biological fluid samples within a complex microfluidic flow cell, meter the samples into many independently controlled microfluidic channels, and avoid the pitfalls that commonly affect the performance of tests using small, disposable microfluidic test cartridges. This invention addresses these needs, among others. [Overview of the project]

[0006] The following is a simplified summary of the invention to provide a basic understanding of some aspects of the invention. This summary is not a comprehensive overview of the invention. It is not intended to identify important / crucial elements of the invention or to describe its scope. Its sole purpose is to present some concepts of the invention in a simplified form as a prelude to the more detailed description to be presented later.

[0007] One aspect of the present invention is a card reading system in which electrical contact between an assay card and a card reader is made through vertical movement of a card interface relative to the assay card. In one embodiment, the assay card reading system includes: (a) a card reader including at least one card interface having a plurality of card interface contacts; (b) an assay card having card contacts; and (c) a vertical actuation mechanism that causes relative vertical movement between the assay card and the card interface to bring at least a portion of the card interface contacts into contact with the card contacts. In one embodiment, the vertical actuation mechanism is connected to the card interface and acts the card interface vertically.

[0008] Another aspect of the present invention is an assay card reading system having a two-stage alignment configuration. In one embodiment, an assay card reading system includes: (a) an assay card having an upper side, a lower side, and one or more edges, and including at least: (i) one or more first alignment reference marks on at least one of the edges; (ii) one or more second alignment reference marks on the upper or lower side; and (b) a card reader including at least: (i) a card bay for receiving assay cards; (ii) one or more first reference mark cooperative members along at least one side of the card bay to engage with the first reference marks and provide initial alignment of the assay card in the card bay; and (iii) at least one card interface above and / or below the card bay, wherein the card interface includes one or more second reference mark cooperative members, and the card interface has at least two states, in a first state the card interface is separated from the card bay, allowing assay cards to be received in the card bay; and in a second state the second reference mark cooperative members engage with the second reference marks on the assay card to provide final alignment of the assay card relative to the card interface.

[0009] A further aspect of the present invention is an assay card reading system that prevents improper insertion of test cards, accommodates cards of variable thickness and length, and / or synergistically holds and ejects cards. In one embodiment, the assay card reading system includes: (a) an assay card having sides and at least one first component of an interlocking mechanism along one of the sides; (b) a card reader including at least: (i) a card bay for receiving assay cards; and (ii) a second component of an interlocking mechanism for interlocking with the first component to hold the assay card in a predetermined position within the card bay, wherein the assay is positioned in the predetermined position by the interlocking mechanism and not by the register surface.

[0010] A further aspect of the present invention is an assay card reading system capable of accommodating cards of different thicknesses. In one embodiment, the assay card reader includes: (a) a card bay for receiving assay cards; (b) a card interface positioned above or below the card bay, wherein the card interface has a plurality of elastic connectors, and the card interface has at least two states, in a first state, the card interface is spaced apart from the card bay and the elastic connectors are at their fully extended length, allowing assay cards to be received into the card bay without contact with the card interface; and in a second state, the elastic connectors are in contact with the assay card and are at least partially compressed.

[0011] Another aspect of the present invention is an assay card reading system having one or more different heating zones with enhanced cooling and / or interlocks to prevent hot cards from being removed and to prevent new cards from being inserted while the reader is too hot. In one embodiment, the card reader includes: (a) a card bay for receiving assay cards; (b) a card interface located above or below the card bay and configured to move vertically, wherein the card interface has at least two states, in a first state in which the card interface is separated from the card bay, allowing assay cards to be received into the card bay, and in a second state in which the card interface is in contact with the assay card; and (c) at least one discrete heating zone within the card interface, which is in contact with the assay card and controls the temperature of the assay card when the card interface is in the second state. In one embodiment, the card interface includes at least two or more heating zones.

[0012] A further aspect of the present invention is, in one embodiment, an improvement in the manufacturability of the card by using a uniform body. In one embodiment, the assay card comprises: (a) first and second layers, the first layer covering the second layer and defining a space between them; (b) microfluidic channels within the space; and (c) the second layer and channels formed within a unified body.

[0013] A further aspect of the present invention is a capillary filament destroyer positioned within a microfluidic channel to destroy capillary filaments. In one embodiment, the card comprises: (a) at least first and second layers, the second layer defining at least one channel, the first layer covering the second layer, thereby defining at least one corner within the channel between the first and second layers; and (b) one or more capillary filament destroyers positioned within the at least one corner to destroy capillary filaments.

[0014] Another aspect of the present invention is a lateral flow enhancer for regulating flow in a microfluidic channel. In one embodiment, the card comprises: (a) a base; (b) at least one microfluidic channel in or on the base for guiding fluid flow, the microfluidic channel being defined by side walls 804 and a width 805 between the side walls; and (c) one or more lateral flow enhancers. Each lateral flow enhancer extends over at least a substantial portion of the width and causes a lateral capillary action perpendicular to the flow, so that the fluid flow flows to the side walls in the width direction of the channel before passing through the lateral capillary enhancer, thereby creating flow throughout the width direction of the channel. In one embodiment, the lateral flow enhancer extends completely over the entire width.

[0015] Another aspect of the present invention is capacitive monitoring of flow in a microfluidic channel for high-resolution metering and / or for the detection of previously wet test cards and / or the reuse of test cards with insufficient capacity. In one embodiment, the card includes: (a) a base; (b) at least one microfluidic channel defined in or on the base, wherein the channel is configured to guide fluid from a port; and (c) two or more capacitive elements arranged in a contiguous manner along the channel, wherein the capacitive elements are electrically connected by conductive traces outside the channel, and a discrete increase in capacitance is detectable as fluid flows from one capacitive element to a contiguous capacitive element. In one embodiment, the two or more capacitive elements include more than three contiguous capacitive elements.

[0016] Other aspects and embodiments of the present invention will be apparent to those skilled in the art in light of this disclosure. [Brief explanation of the drawing]

[0017] [Figure 1] Figure 1 is an external perspective view of one embodiment of the assembled device.

[0018] [Figure 2A] Figure 2A is a front view of one embodiment of the assembled device.

[0019] [Figure 2B] Figure 2B is a vertical cross-sectional view (section AA) passing through the center of one embodiment of the assembled equipment.

[0020] [Figure 3A] Figure 3A is a perspective view of one embodiment of the core module assembly.

[0021] [Figure 3B] Figure 3B is a front view of one embodiment of the core module assembly.

[0022] [Figure 3C] Figure 3C is a downward view of a horizontal cross-section (section A-A) of an embodiment of the core module assembly.

[0023] [Figure 3D] Figure 3D is an upward view of a horizontal cross-section (section B-B) of an embodiment of the core module assembly.

[0024] [Figure 4A] Figure 4A is a top view of an embodiment of the core module assembly showing the cutting line for subsequent vertical cross-sectional views.

[0025] [Figure 4B] Figure 4B is a view of a vertical cross-section (section A-A) through an embodiment of the core module assembly with the platform in the fully open position.

[0026] [Figure 4C] Figure 4C is a view of a vertical cross-section (section B-B) through an embodiment of the core module assembly with the platform in the intermediate position.

[0027] [Figure 4D] Figure 4D is a view of a vertical cross-section (section C-C) through an embodiment of the core module assembly with the platform in the fully closed or clamped position.

[0028] [Figure 4E] Figure 4E is a top view of an embodiment of the core module assembly with two independent card interfaces showing the section line for subsequent vertical cross-sections.

[0029] [Figure 4F] Figure 4F is a view of a vertical cross-section (section D-D) through an embodiment of the core module assembly with two independent card interfaces and the platform in the clamped position.

[0030] [Figure 4G] Figure 4G is a top view of an embodiment of a long assay card that can interface with a rear card interface from a core module assembly, which has two independent card interfaces.

[0031] [Figure 5A] Figure 5A is a side view of one embodiment of a core module assembly showing a latch in a locked position that is not ready to accept the insertion of a test card.

[0032] [Figure 5B] Figure 5B is a horizontal cross-sectional view (section AA) through one embodiment of the core module assembly, showing that the latch is locked and preventing the insertion of a test card.

[0033] [Figure 5C] Figure 5C is a side view of one embodiment of a core module assembly showing the latch unlocked and the test card partially inserted.

[0034] [Figure 5D] Figure 5D is a horizontal cross-sectional view (section BB) through one embodiment of a core module assembly showing a latch acting outward to engage with a partially inserted test card.

[0035] [Figure 5E] Figure 5E is a side view of one embodiment of a core module assembly showing the latch unlocked and the test card fully inserted.

[0036] [Figure 5F] Figure 5F is a horizontal cross-sectional view (cross-section CC) through one embodiment of a core module assembly showing a latch that captures a test card fully inserted through a notch.

[0037] [Figure 6A]Figure 6A is an exploded view of one embodiment of a microfluidic test card showing individual layers.

[0038] [Figure 6B] Figure 6B is an enlarged exploded view of one embodiment of a microfluidic test card, showing individual layers trimmed to focus on a series of comb-shaped capacitive elements.

[0039] [Figure 7] Figure 7 is a top view of one embodiment of a microfluidic test card, including a magnified view of its main features.

[0040] [Figure 8A] Figure 8A is a perspective view of one embodiment of a microfluidic test card, including a magnified view of its main features.

[0041] [Figure 8B] Figure 8B is a top view of one embodiment of a microfluidic test card with a channel support, including an enlarged view to highlight the channel support features.

[0042] [Figure 9] Figure 9 is a graph showing the heating time on a clamp / closed platform and the cooling time on a clamp / closed vs. open platform.

[0043] [Figure 10A] Figure 10A is a graph showing an example of the capacitive response from a test sample traveling across a series of consecutive capacitive elements.

[0044] [Figure 10B] Figure 10B is a graph showing an example of the derivative of the capacitive response from a test sample traveling across a series of consecutive capacitive elements.

[0045] [Figure 10C] Figure 10C shows an example of a test sample traveling through a microfluidic channel across a series of consecutive comb-shaped capacitive elements. [Modes for carrying out the invention]

[0046] In the following paragraphs, the present invention will be described in detail illustratively with reference to the accompanying drawings. Throughout this description, preferred embodiments and examples shown should be considered illustrative rather than limiting. As used herein, “the present invention” refers to any embodiment of the invention described herein and any equivalent. Furthermore, references to various features of “the present invention” throughout this document do not imply that all claimed embodiments or methods must include the features referenced.

[0047] Next-generation point-of-care laboratory alternative platforms are required to be low-cost and compact, yet provide highly multiplexed inspection panels with advanced fluid control, achieving performance equivalent to centralized inspection. However, existing systems have significant limitations that have hindered the achievement of these goals. Low-cost platforms offer only simple fluid structure and basic analysis functions. On the other hand, platforms with advanced functions typically use extremely expensive, large injection-molded inspection panels. Given this situation, there is a need for innovative new strategies to achieve high multiplexing while incorporating advanced fluid control in a low-cost form factor.

[0048] Referring to Figure 1, one embodiment of a small, compact point-of-care testing device is shown in a perspective view. In this embodiment, the device has an external housing 100 through which an assay card 101 is inserted into the device. This portable device also has a battery compartment 102 and a display 103.

[0049] Referring to Figure 2A, an embodiment of the point-of-care device is again shown in a front view. The front of the external housing includes a slot 201 for a test card to enter the card bay. The entry of the test card through the slot is prevented by a large latch 200 on one side and a small latch 202 on the other side. The front end of the test card may have tapered corners on each side, large enough to clear the corresponding latches for the test card to enter the card bay. As seen in the vertical cross-section through the device in Figure 2B, the device includes a core module 203, which is a self-contained mechanical assembly capable of accepting an assay card upon insertion through the slot 201 in the external housing and performing highly multiplexed functions required by an advanced diagnostic platform; a control electronic board 204 that interfaces with both the core module and the display and controls an onboard operating system with a user interface; an optical barcode scanner 205; a cooling fan assembly 206 for cooling the core module during operation; and a replaceable removable battery 207 in a battery compartment.

[0050] Electronic connection between the test card and the card reader

[0051] A major limitation of typical microfluidic devices is the number of electronic connections that can be made between the card reader and the test card. As a result, advanced devices requiring numerous connections per assay channel have limited multiplexing capabilities. For example, high-resolution metering is key to advanced fluid control, but requires multiple sensing points within each microfluidic channel connected to the card reader. Each electrochemical assay also requires at least two electrodes, all connected to the card reader. Current design limitations necessitate a complex weaving of conductive traces on the test card to ensure each conductive trace can be traced to a connector for interface with the test system. However, because trace paths cannot overlap or intersect with each other, there are practical limitations on the number of connections that can be traced to the edge of the test card and interface with typical edge connectors. Few devices utilize island contacts—contacts that interface with the instrument but are not located at the edge of the test card and are not intended for edge connectors. A reliable method is needed to interface with island contacts to enable connections across the entire area of ​​the test card. An object of the present invention is to disclose a compact test system that can provide a reliable interface for multiple independent connections between a card reader and a test card, including island contacts.

[0052] Compared to a typical card reader for a diagnostic platform, the disclosed device, in one embodiment, does not include an edge connector for contact with the test cartridge during insertion. In fact, in one embodiment, no physical connection is initially made between the card reader and the test card when the user inserts it into the card bay. The disclosed system, in one embodiment, includes a card interface and utilizes a mobile platform located above the card bay that accepts the test card. The card interface on the platform, in one embodiment, includes an array having multiple rows of spring-loaded pin connectors (also known as "pogo pins"). The spring connectors are assembled in a grid on a printed circuit board (PCB). Standard PCB manufacturing methods can achieve tolerances of ±100 microns or less for the mounting position of the pin connectors on the grid. In addition to the connectors, in one embodiment, two alignment pins with conical tips are also mounted on the PCB during assembly so as to align with the position of the pin connectors on the array. In one embodiment, the mobile platform has at least two states, in a first state, the platform is positioned away from the card bay, allowing the test card to be inserted into the card bay, and in a second state, the platform is positioned toward the card bay, allowing the pin connector to contact the test card. In one embodiment, two motor-driven lead screws actuate the platform between the first and second states. Pins on the platform can connect to conductive contact pads screen-printed with carbon and / or silver ink in a corresponding grid pattern across the entire test card. In one embodiment, island contacts can be positioned across the entire assay card because contact is made from above the entire test card. In one embodiment, the contact pads on the test card are screen-printed aligned to two alignment holes, which are through-holes cut out from the upper and base layers of the test card. In one embodiment, the connection between the pin connector and the contact pads on the test card is made through openings or holes cut out only in the upper layer of the test card that overlap each contact pad.In other embodiments, connections may be made via vias. In one embodiment, the test card is initially inserted and captured by a latching system, which provides preliminary alignment of the test card to a tolerance of approximately ±1 mm from the nominal position along the X and Y axes. This ensures that the test card is positioned appropriately for the alignment pins on the platform to engage with the alignment holes. Larger misalignments could damage the card reader or the test card if the alignment pins disengage from the holes and pierce the test card. In one embodiment, once the test card is captured, the moving platform descends to allow the alignment pins to engage with the alignment holes, which provides fine adjustment of the test card's position based on the tight mating of the alignment pins in the alignment holes in the base layer of the test card. The alignment holes in the upper layer may be oversized relative to the holes in the base layer, so that misalignments between the base and upper layers of the test card do not affect the alignment of the pins relative to the contact pads on the base. Alignment pins provide alignment between the contact pads on the base and the connector array on the platform, ensuring that each pin connector aligns with the center of the contact pads for contact. Furthermore, the alignment pins may also provide fine alignment between the optical aperture on the test card and the optical sensing element on the card reader. In one embodiment, when the moving platform stops in a second state and the alignment pins are fully engaged, the fine adjustment of the test card's position by the alignment pins aligns the contact pads on the base of the test card and the connector array within a tolerance of approximately ±0.5 mm from the nominal value in both the X and Y axes. This ensures that each spring pin connector contacts the corresponding contact pad based on the relative size of the contact pads. While the connectors on the platform should align with the contact pads on the base of the test card, the upper layer openings or holes also need to be aligned to allow access to the contact pads. The contact pads are nominally located directly below the upper layer openings or holes of the test card.For the lamination of the base and upper layers of the test card, an alignment tolerance of approximately ±0.4 mm in the X and Y axes is sufficient to ensure successful contact. This level of tolerance can be easily achieved in a typical high-speed manufacturing process utilizing machine vision and / or other common alignment techniques during lamination. Upon contact with the connector array, the contact pads on the base can be used to transmit signals from capacitive sensing elements, short-circuit detection elements, assay electrodes, and / or other electronic signals through the pin connectors. Using island contacts dramatically increases the usable space on the test card because a large number of connections can be made across its entire surface area. Furthermore, this approach can significantly reduce trace length, reduce leakage, and improve the quality of signals to the devices. In one embodiment, 46 pin connectors are arranged in two separate thermal heating zones across the entire array on the platform. Thus, the test card will have up to 46 corresponding contact pads and associated openings.

[0053] Two-stage alignment of the test card and card reader.

[0054] Certain functions of a card reader for a diagnostic platform, such as pin connectors or optical detection systems, require precise alignment of the test card after insertion into the card bay. In a typical in vitro diagnostic device, mating of the test card within the card bay physically aligns the test card with the card reader. More specifically, mating of the test card's edge or corner with the side or corner of the card bay provides the alignment. However, in such cases, the alignment tolerance of the system depends on the precise assembly of the card bay with respect to other components within the card reader (such as pin connectors or optical detection systems), which may not be directly facing each other, and also on the isolation tolerance of each individual test card. This becomes cumbersome in highly multiplexed platforms with numerous components requiring precise alignment. Physical alignment of the test card based on mating into the card bay limits the test card dimensions to a universal size. An object of the present invention is to disclose the design of a test system that automatically aligns test cards of various lengths after insertion into the card reader by the user.

[0055] In one embodiment of the disclosed device, alignment between the card reader and the test card is performed in two stages. For example, the test card is first inserted into the card bay in a sliding motion, and latches on each side of the card bay engage with notches on each side of the test card to capture the test card. In one embodiment, the latches engage with the notches to capture the card in its initial position, thereby providing preliminary alignment within nominal ±1 mm along the X and Y axes. This level of alignment is sufficient for conical alignment pins to engage with alignment holes on the test card without the risk of puncturing the test card due to serious misalignment. Subsequently, in one embodiment, a moving platform is lowered, and the alignment pins engage with alignment holes in the base layer of the test card. The tightly tolerated mating of the alignment pins in the alignment holes provides fine adjustment of the position of the test card relative to the platform, including the card interface. In one embodiment, the engagement of the notches by the latches allows for some movement so that fine adjustments by the alignment pins can adjust the position of the latches in the notches. In one embodiment, the first alignment hole provides constraint in two dimensions, while the second alignment hole provides constraint in only one dimension. In one embodiment, the first alignment hole in the test card is circular, and the second alignment hole in the test card is circular at the ends but slightly elongated along one axis, so that the alignment pin is not constrained within the alignment hole along its one axis. Unlike typical devices that align a card reader to a test card based on the mating of the test card in a card bay, the final alignment of the test card in the disclosed system is provided, in one embodiment, solely by alignment pins on a platform including the card interface. Thus, the mechanical position of the card bay during assembly only affects the initial alignment and does not determine the final position of the test card relative to the platform.The platform includes a PCB, which is assembled with alignment pins in a precise alignment manner, along with all other components that interface with and need to be aligned with the test card, such as pin connectors. In one embodiment, an optical detection system is also mounted on the PCB to provide alignment between the optical components and the optical aperture on the test card. A standard PCB assembly method can achieve an alignment tolerance of ±0.100 mm, thereby ensuring that all critical elements of the card reader that interface with the test card are precisely aligned with the alignment pins on the platform. When the alignment pins engage with the alignment holes on the test card, this alignment mechanism ensures that the card interface is precisely aligned based on the alignment to the base layer of the test card. During the manufacturing of the test card, the alignment holes are aligned to within ±0.150 mm of the positions of screen-printed elements such as contact pads. Alignment pins are aligned to the pin connectors on the PCB, so the alignment process ensures that the pin connectors and contact pads are aligned to a tolerance of less than ±0.500 mm, guaranteeing that the pin connectors make contact with the contact pads. Unlike most test systems, alignment does not rely on mating the edges or corners of the test card to the sides or corners of the card bay, neither of which is aligned to any specific feature on individual test cards or to pin connectors on the platform. Alignment using this technique is achieved by utilizing features that can be manufactured with the best possible alignment tolerance. For example, screen printing has an alignment tolerance of within ±0.150 mm, and mounting the pin connectors by PCB assembly on the platform can be achieved within ±0.100 mm. Conversely, the mechanical assembly of the equipment housing and card bays is not precisely aligned to any of these features (such as features on the platform) that require precise alignment. Implementing a manufacturing and assembly process that can align such features to the required tolerances can be prohibitively expensive or even impossible.One embodiment of the connector array includes, for example, 46 pogo pins that form connections with contact pads on a test card. One embodiment of the optical system includes eight optical detectors that align with optical apertures on a test card. The number of features requiring alignment in a highly multiplexed test system motivated the disclosed invention to achieve alignment between the device and many different elements of the entire test card.

[0056] The card reader accepts variable card length and thickness and prevents buckling.

[0057] For a single diagnostic platform to perform a wide range of diagnostic tests, from blood chemistry tests to highly sensitive immunoassays and nucleic acid amplification, it is desirable that the device be flexible enough to accept test cartridges or cards of various form factors. For example, low-cost microfluidic test cards manufactured using a roll-to-roll method typically use thin films (0.3–1 mm) that can be wound and loaded on an automated roll-to-roll production line. While there are advantages to manufacturing test cards using roll film, thin and flexible test cards present certain challenges. Specifically, thin test cards, especially large ones, are prone to bending or buckling during insertion. Some diagnostic tests may require metallized blister packs or injection-molded parts with a larger minimum thickness (0.5–3 mm). Similarly, optimizing the size of test cards to accommodate the number of assays required for a test panel is usually advantageous. Smaller form factors can maximize throughput and reduce manufacturing costs. Complex test panels with numerous reagents and microfluidic channels require more space than simpler tests with fewer reagents and a single microfluidic channel. Furthermore, while some test panels can be run at a single reaction temperature, others, such as nucleic acid amplification, require multiple temperature zones to rapidly shuttle the test sample between various reaction temperatures. An object of the present invention is to disclose a test system design that can utilize test cards with a thickness of at least 0.3–3 mm and a length of at least 40–75 mm, with longer test cards operating across two or more independently controlled and thermally isolated temperature zones. An object of the present invention is to disclose a test system design that can prevent buckling of flexible, thin test cards.

[0058] In one embodiment, the disclosed test system implements a platform including a card interface, which can be moved vertically to a position to accept test cards of varying thicknesses up to at least 3 mm into a card bay, depending on the available space inside the device and the operating range of the spring connector on the card interface. In one embodiment, the test system includes a shutter located behind the front insertion opening of the housing and leading to the card bay. The shutter is biased downward by a leaf spring and configured to block the insertion opening. The shutter may further have a chamfered edge, which engages with the front end of the test card, causing the test card to lift and retract the shutter, allowing test cards of different thicknesses to enter the card bay. In one embodiment, the leaf spring on the shutter can be compressed to allow test cards of the same thickness as the entire height of the entry slot to be inserted through the entry slot. The downward force of the shutter on the test card shutter biases the test card downward, preventing buckling of the test card during insertion. The shutter has the additional function of preventing light from entering the equipment housing. In other embodiments, the shutter may be a roller similarly biased downward by a leaf spring. The rounded shape of the roller is similar to the chamfered edge in that the leading edge of the test card wedge into the roller, lifting the roller against the leaf spring and allowing the test card to be inserted. While the shutter provides a downward force at the front entry of the device, an additional leaf spring descending from a platform above the card bay to the rear of the card bay may also bias the test card downward until it is fully seated in the device, preventing buckling or improper positioning of thin, flexible test cards. These features may be important to allow test cards as thin as 0.3 mm to be inserted into the device. For example, initially, the leaf spring is fully extended and in contact with the bottom surface of the card bay, and therefore engages with test cards of any thickness entering through the entry slot.When the platform is compressed on the test card, the leaf spring can be fully compressed and become flush with the platform surface. The rear leaf spring also provides a downward force on the test card as the platform rises and pulls the alignment pins out of the alignment holes in the test card. Without the rear leaf spring, the test card could get caught on the alignment pins and rise with the rising platform. The absence of an edge connector means there is no shear force necessary to engage the spring connector during insertion, which would otherwise cause the thin test card to bend or buckle.

[0059] In one embodiment, when a test card is in a predetermined position, a moving platform descends onto the test card from above and engages with the test card at alignment pins to optimize the position of the test card relative to an array of pin connectors on the card interface to establish an electronic connection. In one embodiment, the card interface includes spring-biased pin connectors that are initially fully extended when the test card is inserted and are at least partially compressed as the platform converges onto the test card. The spring connectors have an operating range such that they can be flush with a heating plate on the platform. The amount each spring connector is compressed depends on whether all spring connectors engage with the base of the test card through an opening. If all spring connectors enter through the opening and engage with the base, all spring connectors should be compressed to their solid length. If some spring connectors engage with the top of the test card and do not contact the base through the opening, only the spring connectors that are in contact with the top of the test card are fully compressed to their solid length. As a result, the spring connectors that enter the base through the opening are partially compressed by an amount depending on the thickness of the top layer of the test card. The operating range of the spring connector defines the maximum thickness of the upper layer of the test card that can be used when no portion of the spring connector penetrates the opening.

[0060] A typical diagnostic test system utilizes a physical guide and spring to restrain the test card from its front edge during insertion, requiring a universally sized test card. This also emphasizes the assembly of the physical guide and spring within the instrument housing to ensure that the device's alignment tolerances are met. This can be challenging because there is often no physical connection between the guide and the instrument features (e.g., pin connectors or optical components) that the test card will ultimately align with after insertion. Instead, the disclosed test system, in one embodiment, uses a latch to capture the test card through a notch cut into the side of the test card. Because there is no physical restraint based on the front edge of the card, test cards of various lengths can be used. Although there is physical restraint from the side of the test card by the latch, the latch system only provides preliminary alignment of the test card. Final alignment comes from alignment pins on the card interface itself. The alignment pins descend with the platform, fine-tuning the position of the test card so that it aligns with connectors on the card interface and optical systems on the platform. The disclosed system, in one embodiment, also has at least two independent temperature zones. Each zone has one set of thermally isolated heating plates at the front and one set at the rear. Tests requiring dual temperature control are lengthened to extend from the first temperature zone to the second temperature zone. For example, a test card up to 55 mm in length extends across one set of upper and lower heating plates in the first temperature zone, while a test card longer than 55 mm extends into the second temperature zone, so that the temperature of the front of the test card is thermally controlled by the heating plates in the first temperature zone, and the temperature of the rear of the test card extends into the second temperature control zone. If only one temperature zone is required, the test card may be 55 mm or less, and manufacturing throughput is maximized by placing more cards per sheet or roll.

[0061] The card reader prevents the insertion of test cards in the wrong orientation.

[0062] For a diagnostic platform to be simple for untrained users, the goal is to design controls to prevent improper use. For example, the test system should be optimally designed to prevent test cards from being inserted in the wrong orientation. The object of this invention is to disclose a test system design that prevents improper insertion of test cards (upside down or backward), which would not allow proper functioning.

[0063] In one embodiment, the disclosed device utilizes a latching system that captures a test card and also prevents improper insertion of the test card. In addition to capturing the test card by engaging with a notch on the side edge of the test card, the latch also acts as an obstacle to improper insertion of the test card. In other embodiments, the latching and obstacle functions can be achieved by two separate components within the device. The latch is a leaf spring that is compressed when it acts outward from the card bay and then springs back towards the card bay to return to a relaxed state. The leaf spring partially blocks the slot, which is the entrance to the card bay, so that a test card with typical square corners cannot be physically inserted. In one embodiment, the front corner of the test card has a taper. The tapered corner of the test card engages with the leaf spring of the latch, compressing the leaf spring and displacing it outward from the card bay. In this way, the front end of the test card can clear the latch and advance the test card completely into the device. In other embodiments, the shape of the front end of the test card may simply be such that it physically clears the obstacle at the front of the card bay without requiring the obstacle to be physically displaced or actuated. For example, a slit at the front end of the card may be aligned with the obstacle so that the obstacle is cleared only when the front end is properly inserted. In some embodiments, the obstacle may be actuated outward from the card bay by an automatic mechanism when the test card is detected. In one embodiment, in addition to being an obstacle, the primary function of the latch is to engage with a notch on the side of the test card to capture the test card. When the tapered corner clears the leaf spring and displaces it outward in a compressed position away from the card bay, the compressed leaf spring continues to slide along the side edge of the test card as it is inserted and reaches the notch. The leaf spring then rebounds and extends inward, engaging with the notch and capturing the test card in place. This ensures that the test card stops in place during insertion based on the position of the notch. Since the test card is captured only by the side latch and there is no physical backstop to the card bay, test cards of various lengths can be inserted into the card bay.In fact, the test card does not necessarily need to be in contact with the side of the card bay as long as the latch can engage. In some embodiments, the predetermined position for capturing the test card needs to be set so that the alignment pins can properly engage with the alignment holes on the test card. Thus, the latch also provides preliminary alignment. Improper positioning of the test card can cause the alignment pins to pierce the test card, potentially damaging either the test card or the card reader. In addition to capturing the test card and providing preliminary alignment, the leaf spring centers the test card within the card bay. In one embodiment, only the front end of the test card, and not the rear end, has a tapered corner. If the test card is inserted backward, the square corner at the rear end cannot engage with the leaf spring and clear it. Thus, the latch prevents the test card from being inserted backward. Furthermore, the latch and taper on one side of the card bay and the test card are larger than those on the opposite side. A larger latch leaf spring provides a correspondingly larger obstruction and requires a larger taper to engage with the leaf spring and clear it. In one embodiment, the smaller taper on the opposite side of the test card cannot clear the obstruction from the larger latch spring. This prevents the test card from being inserted upside down, and prevents the smaller taper from clearing the larger latch.

[0064] Thermally isolated temperature control zone

[0065] Diagnostic tests often require strictly controlled reaction temperatures to achieve consistent performance. For example, the dynamics of an enzyme in a particular assay may increase with reaction temperature up to an optimal temperature, which is generally close to body temperature (37°C). Other reactions, such as isothermal amplification, perform best at higher temperatures of around 65°C. Some reagents are highly temperature-sensitive and may be inactivated if heated above a certain temperature. In one specific example, heating a saliva or nasal swab sample at 95°C for 5-10 minutes can be used as a method to inactivate the proteinase enzyme introduced to inactivate the nuclease enzyme. This two-step method avoids the need for sample extraction. However, the heat-inactivated sample needs to be rapidly cooled to a low temperature for nucleic acid amplification. For a test system to be practical for use in multiple tests at significantly different reaction temperatures and individual tests requiring rapidly changing reaction temperatures, the test system needs to have multiple thermally isolated temperature zones with large temperature gradients between them. The device also needs to have the ability to cool rapidly to avoid long delays between tests while the system reaches an acceptable temperature for the next test. The test system also needs to be able to prevent the removal of test cards that may become hot, and to prevent the insertion of test cards that may be damaged by high temperatures before the system has cooled down. An object of the present invention is to disclose a test system that incorporates at least two independent temperature zones with a temperature difference of at least 40°C between the zones and can be rapidly cooled from 95°C to 35°C within 3 to 5 minutes. Another object of the present invention is to disclose a method for preventing the insertion or removal of test cards when the device temperature is outside the acceptable range.

[0066] The disclosed test system, in one embodiment, has at least two thermally isolated temperature zones, each having upper and lower aluminum heating plates, each having a closed-loop temperature control system with temperature sensors. The temperature zones are thermally isolated. Thermal isolation is achieved, in one embodiment, by a structural assembly utilizing a plastic support that does not have a thermally conductive material connecting the two zones. The plastic support, also called a plastic side slider, is a thermally insulating (non-conductive) component that holds the platform together and limits heat transfer between the temperature zones. The flexibility of the plastic side slider is also important to allow slight misalignment of the threaded screws of each of the two motors. The flexible slider allows for quick adjustment to prevent jamming of the lead screws and motors that move the platform vertically up and down. If one motor moves faster than the other and becomes out of sync, the plastic side slider can bend slightly to prevent jamming. When the platform bottoms out at the bottom of the threaded screws, the two motors synchronize again. The plastic side slider connects the moving platform and temperature zones without having thermally conductive metal contacts between the zones. In one embodiment, even when heated to 95°C, the second temperature zone can be maintained below 60°C, and both zones can be cooled to the device's standard operating temperature of 35°C within approximately 5 minutes. Airflow within the test system is optimized with a high-flow fan positioned behind or below the assembly. The airflow moves through the center of the assembly between the upper and lower heating plates to maximize cooling. When the moving platform is closed or clamped, there is less airflow between the heating plates. Airflow and cooling rate are increased by maximizing the distance between the two heating plates in the open or unclamped state, where the moving stage is at its furthest position. Referring to Figure 9, heating and cooling times were experimentally tested.After heating the temperature zone to 95°C and then initiating cooling, when the mobile platform was in the open position and the upper heating plate was separated from the lower heating plate to allow maximum airflow between the plates, the temperature zone cooled down to 37°C, up to 45% faster compared to when the mobile platform was in a clamped state with no space between the upper and lower plates.

[0067] In one embodiment, when the mobile platform is in a closed or clamped position during inspection, the test card is compressed between the platform and a stage below the card bay. This compressive force prevents the test card from being removed while it is very hot. The compressive force also protects the alignment pins and pin connectors on the platform. In one embodiment, when the platform moves to its farthest position to maximize cooling, the platform pushes the lock down and engages with the latch system, preventing the latch from operating. The latch lock prevents the insertion of the test card when the system is not ready to accept a new inspection, for example, when the card bay is still very hot. The lock is also connected to the mobile platform by a spring. In one embodiment, when the test system is ready to accept a new test card, the platform moves to a slightly less separated position that puts tension on the spring, thereby disengaging the lock from the latch. This position is considered the first state, as the card reader is ready to accept the test card into the card bay. In one embodiment, the test device can detect the insertion of the test card based on the position of the latch leaf spring, which is monitored by a Hall effect sensor. Hall effect sensors and magnets monitor the physical position of the latch leaf spring during the process of inserting a test card. The sensors can detect when the leaf spring is compressed by engaging with a taper on the test card during insertion, and also when the leaf spring rebounds and returns to its extended position to engage with the notch when the test card is fully inserted. Similarly, they can detect when the latch disengages from the notch during the removal of the test card. Information from the Hall effect sensors regarding the insertion or removal of the test card can be used to determine the timing of locking and unlocking the latch. For example, a moving platform is initially positioned to unlock the latch while the previously used test card is cooling until it is removed, but then moves further apart to lock the latch and prevent the insertion of a new test card until the heating plate has cooled.

[0068] A pneumatic system for controlling active fluid dynamics and preventing overflow from flow cells.

[0069] Advanced active fluid engineering is crucial for precisely controlling metering within flow cells and for resuspending and mixing reagents within flow cells to perform complex diagnostic tests. Active fluid engineering can be achieved with internal (self-contained) or external pneumatic systems. While there are potential advantages to self-containing the pneumatic system within a disposable test card, internal systems generally increase the unit cost of disposable test cartridges and may have functional limitations. In external pneumatic systems for active microfluidics, the system needs to be designed to prevent overflow of sample fluid volume from the disposable test card to the external pneumatic pumping system on the card reader. The object of this invention is to disclose an external pneumatic pumping system for active microfluidics that can prevent overflow of fluid samples.

[0070] The disclosed test system includes, in one embodiment, an air manifold assembly that provides a conduit for transmitting pneumatic pressure from a pump through a valve and then to a gasket that interface directly with a small port on a test card. In one embodiment, the small pneumatic port is led directly from an air channel or conduit that connects to a microfluidic flow cell on the test card. The air manifold assembly houses a pump, valve, and pressure sensor that form an external active pneumatic pumping system on the device. The pump and valve are held in place so that they can be directly surface-mounted to connect to openings on the manifold, and the manifold has internal air channels for the pneumatic system that connect the pump and valve to the gasket. The gasket may be made of a flexible material such as silicone or rubber. Using silicone has the advantage of temperature and chemical stability. The gasket is positioned above or below the test card depending on the location of the air manifold in the device and forms a seal around each of the small pneumatic ports that make connections to the microfluidic channels in the test card. The gasket seal is maintained by the force of a moving platform up to, for example, a force of up to 10 kg or the force required to compress and maintain the airtight seal. Manifolds can be manufactured using different manufacturing processes such as injection molding, CNC milling, or laser cutting.

[0071] In one embodiment, the external pneumatic system is terminated with a silicone gasket that is compressed onto the surface of the test card to form a seal around a small pneumatic port. Therefore, in this embodiment, since the external system is not disposable, it is important to prevent the fluid sample from entering the pneumatic system starting from the gasket. To prevent overflow of the fluid sample on the test card, the fluid sample may be monitored with capacitive sensing, which includes a fill detection, a capacitive sensing element located at the end of the microfluidic channel indicating that the channel is fully filled. In one embodiment, when the fill detection detects fluid at the end of the assay channel, a feedback loop triggers the system to close a valve, shutting off the pneumatic system at the port leading to that channel to prevent overflow. The microfluidic flow cell and the gasket-sealed pneumatic port hole are connected by narrow air lines or air conduits, which, in one embodiment, are at least five times smaller than the microfluidic flow cell to increase flow resistance. This flow resistance limits the filling rate of the air lines to give the system time to shut off the pneumatic system before the fluid sample reaches the external gasket. In some cases, a single pneumatic port can be used to control multiple microfluidic channels within a flow cell. Each channel needs to be fully filled before the pneumatic system leading to its port can be shut off, but it is unlikely that both channels will complete filling at exactly the same time. Therefore, the air line is used as an overflow chamber to ensure that all channels complete filling without overflowing before the pneumatic pump is shut off. For the fastest-filling channels, the length of the air line may be increased to increase flow resistance and overflow capacity. An additional overflow chamber can also be included within the air line to provide more capacity for overfilling before the sample reaches the gasket. Another strategy to prevent channel overfilling is to terminate the channels with a hydrophobic sticker, such as an expanded PTFE membrane, which allows air to pass through but not liquid. PTFE filters can also be incorporated into an external pneumatic system.

[0072] Microfluidic flow cell without PSA film spacer

[0073] Typical diagnostic test specimens containing microfluidic channels, such as glucose test strips, are manufactured with a three-layer structure, consisting of (1) a pressure-sensitive adhesive (PSA) film spacer sandwiched between two flat plastic films, (2) a plastic base film, and (3) a plastic top film. Microfluidic channels are formed by cutting voids in the PSA film spacer, with the voids within the PSA film functioning as spaces continuous with the sample inlet port. The depth of the microfluidic channels forming the flow cell is determined by the thickness of the PSA film spacer. However, the use of PSA films to form microfluidic flow cells has significant limitations, particularly in increasingly complex test card designs. PSA films readily stretch and deform, which can make it difficult to accurately align the films during lamination. This is especially true for highly multilayered test cards with many features that need to be well aligned between multiple layers. The stretching and deformation of PSA films can also alter the dimensions of the microfluidic channels they form, potentially reducing the accuracy and reproducibility of the assay. The integrity of features formed by voids within a PSA film can be difficult or impossible to maintain if substantial portions of the film are cut and removed, making it even more flexible and delicate. For example, many voids are needed within a PSA film to form a complex series of microfluidic channels and create openings for platform connectors. Too many voids can cause the PSA film itself to simply collapse when lamination is attempted. Furthermore, the PSA film must be applied as one completely continuous film during lamination, and therefore features that introduce a break in the continuity of the PSA film cannot be formed in this manner. For example, a small air channel connecting two adjacent microfluidic channels from one flow cell to a single pneumatic port opening forms a discontinuous island. A PSA film that is not continuous with the remaining film after die-cutting will simply collapse after cutting.Such a design can only be manufactured using multiple film layers so that the additional film layers act as supports, but this cannot be done without introducing additional cost and complexity. Each additional layer is a separate surface that must be precisely aligned and laminated during manufacturing. For example, three film layers require two lamination steps, each introducing some degree of misalignment between layers. Each additional layer requires at least one additional lamination step, which is added to the stacking of misalignment tolerances. An object of the present invention is to disclose an in vitro diagnostic test card incorporating microfluidic channels formed in a single, unified body or film without PSA film spacers. Since the microfluidic channels are formed in a plastic body or film, they have good structural integrity. Such test cards can also be manufactured at ultra-high speed in a roll-to-roll process. An object of the present invention is also to disclose a technique for directly bonding the layers of a test card while maintaining the integrity of the seal around the microfluidic channels.

[0074] In one embodiment, the microfluidic test card of the disclosed invention is formed from a structured upper film and base film without a PSA film spacer. Compared to conventional glucose test strips, in which a flow cell is formed by laminating a flat upper film onto a PSA spacer, the flow cell is formed from a single, unified body, and the microfluidic channel voids are formed directly into the structure of the upper film. Instead of forming microfluidic channels from voids in a PSA film spacer, the microfluidic channels are formed directly into the structured surface of the film. The microfluidic flow cell is completed by laminating a structured upper film having channel voids onto a second base film, which is typically flat. The channel voids formed in the upper film are continuous with the sample inlet port, and when the test card is laminated onto the flat base, it forms a flow cell. The upper film can be structured by making an impression during film extrusion in the molten phase or by thermal embossing a pattern onto a pre-formed thermoplastic film. Individual structured components may also be formed by injection molding, but a process for producing a roll of structured film is preferred for high-speed manufacturing. In addition to creating voids for microfluidic channels, the process of structuring the top film can also be used to form other features such as ridges and grooves, or to modify the effective contact angle of the channel surface by incorporating patterns into the plastic surface of the flow cell. The flat base layer of the test card is typically a heat-stabilized polyester (PET) film optimized as a screen-printing surface. In other embodiments, the structured top film may be additionally formed by printing on the surface of the flat film to create voids for microfluidic channels. The physical structure can be formed on the flat film by UV-curable acrylic or other screen-printable inks with different print heights. In other embodiments, surface treatments can be implemented to modify the contact angle along the interface between the two films and control the flow properties, as can structures such as ridges and grooves modify the flow properties.

[0075] Microfluidic channel voids can be formed on the surface of plastic films using various techniques. Extrusion printing and thermal embossing can directly form structures on thermoplastic films, including rolls of film for roll-to-roll processes. In embossing, the structure is formed on a pre-formed film. In extrusion, the structure is formed on the film in the molten phase during the extrusion process. Injection molding can also form structured components by forcing molten plastic from heated plastic resin into a mold. However, components formed by injection molding are inaccessible in roll form. Currently, the minimum thickness of components produced by injection molding using high-flow resins is typically around 0.5 mm. Thinner components may be difficult to injection mold due to the high injection force required to fill the mold. In comparison, roll films can be extruded to thicknesses of 50 microns or less. The thermoplastic polymers used to manufacture the top film must be biocompatible and optically transparent in the case of test cards requiring optical detection. Common biocompatible polymers used in diagnostic test cartridges include polycarbonates and cyclic olefin copolymers. Tritan copolymer is a new thermoplastic resin that is relatively inert and optically transparent. For extrusion and embossing, structured films need to be formed from thermoplastic materials with relatively low working temperatures. For embossing, the plastic also needs to have low shape memory properties. PET has a very high working temperature and is not suitable for these techniques to form structured films.

[0076] In a typical three-layer microfluidic device, the PSA film serves both as a spacer to form the voids in the microfluidic channel and as a bonding agent between the top and base films. When the structured film forms the microfluidic channel, the channel depth is primarily determined by the depth of the impression made to the film, which can be manufactured with high reproducibility. However, the thickness of the bonding layer between the two films also affects the effective channel depth, if necessary. Optimally, the two films can be bonded with no bonding agent or a very thin layer of bonding agent, minimizing the impact on the variability of the channel depth. The test card design should allow for a minimum sealing area of ​​approximately 250–500 microns around the channel and between the channel and the edge of the card. The adhesive used for lamination and bonding must not disrupt the surface chemistry of the microfluidic channel or interfere with the assay chemistry. The adhesive must also not obstruct elements of the microfluidic channel or pneumatic system. For example, a narrow air line that transmits air pressure from a pneumatic port to a microfluidic channel can be obstructed even by a small amount of adhesive overflowing into the line during lamination. In some embodiments, the structured upper film and base film are made of similar materials such that the two similar surfaces can be directly bonded between them by thermal or acoustic energy without the need for an additional bonding medium. In other embodiments, a thin layer (5–50 microns) of bonding agent is applied between the two plastic film layers to allow a strong bond to form between the plastic films during lamination. In some embodiments, the bonding agent may be screen-printable so that it can be applied in a precise manner to avoid overflowing into microfluidic channel voids or interacting with potentially incompatible reagents. Screen printing has already been used in diagnostics to form other important features on the base layer of test cards, such as assay electrodes or conductive traces. Screen printing the adhesive to the base layer also ensures that narrow air lines are not obstructed during lamination. In one embodiment, narrow air lines may also be additionally formed with screen-printed adhesive applied to the flat surface of the film.The bonding agent may be an adhesive such as a thermoplastic hot-melt adhesive, a thermosetting adhesive, or an acrylic adhesive. The adhesive can be applied by screen printing, or by surface coating instead, or by roller application. Poor application methods can lead to contamination of the flow cell surface or interaction between the adhesive and incompatible reagents. In some embodiments, the adhesive is a biocompatible hot-melt encapsulating agent that can function as both an adhesive and a dielectric ink. Conductive traces in electrochemical diagnostic test strips are typically covered with dielectric inks. However, dielectric inks are usually UV-curing and cannot be thermally reflowed to form a bond between two opposing surfaces. Hot-melt encapsulating agents that function as dielectric inks can be thermally reflowed at relatively low temperatures (60-120°C), depending on the type of thermoplastic used, and pressure is applied to form a bond between two plastic film surfaces. Screen-printable dielectrics are typically formulated to be chemically inert for use in glucose test strips. In other embodiments, a screen-printable acrylic adhesive may function as the bonding agent. The acrylic adhesive is screen-printed onto a base film and then exposed to UV light to activate it as a pressure-sensitive adhesive. This is preferable because it does not require heat for lamination. Thermal lamination can affect the stability of some assay reagents. The acrylic monomers in UV-curing adhesives may or may not be compatible with assay reagents, depending on their chemical composition and application conditions.

[0077] With only a thin layer of adhesive forming the bonding medium between the upper and base films, uniform pressure applied during lamination is crucial for the integrity of the interlayer seal. Furthermore, surface imperfections or defects in the adhesive layer may require additional pressure to overcome non-uniformity, or even potentially lead to seal failure. It has been found that by introducing a pattern of closed or discontinuous cells on the sealing surface of the film outside the flow cells, the overall sealing surface area can be reduced, effectively increasing the pressure and thus significantly reducing the amount of force that needs to be applied to the majority of the upper film during lamination, thereby creating a highly integrity seal. In one embodiment, the cells are closed, meaning they do not communicate with the microfluidic channels, and any fluid that might unintentionally leak from the microfluidic channels into a closed cell will not subsequently flow into adjacent closed cells. The effects of surface defects causing non-uniformity in the adhesive layer are eliminated by the presence of closed cells covering at least a portion of the sealing area. Adhesive displaced due to non-uniformity caused by defects can occupy space within the closed cells. Adhesive can also reflow and displace by applying high pressure during lamination. The reflowed adhesive can also occupy space within the closed cells instead of being displaced into the microfluidic channels of the flow cell. In one embodiment, a hexagonal honeycomb pattern was formed across the entire sealing surface of the top film. The hexagonal pattern disrupts the flat bonding surface, reducing its overall surface area. The cells help isolate sealing defects after lamination and prevent leaks from propagating to the edges of the test card. Each closed cell is an independent area that can be sealed to contain leaks because fluid cannot flow between the closed cells. The hexagonal shape is the optimal cell shape because it packs most densely, but other patterns and shapes can also be used to reduce the required sealing pressure and improve seal integrity. In one embodiment, a structured top film with a complex microfluidic flow cell (80 microns) and a honeycomb pattern of closed hexagonal cells outside the microfluidic flow cell was injection molded and sealed to a polyester base film. High-integrity seals were achieved with both screen-printed dielectric ink and UV-curable acrylic adhesive.

[0078] Supports to prevent the collapse of microfluidic channels

[0079] When the voids of microfluidic channels are formed as spaces between a structured upper film and a flat base film, it is preferable that the channels are not collapsible or otherwise deformable. Variability in channel volume due to collapse or deformation directly leads to problems with assay reproducibility. Furthermore, depending on the type of adhesive used to form the seal, collapsed channels may become permanently closed if the adhesive remains activated and overflows into the channels. An object of the present invention is to disclose features that can be formed on the structured upper film during processing to maintain the height of the space forming the microfluidic channels in order to prevent collapse.

[0080] The collapse of channel voids created by structured top and base films depends on the channel dimensions, the material properties of the film, and the film thickness. Channel voids over larger surface areas and those created with thin films using low-density thermoplastic resins are more likely to collapse or deform from pressure applied by the user, device, or during the manufacturing process. Channels formed by extrusion printing and thermal embossing are particularly prone to collapse because the manufacturing process generates a positive impression on the opposite side of the film. Depending on the material properties of the film, some pressure applied to the positive impression can cause the embossed structure to collapse or deform. Conversely, if the structure of the top film is created by injection molding, no positive printing is formed. To prevent the collapse of negative printing, structural supports can be formed and strategically placed within the channel to prevent collapse and maintain channel volume. Supports can be formed by the same processes used to form the structured surface of the film, such as extrusion printing, thermal embossing, or injection molding. Supports effectively function as supports to prevent displacement of the printed pattern, which could lead to partial or complete collapse of the channel. Supports can significantly affect the flow characteristics and resistance to flow within microfluidic channels. Therefore, the size and placement of supports must be optimized for each channel design to achieve appropriate fluid performance. Furthermore, narrow channel regions may not require supports to maintain structural integrity, as large forces are needed to cause deformation.

[0081] Ridge trap for capillary filaments

[0082] A problem with microfluidic channels is the formation of Concus-Finn capillary filaments. Depending on the contact angle and shape of the flow cell surface, Concus-Finn filaments can form at sharp corners of the interface between two plastic films forming a microfluidic channel, potentially causing obstructions in the microfluidic system. The object of the present invention is to disclose design features in a microfluidic test card that can prevent obstructions caused by the formation and release of such filaments.

[0083] Because Concus-Finn capillary filaments form at the interface between two stacked film layers, introducing a pattern of sharp ridges called ridge filament traps and aligning them at the interface between the two films can trap the Concus-Finn capillary filaments and prevent their diffusion into narrow areas of microfluidic devices that could otherwise be easily obstructed. For example, a narrow air line propagating pressure from a pneumatic system could otherwise be easily blocked, potentially leading to a complete loss of the active fluid engineering function of the test card. Ridge filament traps have been perfectly effective in trapping filaments and preventing loss of fluid function when filaments form in the device under test. Ridge filament traps can be formed using the same processes used to structure the top film, such as extrusion printing, embossing, or injection molding. Ridges may also be additionally printed on the surface within the channel. The traps are essentially formed by creating a repeating pattern of sharp angles at the interface between the two films. The sharp repeating pattern may be a square wave pattern, a sawtooth wave pattern, a triangular wave pattern, other irregular sharp patterns, or a combination thereof. The repeating pattern of sharp-angled ridges typically needs to be repeated at a pitch of 0.1–0.5 mm. The repetition adds additional redundancy for trapping capillary filaments. An alternative to using structural ridges as traps is to introduce alternating patterns with surface treatments that alter the contact angle along the interface between the two films. This repeating pattern can have the same effect as a physical ridge trap to trap filaments formed at the interface between the two films in a microfluidic channel.

[0084] Microfluidic weirs that control the flow of fluid fronts

[0085] If the fluid front of a sample does not travel uniformly across the entire width of a microfluidic channel, air pockets may form. Non-uniformity of flow across a microfluidic channel can be caused by slight differences in surface energy that affect the contact angle and wetting properties on the channel's surface. For example, a sample may preferentially travel through more hydrophilic areas. It is not uncommon for a fluid sample to preferentially travel along the sides of a flow cell, bypassing the center of the flow cell, due to differences in surface energy. This can trap air and form bubbles in the center of the channel. Differences in surface energy are common, whether due to the use of different materials, surface defects, scratches, or dust particles. The introduction of bubbles into a microfluidic channel can affect the accuracy and precision of microfluidic-based testing, especially if the bubbles form over critical elements on the test card, such as reagent zones or electrodes. They also affect the volume of fluid sample within the channel. An objective of this invention is to disclose features within microfluidic channels for preventing non-uniform flow.

[0086] In one embodiment, disclosed features formed on the surface of a microfluidic channel can control the uniformity of the test sample flow and prevent the formation of air pockets. A microfluidic weir or baffle is a short, repeating element patterned on the surface of a flow cell across its entire width perpendicular to the direction of flow. The microfluidic weir generates capillary pressure perpendicular to the direction of flow, causing the fluid front to preferentially fill the entire width of the channel laterally before the fluid continues to advance. The difference in surface energy across the entire width of the channel, which could otherwise cause non-uniformity of the forward flow, is far less than the pressure introduced by the weir to guide the lateral flow. Thus, the channel is filled laterally before the fluid front advances from one microfluidic weir to the next. This prevents the formation of air pockets, which could affect the performance of the test. In one embodiment, the microfluidic weir is a straight or curved band or groove across the entire width of the flow cell. In some embodiments, the band may be curved to follow the shape of the fluid front meniscus. In some embodiments, the bands or grooves may be 5–25% of the total channel height sufficient to generate the required capillary pressure difference, although some effect may be observed even with band heights less than 5% of the channel height. The weirs are formed in a repeating pattern along the long axis of the channel at a pitch of 50–500 microns. Short repeating elements can be formed in the structured layer of the microfluidic channel by injection molding, extrusion printing, or embossing. Short repeating elements may also be additionally printed on the surface within the channel before lamination. In other embodiments, lateral capillary pressure may be created by applying a surface treatment within the channel that follows a similar repeating pattern along the long axis of the flow cell. Just as physical weirs improve flow uniformity by generating capillary pressure that leads to preferential lateral filling, surface treatments can produce such an effect by altering the surface energy across the entire width of the channel.

[0087] Reagent zone and reagent mixing

[0088] Assay reagents are typically dried within a microfluidic channel for resuspending by a buffer fluid from a test sample or buffer pack. The reagents must be deposited on the surface within the microfluidic channel and dried without wetting beyond the channel's boundaries. During the operation of the assay, the test sample must pass through the dried reagent cake to resuspend the reagents uniformly and with high reproducibility. An object of the present invention is to disclose a reagent deposition zone that constrains the position of the reagents during drying. Another object of the present invention is to disclose a design that improves the uniformity of reagent resuspendion.

[0089] In one embodiment, the test card of the disclosed system includes a reagent deposition zone typically located upstream of the analytical detection element of the system, where dried reagents can be deposited and dried in place so that they can be resuspended by the test sample passing through the microfluidic channel. The reagents are deposited either directly on the bare plastic surface of the channel's base layer or within the reagent zone on a hydrophilic coated surface so that the reagents spread throughout the reagent zone. In one embodiment, the reagent zones are demarcated by screen-printed carbon ink, which is a more hydrophobic surface that can restrain the diffusion of the reagents outside the reagent zones. If the reagents diffuse outside the deposition zones and beyond the channel's region, less reagent will be resuspended within the channel, potentially affecting the assay. The reagent deposition zones are kept entirely within the microfluidic channel to ensure consistency and reproducibility of reagent resuspendion. In one embodiment, the coffee ring effect, where there is more reagent at the edges of the reagent cake than in the center, is retained within the deposition zones of the microfluidic channel. Each microfluidic channel has a reagent mixing zone and an analytical region, which are separated by a constriction called the mixing zone neck. The mixing zone neck is designed to improve the uniformity of reagents in the sample as the entire fluid sample volume passes through this narrow region before entering a wider analytical area, thereby enabling mixing. This helps prevent heterogeneity across the entire channel width when there are differences in resuspension throughout the flow cell.

[0090] High-resolution metering using capacitive monitoring

[0091] A key factor in providing high levels of performance in advanced in vitro diagnostic systems is high-resolution metering, which actively controls the precise position of the test sample within a microfluidic channel. High-resolution metering can also control complex operations within the microfluidic channel, such as reagent mixing, which requires real-time monitoring of the precise position of the forward fluid front within the channel to provide feedback to the pneumatic control loop in the test system. The object of this invention is to disclose a method for accurate real-time high-resolution monitoring of the precise position of a fluid sample with forward and backward flow within a microfluidic channel.

[0092] High-resolution metering in the disclosed test system is achieved in one embodiment by a control loop based on feedback from a capacitive monitoring system. In one embodiment, capacitive monitoring of the liquid front utilizes a series of capacitive elements coupled together to provide discrete capacitive signals as the test sample moves from one capacitive element to the next in a series within a microfluidic channel, screen-printed onto a base layer of a test card with silver conductive ink covered with dielectric ink. In one embodiment, the capacitive elements are first screen-printed with conductive silver ink, then completely covered with a layer of dielectric ink to prevent direct wetting of the conductive ink by the liquid sample. Direct wetting of the conductive ink would cause a short circuit, which would otherwise not allow detection of dynamic wetting and dewetting of the capacitive elements in the series. By encapsulating the conductive ink, the capacitive elements detect both forward and backward flow (wetting and dewetting) of the test sample across the same capacitive elements in the series, as required by a closed-loop system that can control reagent mixing (forward and backward liquid pumping). If conductive silver ink is exposed anywhere inside or outside the microfluidic channel, it is covered with carbon ink to prevent silver contamination of the fluid sample. In one embodiment, the capacitive elements are comb-shaped elements adjacent to at least one reference element. The reference element is charged with a fixed voltage in close proximity to at least one capacitive element in series. In one embodiment, the series of comb-shaped capacitive elements are coupled together by conductive traces to form a ladder of capacitive elements. Each comb-shaped element in the series forms one step on the capacitive ladder, and large discrete changes in capacitance are detected when a liquid test sample wets the successive capacitive elements in the series. In one embodiment, the capacitive elements in the series are appropriately spaced so that the meniscus of a moving liquid front passes through only one capacitive element at a time. This causes large discrete changes ("jumps") in the capacitive signal instead of a continuous but non-discrete increase in the capacitance signal when the fluid front moves from one element to the next.Discrete signals allow for clear distinction of the position of the fluid front as it moves between stages on a capacitive ladder. The increase in a non-discrete, continuous capacitive signal requires calibration to determine the relative position of the liquid front, which is not necessary with discrete signal changes. A series of capacitive elements can be used to determine the precise position of a test sample based on the relative position between two adjacent capacitive elements. Higher resolution is achieved by placing the capacitive elements closer together, but the elements must be sufficiently separated so that the liquid front meniscus does not contact both elements simultaneously. In one embodiment, a short-circuit detection element, which is a conductive silver element not covered with a dielectric, is placed at the end of the capacitive ladder, and the capacitive ladder is short-circuited when the test sample covers all capacitive elements in the series and the last short-circuit detection element. The short-circuit detection element provides a static signal at least until it is completely dry.

[0093] Detection of previously used test cartridges

[0094] The diagnostic platform should preferably be simple and easy to use, even for untrained users, without the risk of unexpected errors. This is a requirement for diagnostic tests on point-of-care platforms to be CLIA-exempt in the United States. Two major sources of error are the accidental reuse of previously used diagnostic tests and the application of insufficient test samples. The object of the present invention is to disclose a method for detecting the reuse of a previously wet test card or a test card with insufficient capacity.

[0095] The disclosed test system, in one embodiment, uses a capacitive element and a short-circuit detection element. The short-circuit detection element, unlike the capacitive sensing element, is a carbon conductive ink element that is not covered with a dielectric. While the capacitive element provides a reversible signal as the fluid sample passes back and forth through the element, the signal provided when wetting the short-circuit detection element is semi-permanent. When the short-circuit detection element is wetted by the fluid sample, the conductive trace causes a short circuit. Depending on the shape of the sensor, the short circuit may persist even if the test card is partially dry, allowing detection of a previously used test strip. The short-circuit detection element and the capacitive sensing element may also be positioned along the main microfluidic feeder channel to monitor the position of the sample fluid and prevent the test card from drying out with insufficient volume. If each assay has its own microfluidic channel filled from the main feeder channel, active fluid engineering can be designed to feed the sample volume from the main channel to one assay channel at a time, stopping the filling of the assay channel when the main feeder channel has run out of sample volume or has insufficient volume to fully fill the next assay channel. The goal is to prevent partial filling of assay channels, which would result in an invalid assay. If the weighing system can ensure that sufficient volume is available in the main feeder channel before beginning to fill the assay channels, this prevents partial filling of channels that would invalidate assay results. As long as no test sample is present in the specific channel leading to partial filling of the assay, additional test samples can then be applied so that the remaining microfluidic channels are filled and the test panel can be completed without invalidating individual results.

[0096] Selected embodiments of the present invention are described in detail here with reference to the drawings. However, it should be understood that these embodiments are for illustrative purposes only, and other embodiments exist within the scope of the claims.

[0097] Referring to Figure 1, one embodiment of a small, compact point-of-care testing device is shown in a perspective view. In this embodiment, the device has an external housing 100 through which an assay card 101 is inserted into the device. This portable device also has a battery compartment 102 and a display 103.

[0098] Referring to Figure 2A, an embodiment of the point-of-care device is again shown in a front view. The front of the external housing includes a slot 201 for the test card to enter the card bay. The entry of the test card through the slot is prevented by a large latch 200 on one side and a small latch 202 on the other side. The front end of the test card must have tapered corners on each side, large enough to clear the corresponding latches for the test card to enter the card bay. As seen in the vertical cross-section through the device in Figure 2B, the device includes a core module 203, which is a self-contained mechanical assembly capable of accepting an assay card upon insertion through the slot 201 in the external housing and performing the highly multiplexed functions required by an advanced diagnostic platform; a control electronic board 204 that interfaces with both the core module and the display and controls an onboard operating system with a user interface; an optical barcode scanner 205; a cooling fan assembly 206 for cooling the core module during operation; and a replaceable removable battery 207 in a battery compartment.

[0099] Referring to Figures 3A-3D, embodiments of the core module are shown in perspective, front view, and two horizontal cross-sections from different viewpoints. The core module is a self-contained assembly of components that form the card bay and mobile platform, and also includes a pneumatic system and other analytical components for performing diagnostic inspections. The same core module can be incorporated into different external housing designs, with or without a display. The components of the core module are assembled together into a structural assembly between the superstructure support 300 and the substructure support 304. The superstructure support provides structural integrity to the mobile platform, and is formed by two aluminum heating plates 322 and 323 for the front and rear heating zones, respectively, as well as a main platform printed circuit board 312 to which alignment pins 318, spring connector pins 319, optical LEDs 320, and a rear leaf spring 321 are mounted. The substructure support provides structural integrity to the base of the card bay. The base of the card bay is formed by two aluminum heating plates 314 and 316 for the front and rear heating zones, respectively, which also include a gasket 315 that connects to a pneumatic system via a conduit and an optical sensor 317 adjacent to an optical LED on the upper platform. The platform moves vertically up and down on lead screws 310 on each side, driven by left 303 and right 307 motors. The aluminum plates from the platform are coupled to the lead screws via plastic side slides 308 on each side, and there are also guide pins 301 that provide additional support to the moving platform mechanism. Entry into the card bay is partially prevented by a large latch 200 on the left side and a small latch 202 on the right side. The latches engage with the tapered corners of the test card and act outward to allow insertion of the test card. The operation of the latches can be locked by a latch lock 309. The engagement of the latches by the latch lock depends on the position of the moving platform. The lock engages with the latch when the platform is at its widest position and is then released by the latch spring 311 when the platform moves to a less separated position.During the insertion of the test card, the test card is initially biased downward by a rolling shutter 306 having a leaf spring 302 that is close to the slot entry, and then biased by a rear leaf spring 321. The pneumatic fluid engineering manifold 305, which houses the pump and valve system, is located below the card bay and connected to a gasket through a series of conduits. Finally, the core module assembly interfaces with a ribbon cable from the control electronics board through a connector 313.

[0100] Referring to Figures 4A–4D, a series of vertical cross-sectional views of one embodiment of the core module assembly illustrate the dynamic movement of the platform, which has alignment pins and spring connector pins that engage with the test card in the card bay. In Figure 4B, the platform remains in a fully separated position after the insertion of the test card 101. The latches capture the test card and provide preliminary alignment so that the alignment pins 318 on the card interface are well aligned with the alignment holes and the conical tips of the alignment pins engage with the alignment holes. In Figure 4C, the moving platform descends to an intermediate position on the lead screw 310 so that the alignment pins engage with the alignment holes and provide fine alignment between the test card and the card interface. In Figure 4D, the moving platform descends further to a fully clamped or closed position so that the spring connector pins 319 also engage with the test card and are compressed. The rear leaf spring 321 is also compressed so that it can be flush with the aluminum heating plate when the platform is in the fully clamped position.

[0101] Referring to Figures 4E-4G, an embodiment of a core module assembly with two independent card interfaces is shown with a long assay card 101. The front card interface of the module has alignment pins 318, and the rear card interface of the module has alignment pins 403. Depending on the design of the long assay card, the assay card can be aligned to either the front or rear card interface. In the embodiment shown in Figure 4G, an oversized through-hole 404 is aligned with the alignment pins from the front card interface, and alignment holes 800 and 801 are aligned with the alignment pins from the rear card interface. Therefore, when the card interface is actuated to the clamp position and lowered, the rear alignment holes of the long assay card engage with the rear alignment pins. Conversely, there is no engagement between the oversized through-holes and the alignment pins of the front card interface.

[0102] Referring to Figures 5A–5F, a series of side views and corresponding horizontal sections of an embodiment of the core module assembly illustrate the insertion and capture of a test card in the card bay by a latching system. In Figures 5A and 5B, the test card 101 is outside the device and ready for insertion, but the large latch 200 and small latch 202 are locked in place by the latch lock 309. Therefore, the latches cannot be moved outward, and the test card cannot be inserted. The latch lock is engaged by lever action when the moving platform is in its farthest position. In Figures 5C and 5D, the latch lock is released from the latch by the latch spring 311 along with the moving platform in a less farthest position. The front end of the test card clears the latches at the large taper 501 on the left corner and the small taper on the right corner, displacing them outward for initial insertion. The outwardly displaced latches can slide along the side edges of the test card until it is fully inserted. In Figures 5E and 5F, the test card is fully inserted, and the latches act inward to engage with the notches, capturing the card in place. The large latch engages with the large notch 503, and the small latch engages with the small notch 504. The test card is captured in place so that it is pre-aligned within the card bay. For example, the test card covers the gasket at the base of the card bay. However, fine alignment is then achieved by alignment pins and alignment holes.

[0103] Referring to Figure 6, an exploded view of an embodiment of a microfluidic test card for the proposed platform shows its layers and corresponding features. The test card is formed of a structured upper film 600 and a base film 606, and is adhesively sealed with screen-printed acrylic 601. Other elements on the base of the test card are formed of screen-printed silver 605, carbon 604, silver chloride 603, and dielectric ink 602. The test card also has large 503 and small 504 notches, large 501 and small 502 tapers, and alignment holes. There is one circular alignment hole 608 and one one-dimensional elongated alignment hole 607.

[0104] Referring to Figure 6B, two consecutive silver conductive elements 613 and 614 are shown within the microfluidic channel. The conductive elements are connected by conductive traces 615 outside the microfluidic channel, and the conductive elements are also adjacent to a single comb-shaped reference element 616. The conductive elements are covered with a dielectric 617 within the microfluidic channel. This ensures that the capacitive elements do not come into direct contact with the fluid sample.

[0105] Referring to Figures 7 and 8, embodiments of the microfluidic test card are shown from a top view and a perspective view, respectively, along with enlarged views of key features. The test card has a sample inlet port 700 for applying a test sample to the microfluidic channel. Sample application can be immediately detected by a short-circuit detection element 702 just below the inlet port. At the end of each microfluidic channel is a gasketed port 711. During inspection, a gasket from the test system seals around the gasketed port, connecting an external pneumatic system to the microfluidic channel on the test card. The microfluidic channel also includes a structural weir 708 to prevent the formation of air pockets and a ridge filament trap 709 to capture capillary filaments that might otherwise obstruct the microfluidic system. In certain areas, an optical aperture 705 aligns with an optical sensor and optical LED on the instrument platform. The optical aperture also includes a weir to prevent the formation of air pockets within the optical aperture. A honeycomb of closed hexagonal cells 703, separated by cell walls 704, covers the sealing surface of the microfluidic test card outside the microfluidic channel. The closed cell reduces the surface area for sealing and prevents leakage. The card contact pads 706 on the base of the test card form island contacts for making electrical connections with the card interface on the device. Openings or holes overlap the card contacts so that spring connector pins on the card interface can contact the card contact pads through the openings. In some areas, individual openings 710 provide access to only one individual contact pad. In other areas, multi-contact openings 707 can provide access to multiple adjacent contact pads. In one embodiment, the test card also includes two alignment holes, one having a circular shape 608 and one having an elongated shape 607.

[0106] Referring to Figure 8B, an embodiment of a microfluidic test card is shown with channel supports 807 within a microfluidic channel. The microfluidic channel supports are spaced apart to prevent deformation or collapse of the microfluidic channel while not interfering with the channel's flow characteristics.

[0107] Referring to Figure 9, heating and cooling times were tested. After heating the temperature zone to 95°C and then initiating cooling, the temperature zone cooled down to 37°C, up to 45% faster compared to when the moving platform was in a clamped position with no space between the upper and lower plates, when the moving platform was in an open position and the upper heating plate was separated from the lower heating plate to allow maximum airflow between the plates.

[0108] Referring to Figure 10, a series of five consecutive capacitive elements is shown within a microfluidic channel, arranged in a comb-like pattern with a reference element. As the test sample moves across each of the five consecutive capacitive elements, a further increase in capacitance is observed, which corresponds to a discrete signal when monitoring the corresponding derivative of the capacitance. The position of the fluid sample can be determined based on the discrete signal indicating that the fluid front is between two capacitive elements. Thus, the resolution of the position monitoring depends on how close the capacitive elements are to each other.

[0109] Accordingly, one aspect of the present invention is a card reading system 104 in which an electrical contact between an assay card 101 and a card reader 104 is made through the vertical actuation of a card interface relative to the assay card. In one embodiment, the assay card reading system includes: (a) a card reader comprising at least one card interface 325, wherein the card interface has a plurality of card interface contacts 319; (b) an assay card having card contacts 706; and (c) a vertical actuation mechanism 402 that causes a relative vertical movement between the assay card and the card interface in order to bring at least a portion of the card interface contacts into contact with the card contacts. In one embodiment, the vertical actuation mechanism is connected to the card interface and acts the card interface vertically.

[0110] In one embodiment, the assay card reading system includes: (a) a card reader including at least: (i) a card bay 324 for receiving assay cards; (ii) at least one card interface positioned either above or below the card bay and configured to move vertically, wherein the card interface has a plurality of card interface contacts, and the card interface has at least two states, in a first state 400, the card interface is separated from the card bay, allowing assay cards to be received into the card bay, and in a second state 401, one or more card interface contacts of the card interface are in contact with an assay card; (b) an assay card having card contacts, wherein when the card interface is in the second state, at least a portion of the card interface contacts are in contact with the card contacts.

[0111] In one embodiment, when the assay card is inserted into the card bay, no contact is made between the card interface contacts and the card contacts. In one embodiment, no electronic connection is made between the assay card reader and the assay card via the card edge connector.

[0112] In one embodiment, the assay card reader includes a housing 100 that defines a slot 201 for providing access to a card bay.

[0113] In one embodiment, the assay card includes at least a first layer 600 and a second layer 606, the card contacts are located on the second layer, and a plurality of openings 710 in the first layer provide access to the card contacts. In one embodiment, at the second position, at least a portion of the card interface connector passes through an opening in one layer of the assay card to contact the card contacts. In one embodiment, when the assay card is in the card bay, the first layer is facing upward.

[0114] In one embodiment, the card interface further includes two or more alignment pins 318 that are received in two or more alignment holes 800 and 801 in the assay card in a second state. In one embodiment, the alignment pins are conical. In one embodiment, the assay card includes at least a first layer and a second layer, and each alignment hole includes first layer alignment holes 609 and 610 in the first layer and second layer alignment holes 607 and 608 in the second layer, and the first layer alignment holes and the second layer alignment holes are arranged to create through holes, and the first layer alignment holes are larger than the second layer alignment holes. In one embodiment, the first alignment pin 318 of the alignment pin corresponds to the first alignment hole 801 of the alignment hole, and the second alignment pin 326 of the alignment pin corresponds to the second alignment hole 800 of the alignment hole, and the first alignment pin and alignment hole prevent relative movement in two dimensions between the card interface and the assay card, while the second alignment pin and alignment hole prevent relative movement only in one dimension between the card interface and the assay card. In one embodiment, the first alignment pin and first alignment hole are circular, the second alignment pin is circular, and the second alignment hole is elongated in one dimension.

[0115] In one embodiment, the assay card comprises at least a first layer and a second layer, the card contacts are located on the second layer, a plurality of openings in the first layer provide access to the card contacts, and a tight tolerance fit between alignment pins and alignment holes positions the card interface connector relative to the card contacts, thereby allowing the card interface contacts to pass through the openings in the assay card and contact the card contacts.

[0116] In one embodiment, the card interface contact is a biased pin connector, such as a spring pin connector 319. In one embodiment, the card contact is a conductive pad 706. In one embodiment, the card contact is an island contact. An island contact is a small, isolated area of ​​conductive material that makes electrical contact with other contacts and is typically surrounded by insulating material to insulate it from the rest of the device. In one embodiment, the conductive pad is a screen-printed electrode.

[0117] In one embodiment, the card interface includes heating elements 314, 316, 322, and 323, and when the card interface is in a second state, the heating plates control the temperature in the card bay. In one embodiment, the assay card reader includes one or more pumps 305, and the assay card includes a microfluidic channel 802 having one or more gasketed ports 711, and the card interface compresses the assay card in the second state, sealing the gasketed ports, and one or more pumps are used to pressurize or depressurize the microfluidic channel.

[0118] Another aspect of the present invention is an assay card reader. In one embodiment, the card reader includes (a) a housing 100, (b) a card bay at least partially located within the housing, and (c) electrical contacts, the electrical contacts being operable and having at least two states, the first state being for accepting an assay card into the card bay, in which the electrical contacts do not form an electrical connection with the assay card, and the second state being in which the electrical contacts are operable and form an electrical connection with the assay card in the card bay. In one embodiment, the card reader further includes a card interface including at least electrical contacts.

[0119] Another aspect of the present invention is an assay card. In one embodiment, the assay card includes (a) a base 606 having a planar surface, (b) card contacts on the planar surface, and (c) microfluidic channels 802 in or on the base.

[0120] Another aspect of the present invention is a method for analyzing a sample using a card reader having an electrical contact, the electrical contact being operable and having at least a first state and a second state. In one embodiment, the method includes (a) depositing the sample onto an assay card, (b) accepting the assay card while the electrical contact is in the first state, (c) activating the electrical contact to the second state so that the electrical contact forms an electrical connection with the island contact of the assay card, (d) analyzing the sample, and outputting the results of the analysis. In one embodiment, step (a) is performed before step (b). In one embodiment, step (c) is performed before step (a).

[0121] Accordingly, one aspect of the present invention is an assay card reading system having a two-stage alignment configuration. In one embodiment, the assay card reading system includes (a) an assay card 101 having an upper side 611, a lower side 612, and one or more edges 712, 713, 714, and includes at least (i) one or more first alignment references 503 and 504 on at least one edge, and (ii) one or more second alignment references 800 and 801 on the upper or lower side, and (b) the card reader includes at least (i) a card bay for receiving assay cards, and (ii) at least one side of the card bay to engage with the first references and provide initial alignment of the assay card in the card bay (iii) comprising one or more first reference cooperating members 200 and 202 along the card bay, and at least one card interface above and / or below the card bay, the card interface comprising one or more second reference cooperating members 318 and 326, the card interface having at least two states, the first state being that the card interface is away from the card bay, allowing the assay card to be received into the card bay, and the second state being that the second reference cooperating member engages with the second reference on the assay card to provide final alignment of the assay card relative to the card interface. In one embodiment, there is compliance between the mating of the first reference and the first reference cooperating member, allowing relative movement between them during final alignment of the assay card by the second reference and the second reference cooperating member. In one embodiment, final alignment does not depend on alignment between the alignment surface of the assay card and the alignment surface of the card bay.

[0122] In one embodiment, the connector on the card interface and the contact pads on the assay card are aligned during final alignment. In one embodiment, the photodetector 317 and / or light source 320 on the card interface and the optical aperture 705 on the assay card are aligned during final alignment.

[0123] In one embodiment, the first alignment reference is located on only one or more side edges of the assay card. In one embodiment, the first reference is located on both side edges of the assay card. In one embodiment, the first reference and the first reference cooperating member are notches 503 and 504 and latches 200 and 202. In one embodiment, the first reference is a notch and the first reference cooperating member is a latch. In one embodiment, the second reference and the second reference cooperating member are alignment pins 318 and 326 and alignment holes 800 and 801. In one embodiment, the second reference is an alignment hole and the second reference cooperating member is an alignment pin.

[0124] In one embodiment, at least some of the alignment pins are conical 318 and 326, the assay card comprises a first layer and a second layer, and the alignment holes 609 and 610 on the first layer are larger than the alignment holes 607 and 608 on the second layer.

[0125] In one embodiment, the card bay has x, y, and z directions, and in initial alignment, the assay card is aligned with the card bay along the X and Y axes 327 and 328. In one embodiment, the initial alignment of the assay card along the x and y axes by the first reference and the first reference cooperating member allows engagement between the second reference and the second reference cooperating member. In one embodiment, the initial alignment of the assay card along the x and y axes by the first reference and the first reference cooperating member allows engagement of the alignment pins on the card interface with the alignment holes on the assay card. In one embodiment, the initial alignment of the assay card aligns the second reference and the second reference cooperating member within 1 mm along the x and y axes. In one embodiment, the final alignment of the assay card by the second reference and the second reference cooperating member aligns the card interface and the assay card within at least 0.5 mm along the x and y axes. In one embodiment, the final alignment of the assay card along the x and y axes by the second reference and second reference cooperating member aligns the connector on the card interface with the contact pads on the assay card. In one embodiment, the final alignment of the assay card along the x and y axes by the second reference and second reference cooperating member aligns the photodetector and / or light source on the card interface with the optical aperture on the assay card.

[0126] In one embodiment, the assay card is received into the card bay through a slot to engage the first criterion with the first criterion cooperative member.

[0127] In one embodiment, the card reader includes one or more elastic members 321 that bias the assay card upward or downward to hold it against the surface of the card bay, thereby allowing a second reference cooperating member on the card interface to disengage from the second reference on the assay card when the card interface returns from a second state to a first state, thereby allowing the assay card to be removed from the card bay. In one embodiment, the elastic member biases the assay card upward or downward so that an alignment pin on the card interface disengages from an alignment hole on the assay card. In one embodiment, the elastic member is a leaf spring 321.

[0128] In one embodiment, at least one card interface includes two or more card interfaces 405 and 406, each card interface having an independent second reference cooperating member for aligning with the assay card independently of other card interfaces. In one embodiment, the assay card has a second reference corresponding to one specific card interface 800 and 801 and larger through holes 404 corresponding to all other card interfaces, thereby aligning only one of the card interfaces with the assay card. In one embodiment, the assay card has multiple sets of second references, each set corresponding to one specific card interface. In one embodiment, the second references are alignment holes, and each set of alignment holes is of a different size. In one embodiment, one of the card interfaces is located above the card bay and one of the card interfaces is located below the card bay.

[0129] Accordingly, another aspect of the present invention is an assay card reading system that prevents improper insertion of test cards, accommodates cards of variable thickness and length, and / or synergistically presses down and ejects the cards. In one embodiment, the assay card reading system includes (a) an assay card having sides and having at least one first component 503 and 504 of an interlocking mechanism 505 along one of the sides, and (b) a card reader, the card reader including at least (i) a card bay for receiving assay cards, and (ii) second components 200 and 202 of the interlocking mechanism 505 for interlocking with the first component to hold the assay card in place within the card bay, wherein the assay is positioned in place by the interlocking mechanism and not by a alignment surface.

[0130] In one embodiment, the interaction mechanism is at least one of a latch / catch, a cantilever snap fit, a detent, a key and slot, or a magnetic interaction. In one embodiment, the interaction mechanism is latches / catches 200, 202, 503, and 504. In one embodiment, the first component is a catch, and the second component is latches 200 and 202. In one embodiment, the latch is a leaf spring 200 and 202 that engages with the catch. In one embodiment, the catch is a notch 504 and 504. In one embodiment, the first component is a hole, and the second component is a spring-loaded pin that interacts with the hole.

[0131] In one embodiment, the assay card is one of a family of assay cards of different lengths, each assay card of the family having a first component on one side of the assay card, and the predetermined position is defined by the engagement of the first component and a second component. In one embodiment, each assay card of the family is provided with at least one alignment fiducial 800 and 801, the fiducial being positioned at the same distance from the first component along the insertion axis 328 for each assay card of the family. In one embodiment, the assay cards of the family are 20-150 mm, 40-100 mm, or 50-75 mm in length and are held at the same predetermined position relative to the position of the first component.

[0132] In one embodiment, the mutual engagement of the first and second components provides initial alignment of the assay card within the card bay. In one embodiment, the second component is actuated to engage with the first component.

[0133] In one embodiment, the operation is monitored by one or more sensors 329 to detect the insertion or removal of the assay card.

[0134] In one embodiment, the lock 309 prevents the operation of the second component. In one embodiment, the assay card reader further includes a card interface above or below the card bay connected to the lock, the card interface being configured to move perpendicular to the card bay, so that the vertical position of the card reader determines whether the lock is engaged or disengaged from the second component.

[0135] In one embodiment, the first component includes a first component on each side of the assay card, and at least one second component includes two second components, each positioned on a different side of the assay card in the card bay.

[0136] In one embodiment, the card reader includes one or more elastic members 321 that bias the assay card upward or downward to align the assay card to a specific vertical position within the card bay. In one embodiment, the elastic member is a leaf spring 321.

[0137] In one embodiment, the assay card has a polarized front edge 712 that allows the assay card to be inserted into the card bay in only one direction. In one embodiment, the assay card reader has obstacles 200 and 202 that prevent the front edge from being inserted into the card bay. In one embodiment, the front edge has a shape that clears the obstacle, and the rear edge has a different shape that does not clear the obstacle. In one embodiment, the shape of the front edge is asymmetrical, with one side clearing the obstacle but the other side not. In one embodiment, each corner of the front edge has tapers 501 and 502 of different sizes to clear obstacles of different sizes.

[0138] In one embodiment, the obstruction is a fixed or displaceable obstruction. In one embodiment, the displaceable obstruction is a second component. In one embodiment, during the insertion of the assay card, the second component is contacted by the tapered corner of the assay card and displaced away from the card bay to allow clearance for the leading edge of the assay card, and then acts back toward the card bay to engage with the catch when the assay card is fully inserted.

[0139] In one embodiment, one or more locks 309 prevent displacement of the second component. In one embodiment, the assay card reader further includes a card interface above or below the card bay connected to the lock, the card interface being configured to move perpendicular to the card bay, so that the vertical position of the card reader determines whether the lock is engaged or disengaged from the second component.

[0140] In one embodiment, the assay card includes at least one alignment reference, the alignment reference being at a specific distance from a first component along the insertion axis.

[0141] Accordingly, one aspect of the present invention is an assay card reading system that can accommodate cards of different thicknesses. In one embodiment, the assay card reader includes (a) a card bay for receiving assay cards and (b) a card interface positioned above or below the card bay, wherein the card interface has a plurality of elastic connectors 319, and the card interface has at least two states, the first state being that the card interface is away from the card bay and the elastic connectors are at a fully extended length 319, allowing assay cards to be received into the card bay without contact with the card interface, and the second state being that the elastic connectors are in contact with the assay card and are at least partially compressed 407. In one embodiment, the elastic connectors are spring connectors. In one embodiment, the spring connectors are spring-loaded pins.

[0142] In one embodiment, the spring connector is at least partially compressed when the card interface is activated to a second state. In one embodiment, the spring connector is compressed from its fully extended length when the card interface is in a first state to a contact length of 407 when the card interface is in a second state, and the degree of compression of the spring connector varies to accommodate various assay card thicknesses.

[0143] In one embodiment, the card reader is configured to accept assay cards having a thickness of 0.1 to 5 mm, or 0.2 to 4 mm, or 0.3 to 3 mm. In one embodiment, the card reader is configured to accept assay cards in which the ratio of the thickness of the thinnest assay card to the thickest assay card varies by at least 1:50, or 1:40, or 1:30, or 1:20, or 1:10.

[0144] In one embodiment, the card reader includes at least one or more elastic members 306 and 321 that bias the assay card upward or downward to hold the assay card without displacement within the card bay when the assay card is inserted. In one embodiment, the elastic member is a leaf spring 321. In one embodiment, the elastic member is configured to be pushed upward by the leading edge of the assay card upon insertion, thereby allowing the assay card to be received into the card bay beyond the elastic member. In one embodiment, the leaf spring is configured to be fully compressed and flush with the card interface, thereby allowing the insertion of assay cards of different thicknesses. In one embodiment, the solid length of the leaf spring is configured to accept assay cards with a thickness of 0.1 to 5 mm, or 0.2 to 4 mm, or 0.3 to 3 mm.

[0145] In one embodiment, at least one elastic member is configured as a shutter 306 positioned adjacent to the slot. In one embodiment, the shutter at least partially blocks light from entering the slot. In one embodiment, the shape of the shutter is configured to be pushed upward by the leading edge of the assay card inserted through the slot of the card reader.

[0146] In one embodiment, the card interface includes heating elements 314, 316, 322, and 323, and the elastic connector is sufficiently compressed in the second state so that the heating elements come into contact with the assay card.

[0147] Another aspect of the present invention is an assay card reading system having one or more different heating zones with enhanced cooling and / or interlocks to prevent hot cards from being removed or new cards from being inserted while the reader is too hot. In one embodiment, the card reader includes (a) a card bay for receiving assay cards, and (b) a card interface located above or below the card bay and configured to move vertically, wherein the card interface has at least two states, in a first state the card interface is away from the card bay, allowing assay cards to be received into the card bay, and in a second state the card interface is in contact with an assay card, and (c) at least one discrete heating zone 314, 316, 322, 323 within the card interface, which is in contact with the assay card and controls the temperature of the assay card when the card interface is in the second state. In one embodiment, the card interface includes at least two or more heating zones 314, 316, 322, 323.

[0148] In one embodiment, the heating zones are thermally isolated. In one embodiment, thermal isolation is achieved by constructing a card interface with a support 308 between the heating zones made of a material with low thermal conductivity. In one embodiment, the support is made of a polymer material such as rubber or plastic.

[0149] In one embodiment, the heating zones are arranged such that shorter assay cards are exposed only to the first heating zones 316 and 322, while longer assay cards may be exposed to one or more additional heating zones 314 and 323.

[0150] In one embodiment, the heating zone includes metal heating plates 314, 316, 322, 323 and a closed-loop temperature control system. In one embodiment, the heating plates are made of aluminum. In one embodiment, the heating zone includes upper heating plates 322 and 323 above the card interface and lower heating plates 314 and 316 below the card bay.

[0151] In one embodiment, the compressive force of the card interface on the assay card in the second state prevents the assay card from being removed while it is heated. In one embodiment, the reader is configured to actuate the card interface to the first state, releasing the compressive force of the card interface on the assay card and allowing the assay card to be removed. In one embodiment, the reader is configured to actuate the card interface to the third state 506, in which the upper heating plate separates from the lower heating plate to maximize airflow between the heating plates for faster cooling. In one embodiment, the system further includes an airflow system 206 having a high-flow fan configured to move air between the upper and lower heating plates to maximize cooling.

[0152] In one embodiment, one or more obstruction elements 200 and 202 partially block entry into the card bay, and the obstruction elements are locked during the cooling phase to prevent insertion of the assay card until the heating zone has cooled to a set temperature. In one embodiment, the system further includes a locking mechanism 309 for locking the obstruction elements in place, and the card interface is coupled to the locking mechanism so that a specific vertical position 506 of the card interface engages with the locking mechanism. In one embodiment, in a third state, the card interface moves vertically to a predetermined vertical position further away from the card bay than its position in the first state, and engages with the locking mechanism to fix the obstruction elements in place during cooling. In one embodiment, after cooling, the card interface returns vertically to the first state, so that the locking mechanism disengages from the obstruction elements and allows insertion of the assay card. In one embodiment, the obstruction elements are a second component of the engagement mechanism.

[0153] In one embodiment, the card interface is connected to the locking mechanism by a spring 311, so that when the card interface moves vertically from the third state to the first state, the spring disengages the locking mechanism.

[0154] One aspect of the present invention is an improvement in the manufacturability of the card by using a unified body in one embodiment. In one embodiment, the assay card includes (a) first and second layers, the first layer covering the second layer and defining a space 802 between them, (b) microfluidic channels in the space, and (c) the second layer and channels being formed by a unified body 600.

[0155] In one embodiment, the unified body is embossed. In one embodiment, the unified body is imprinted. In one embodiment, the unified body is injection molded. In one embodiment, the unified body is integrally formed. In one embodiment, the unified body is integrally molded.

[0156] In one embodiment, the card further includes at least one of a cell wall 704 for defining a cell in space, a lateral flow promoter 708 along a channel, or an edge structure 709 along a channel, wherein the cell wall, lateral flow promoter, or edge structure is formed within a unified body. In one embodiment, the card further includes a cell wall, which is formed within a unified body.

[0157] In one embodiment, the card further includes an edge structure, the edge structure being formed within a unified body. In one embodiment, the card further includes a lateral flow enhancer, the lateral flow enhancer being formed within a unified body.

[0158] In one embodiment, the lateral flow enhancer is deposited by printing or other means on a uniform body along the channel. In one embodiment, the lateral flow enhancer is a surface treatment.

[0159] In one embodiment, the first and second layers are joined by adhesive. In another embodiment, the first and second layers are integrated by welding.

[0160] One aspect of the present invention is the improvement of the manufacturability of a card by forming cells between an upper and a lower part. In one embodiment, the card includes (a) first and second layers, at least a portion of which are spaced apart to create a space 802 between them; (b) at least one port defined within the first layer; (c) at least one microfluidic channel defined within the space and fluidly connected to at least one port; and (d) a cell wall 704 between the first and second layers, which defines a plurality of cells 703 within the space.

[0161] In one embodiment, at least a portion of the cell is a closed cell 703 that does not communicate fluidly in order to prevent fluid communication between closed cells. In one embodiment, the microfluidic channel is at least partially defined by a portion of the cell wall. In one embodiment, at least a portion of the closed cell does not communicate fluidly with the microfluidic channel.

[0162] In one embodiment, at least a portion of the closed cell is hexagonal 703.

[0163] In one embodiment, the card further includes an adhesive 601 between the first layer and the second layer. In one embodiment, at least a portion of the closed cell contains a portion of the adhesive 601. In one embodiment, the adhesive is UV-curable. In one embodiment, the UV-curable adhesive contains acrylic. In one embodiment, the adhesive is heat-activated. In one embodiment, the adhesive is a screen-printed electrode.

[0164] In one embodiment, the first and second layers are welded either thermally or ultrasonically. In one embodiment, at least a portion of the closed cell contains a portion of the reflow material 601.

[0165] In one embodiment, at least a portion of the microfluidic channel includes a channel support 807 configured to prevent deformation between the first and second layers.

[0166] One aspect of the present invention is a capillary filament breaking element 709 positioned within a microfluidic channel to break capillary filaments. In one embodiment, the card includes (a) at least first and second layers, the second layer defining at least one channel, the first layer covering the second layer, thereby defining at least one corner 803 within the channel between the first and second layers, and (b) one or more capillary filament breaking elements positioned within at least one corner to break capillary filaments.

[0167] In one embodiment, the capillary filament breaking element is an edge structure 709. In one embodiment, the edge structure is a ridge 709. In one embodiment, there are multiple edge structures.

[0168] In one embodiment, the multiple edge structures are patterned. In one embodiment, the pattern is at least one of a square wave pattern 708, a sawtooth wave pattern, a triangular wave pattern, or an irregular pattern. In one embodiment, the pattern is a combination of two or more of the square wave pattern, sawtooth wave pattern, triangular wave pattern, or irregular pattern. In one embodiment, the pattern is a repeating pattern. In one embodiment, the repeating pattern has a pitch of 0.1 to 0.5 mm.

[0169] In one embodiment, the capillary filament breaking element includes a surface treatment that modifies the surface energy and wettability. In one embodiment, the surface treatment is a band having a modified contact angle. In one embodiment, there are alternating bands with varying contact angles in a repeating pattern.

[0170] One aspect of the present invention is a lateral flow enhancer 708 for regulating flow in a microfluidic channel. In one embodiment, the card includes (a) a base 600, (b) at least one microfluidic channel in or on the base for conducting fluid flow, the microfluidic channel being defined by a side 804 and a width 805 between the side halves, and (c) one or more lateral flow enhancers, each lateral flow enhancer extending over at least a substantial portion of the width to generate a lateral capillary action perpendicular to the flow, thereby causing the fluid flow to flow laterally to the side halves and fill the width of the channel before continuing to pass through the lateral capillary enhancing element, thereby resulting in flow over the entire width. In one embodiment, the lateral flow enhancer extends completely over the entire width.

[0171] In one embodiment, the lateral flow enhancer is at least one of a structural element 708, a surface treatment, or a combination thereof. In one embodiment, one or more of the lateral flow enhancers are weirs 708 that increase resistance to flow along the channel. In one embodiment, the weir includes a straight or curved band 708. In one embodiment, the microfluidic channel has a channel height 806, and the band has a band height of 5% to 25% of the channel height.

[0172] In one embodiment, the lateral flow enhancer includes a surface treatment that modifies the surface energy and wettability. In one embodiment, the surface treatment is deposited on the surface of the microfluidic channel by printing or other means.

[0173] In one embodiment, the lateral flow accelerator is arranged along the microfluidic channel at a pitch of 50 to 500 microns. In one embodiment, the lateral flow accelerator is arranged in a repeating pattern. In one embodiment, the repeating pattern is such that the lateral flow accelerator is arranged at equal intervals from one another along the channel. In one embodiment, the repeating pattern is such that there are alternating bands of varying contact angles due to surface treatment.

[0174] In one embodiment, the channel is formed by first and second layers, and the lateral flow accelerator is formed within or on one of the layers. In one embodiment, the lateral flow accelerator is printed or otherwise deposited on at least one of the first or second layers.

[0175] In one embodiment, a lateral flow enhancer is formed within a second layer, the second layer being selected from a Tritan copolymer, polycarbonate, or cyclic olefin copolymer. In one embodiment, the second layer is a Tritan copolymer.

[0176] One aspect of the present invention is capacitive monitoring of flow in a microfluidic channel for high-resolution metering, and / or for detecting the reuse of a previously wet test card, and / or for detecting a test card with insufficient volume. In one embodiment, the card includes (a) a base, (b) at least one microfluidic channel defined in or on the base, wherein the channel is configured to conduct fluid from a port, and (c) two or more capacitive elements 613 and 614 arranged in a contiguous manner along the channel, wherein the capacitive elements are electrically connected by conductive traces 615 outside the channel, thereby enabling the detection of a discrete increase in capacitance as fluid flows from one capacitive element to a contiguous capacitive element. In one embodiment, the two or more capacitive elements include three or more contiguous capacitive elements.

[0177] In one embodiment, the capacitive elements are arranged adjacent to at least one reference element 616 set to a fixed voltage. In one embodiment, the capacitive elements and adjacent reference elements are arranged in a comb-like manner. In one embodiment, a series of capacitive elements share a reference element.

[0178] In one embodiment, the capacitive element includes conductive elements 613 and 614 covered with a dielectric 617 in a channel. In one embodiment, the conductive element is silver. In one embodiment, the conductive element is an electrode screen-printed with silver ink. In one embodiment, the silver conductive element is at least partially covered with carbon. In one embodiment, the dielectric is the dielectric ink of the screen-printed electrode.

[0179] In one embodiment, the channel includes at least one exposed conductive element 702 that is not covered with a dielectric, thereby causing a short circuit when the exposed conductive element is in contact with the fluid sample. In one embodiment, the exposed conductive element is located continuously downstream from the capacitive element, and the capacitive element and the at least one exposed conductive element are also electrically connected by a conductive trace on the outside of the flow cell.

[0180] In one embodiment, the distance between adjacent capacitive elements is greater than the size of the fluid meniscus in the channel. In one embodiment, the ratio of the distance between adjacent capacitive elements to the thickness of each capacitive element is in the range of 3 to 50, or 5 to 30, or 10 to 20.

[0181] These and other advantages may be realized according to specific embodiments and other modifications. It should be understood that the above description is intended to be illustrative and not limiting. Many other embodiments and modifications within the spirit and scope of the claims will be apparent to those skilled in the art who have considered the above description. Accordingly, the scope of the invention should be determined by reference to the appended claims, along with the entire scope of equivalents to which such claims are entitled.

Claims

1. Assay card reading system, at least, A card bay for accepting assay cards, A card interface comprising at least one card interface positioned above or below the card bay and configured to move vertically, wherein the card interface has a plurality of card interface contacts and has at least two states, in a first state the card interface is separated from the card bay, allowing an assay card to be received into the card bay, and in a second state one or more card interface contacts of the card interface are in contact with the assay card, A card reader including, The assay card includes having card contacts, wherein when the card interface is in the second state, at least a portion of the card interface contacts contacts the card contacts. Assay card reading system.

2. The assay card system according to claim 1, wherein the assay card comprises at least a first layer and a second layer, the card contacts are located on the second layer, and a plurality of openings in the first layer provide access to the card contacts.

3. The assay card system according to claim 2, wherein at least a portion of the card interface connector passes through an opening in one layer of the assay card to contact the card contacts at the second position.

4. The assay card system according to claim 2, wherein the first layer is facing upward when the assay card is in the card bay.

5. The assay card system according to claim 1, wherein the card interface further comprises two or more alignment pins that are received in two or more alignment holes in the assay card in the second state.

6. The assay card system according to claim 5, wherein the alignment pin is cone-shaped.

7. The assay card system according to claim 6, wherein the assay card comprises at least a first layer and a second layer, and each of the alignment holes comprises a first layer alignment hole in the first layer and a second layer alignment hole in the second layer, the first layer alignment hole and the second layer alignment hole are arranged to form a through hole, and the first layer alignment hole is larger than the second layer alignment hole.

8. The assay card system according to claim 5, wherein the assay card comprises at least a first layer and a second layer, the card contacts are located on the second layer, a plurality of openings in the first layer provide access to the card contacts, and a tight tolerance fitting between the alignment pins and alignment holes positions the card interface connector to the card contacts so that the card interface contacts pass through the openings in the assay card and make contact with the card contacts.

9. The assay card system according to claim 5, wherein the first alignment pin of the alignment pin corresponds to the first alignment hole of the alignment hole, the second alignment pin of the alignment pin corresponds to the second alignment hole of the alignment hole, the first alignment pin and the alignment hole prevent relative movement in two dimensions between the card interface and the assay card, and the second alignment pin and the alignment hole prevent relative movement only in one dimension between the card interface and the assay card.

10. The assay card system according to claim 9, wherein the first alignment pin and the first alignment hole are circular, the second alignment pin is circular, and the second alignment hole is elongated in one dimension.

11. The assay card system according to claim 1, wherein the card interface contact is a biasing pin connector.

12. The assay card system according to claim 1, wherein the card contact is a conductive pad.

13. The assay card system according to claim 1, wherein at least a portion of the card contacts are island contacts.

14. The assay card system according to claim 12, wherein the conductive pad is screen printed.

15. The assay card system according to claim 1, wherein no contact is formed between the card interface contact and the card contact during insertion of the assay card into the card bay.

16. The assay card system according to claim 1, wherein no electronic connection is made between the assay card reader and the assay card via a card edge connector.

17. The assay card system according to claim 1, wherein the card interface includes a heating plate, and when the card interface is in the second state, the heating plate controls the temperature in the card bay.

18. The assay card system according to claim 1, wherein the assay card reader includes one or more pumps, the assay card includes one or more microfluidic channels having gasketed ports, the card interface compresses the assay card in the second state to seal the gasketed ports, and the one or more pumps are used to pressurize or depressurize the microfluidic channels.

19. The assay card system according to claim 1, wherein the assay card reader includes a housing that defines a slot for providing access to the card bay.

20. It is an assay card reader, Housing and A card bay that is at least partially located within the housing, An electrical contact, wherein the electrical contact is operable and has at least two states, in a first state, it is in a state for receiving an assay card in the card bay, and the electrical contact does not make an electrical connection with the assay card, and in a second state, the electrical contact is activated and makes an electrical connection with the assay card in the card bay, Assay card reader, including...

21. The assay card reader according to claim 20, wherein in the second state, the electrical contact passes through an opening in one layer of the assay card to contact the card contact of the assay card.

22. The assay card reader according to claim 20, further comprising a card interface including at least the electrical contacts.

23. The assay card reader according to claim 22, wherein the card interface further comprises two or more alignment pins that are received in two or more alignment holes in the assay card in the second state.

24. The assay card reader according to claim 23, wherein the alignment pin is cone-shaped.

25. The assay card reader according to claim 22, wherein the card interface includes a heating plate, and when the card interface is in the second state, the heating plate controls the temperature in the card bay.

26. The assay card reader according to claim 22, wherein the assay card reader includes one or more pumps, the assay card includes one or more microfluidic channels having gasketed ports, the card interface compresses the assay card in the second state to seal the gasketed ports, and the one or more pumps are used to pressurize or depressurize the microfluidic channels.

27. The assay card reader according to claim 20, wherein the electrical contact is a biasing pin connector.

28. The assay card reader according to claim 27, wherein no contact is formed between the electrical contact and the card contact while the assay card is being inserted into the card bay.

29. The assay card reader according to claim 20, wherein no electronic connection is made between the assay card reader and the assay card via a card edge connector.

30. The assay card reader according to claim 20, wherein the assay card reader includes a housing that defines a slot for providing access to the card bay.

31. It is an assay card, A base having a planar surface, The card contact on the planar surface, A microfluidic channel within or on the base, Assay card, including

32. The assay card according to claim 31, wherein the assay card comprises at least a first layer and a second layer, the card contacts are located on the second layer, and a plurality of openings in the first layer provide access to the card contacts.

33. The assay card according to claim 32, wherein a portion of the card interface connector passes through the opening and contacts the card contacts.

34. The assay card according to claim 31, further comprising two or more alignment holes for receiving two or more alignment pins of a card interface.

35. The assay card according to claim 34, wherein the assay card comprises at least a first layer and a second layer, and each of the alignment holes comprises a first layer alignment hole in the first layer and a second layer alignment hole in the second layer, the first layer alignment hole and the second layer alignment hole are arranged to form a through hole, and the first layer alignment hole is larger than the second layer alignment hole.

36. The assay card according to claim 35, wherein the first alignment pin of the alignment pin corresponds to the first alignment hole of the alignment hole, the second alignment pin of the second alignment pin corresponds to the second alignment hole of the alignment hole, the first alignment pin and the alignment hole prevent relative movement in two dimensions between the card interface and the assay card, and the second alignment pin and the alignment hole prevent relative movement only in one dimension between the card interface and the assay card.

37. The assay card according to claim 36, wherein the first alignment pin and the first alignment hole are circular, the second alignment pin is circular, and the second alignment hole is elongated in one dimension.

38. The assay card according to claim 31, wherein the card contact is a conductive pad.

39. The assay card according to claim 31, wherein at least a portion of the card contacts are island contacts.

40. The assay card according to claim 38, wherein the conductive pad is screen printed.

41. A method for analyzing a sample using a card reader having electrical contacts, wherein the electrical contacts are operable and have at least a first state and a second state, and the method (a) Depositing the sample onto the assay card, (b) Accepting the assay card while the electrical contacts are in the first state, (c) By operating the electrical contact to the second state, the electrical contact makes an electrical connection with the island contact of the assay card, (d) Analyzing the sample, (e) Outputting the results of the above analysis, Methods that include...

42. The method according to claim 41, wherein step (a) is performed before step (b).

43. The method according to claim 41, wherein step (c) is performed before step (a).

44. Assay card reading system, A card reader including at least one card interface having multiple card interface contacts, Assay card having card contacts, A vertical actuation mechanism is included to cause relative vertical movement between the assay card and the card interface in order to bring at least a portion of the card interface contacts into contact with the card contacts, Assay card reading system.

45. The assay card reading system according to claim 44, wherein the vertical operating mechanism is connected to the card interface and operates the card interface.

46. It is an assay card, First and second layers, wherein at least a portion of the first and second layers is separated to create a space between them, At least one port defined within the first layer, A microfluidic channel defined within the space and fluidically coupled to at least one port, A cell wall between the first and second layers, which defines a plurality of cells within the space, Assay card, including

47. The assay card according to claim 46, wherein at least a portion of the cells are closed cells that do not communicate fluidly so as to prevent fluid communication between the closed cells.

48. The assay card according to claim 47, wherein the microfluidic channel is at least partially defined by a portion of the cell wall.

49. The assay card according to claim 48, wherein at least a portion of the closed cell is not in fluid communication with the microfluidic channel.

50. The assay card according to claim 47, wherein at least a portion of the closed cell is hexagonal.

51. The assay card according to claim 47, further comprising an adhesive between the first layer and the second layer.

52. The assay card according to claim 51, wherein at least a portion of the closed cells contains a portion of the adhesive.

53. The assay card according to claim 51, wherein the adhesive is UV curable.

54. The assay card according to claim 53, wherein the UV-curable adhesive contains acrylic.

55. The assay card according to claim 51, wherein the adhesive is thermally activated.

56. The assay card according to claim 51, wherein the adhesive is screen printed.

57. The assay card according to claim 47, wherein the first layer and the second layer are welded together by thermal or ultrasonic means.

58. The assay card according to claim 57, wherein at least a portion of the closed cell contains a portion of the reflowed material.

59. The assay card according to claim 46, wherein at least a portion of the microfluidic channel includes a channel support configured to prevent deformation between the first and second layers.

60. It is an assay card, Bass and, A microfluidic channel in or on the base for guiding fluid flow, wherein the microfluidic channel is defined by the width of its sides and the width between its sides, One or more lateral flow promoters, each of which extends over at least a substantial portion of the width and generates a lateral capillary action perpendicular to the flow, thereby causing a flow lateral to the sides to fill the width of the channel before the fluid flow continues to pass through the lateral flow promoter, thereby resulting in a flow over the entire width. Assay card, including

61. The assay card according to claim 60, wherein the lateral flow promoter extends completely across the entire width.

62. The assay card according to claim 60, wherein the lateral flow promoter is at least one of a structural element, a surface treatment, or a combination thereof.

63. The assay card according to claim 60, wherein one or more lateral flow promoters are weirs that increase resistance to the flow along the channel.

64. The assay card according to claim 63, wherein the weir includes a straight or curved band.

65. The assay card according to claim 64, wherein the microfluidic channel has a channel height, and the band has a band height of 5% to 25% of the channel height.

66. The assay card according to claim 60, wherein the lateral flow enhancer includes a surface treatment that modifies the surface energy and wettability.

67. The assay card according to claim 66, wherein the surface treatment is printed or otherwise deposited on the surface of the microfluidic channel.

68. The assay card according to claim 60, wherein the lateral flow promoter is arranged along the microfluidic channels at a pitch of 50 to 500 microns.

69. The assay card according to claim 68, wherein the lateral flow promoter is arranged in a repeating pattern.

70. The assay card according to claim 69, wherein the repeating pattern is such that the lateral flow promoters are equally spaced along the channels.

71. The assay card according to claim 69, wherein the repeating pattern is an alternating band of varying contact angles from the surface treatment.

72. The assay card according to claim 60, wherein the channel is formed by first and second layers, and the lateral flow promoter is formed in or on one of the layers.

73. The assay card according to claim 72, wherein the transverse flow promoter is printed or otherwise deposited on at least one of the first or second layers.

74. The assay card according to claim 72, wherein the transverse flow promoter is formed in the second layer, and the second layer is selected from one of Tritan copolymer, polycarbonate, or cyclic olefin copolymer.

75. The assay card according to claim 74, wherein the second layer is a Tritan copolymer.

76. It is an assay card, At least first and second layers, wherein the second layer defines at least one channel, and the first layer covers the second layer, thereby defining at least one corner within the channel between the first and second layers, One or more capillary filament destroyers positioned within at least one corner to destroy the capillary filaments, Assay card, including

77. The assay card according to claim 76, wherein the capillary filament disruptor has an edge structure.

78. The assay card according to claim 77, wherein the edge structure is a ridge.

79. The assay card according to claim 77, wherein the aforementioned edge structure is multiple.

80. The assay card according to claim 79, wherein the plurality of edge structures are located within the pattern.

81. The assay card according to claim 80, wherein the pattern is at least one of a square wave pattern, a sawtooth wave pattern, a triangular wave pattern, or an irregular pattern.

82. The assay card according to claim 81, wherein the pattern is a combination of two or more square wave patterns, sawtooth wave patterns, triangular wave patterns, or irregular patterns.

83. The assay card according to claim 80, wherein the pattern is a repeating pattern.

84. The assay card according to claim 83, wherein the repeating pattern has a pitch of 0.1 to 0.5 mm.

85. The assay card according to claim 76, wherein the capillary filament disruptor includes a surface treatment that alters the surface energy and wettability.

86. The assay card according to claim 85, wherein the surface treatment is a band having a modified contact angle.

87. The assay card according to claim 86, having alternating bands of contact angles that vary in a repeating pattern.

88. The assay card according to claim 76, wherein the first and second layers are adhesively joined together.

89. Assay card reading system, Having an upper side, a lower side, and one or more edges, at least, One or more first alignment reference marks located at least one above the aforementioned edge, One or more second alignment reference marks on the upper or lower side, Assay card including, at least, A card bay for accepting assay cards, One or more first fiducial cooperating members are arranged along at least one side of the card bay and engage with the first alignment fiducial to perform initial alignment of the assay card within the card bay, A card interface located above and / or below the card bay, wherein the card interface comprises one or more second fiducial cooperating members, and has at least two states, namely, a first state in which the card interface is positioned apart from the card bay and allows an assay card to be received in the card bay, and a second state in which the second fiducial cooperating members engage with the second alignment fiducial on the assay card to perform final alignment of the assay card with respect to the card interface, A card reader including, Assay card reading system, including [specific component].

90. The assay card reading system according to claim 89, wherein during the final alignment of the assay card by the second reference mark and the second reference mark cooperative member, there is compliance that allows relative movement between the first reference mark and the first reference mark cooperative member during mating.

91. The assay card reading system according to claim 89, wherein the connector on the card interface and the contact pads on the assay card are aligned during final alignment.

92. The assay card reading system according to claim 89, wherein the optical detector and / or optical light source on the card interface and the optical aperture on the assay card are aligned during final alignment.

93. The assay card reading system according to claim 89, wherein the first alignment reference marks are located only on one or more side edges of the assay card.

94. The assay card reading system according to claim 89, wherein the final alignment does not depend on the alignment between the alignment surface of the assay card and the alignment surface of the card bay.

95. The assay card reading system according to claim 89, wherein the first reference mark and the first reference mark cooperating member are a notch and a latch.

96. The assay card reading system according to claim 95, wherein the first reference mark is a notch and the first reference mark cooperating member is a latch.

97. The assay card reading system according to claim 89, wherein the first reference mark is located on both side edges of the assay card.

98. The assay card reading system according to claim 89, wherein the second reference mark and the second reference mark cooperating member are an alignment pin and an alignment hole.

99. The assay card reading system according to claim 89, wherein the second reference mark is an alignment hole and the second reference mark cooperating member is an alignment pin.

100. The assay card reading system according to claim 99, wherein the alignment pin is at least partially conical.

101. The assay card reading system according to claim 100, wherein the assay card comprises a first layer and a second layer, and the alignment holes on the first layer are larger than the alignment holes on the second layer.

102. The assay card reading system according to claim 89, wherein the card bay has x, y, and z directions, and in the initial alignment, the assay card is aligned with the card bay along the X and Y axes.

103. The assay card reading system according to claim 102, wherein the initial alignment of the assay card along the x and y axes by the first reference mark and the first reference mark cooperative member enables engagement between the second reference mark and the second reference mark cooperative member.

104. The assay card reading system according to claim 103, wherein the initial alignment of the assay card along the x and y axes by the first reference mark and the first reference mark collaborating member enables the engagement of the alignment pins on the card interface with the alignment holes on the assay card.

105. The assay card reading system according to claim 104, wherein the initial alignment of the assay card aligns the second reference mark and the second reference mark collaborating member within 1 mm along the x and y axes.

106. The assay card reading system according to claim 102, wherein the final alignment of the assay card by the second reference mark and the second reference mark collaborating member aligns the card interface and the assay card along the x and y axes by at least 0.5 mm.

107. The assay card reading system according to claim 102, wherein the final alignment of the assay card along the x and y axes by the second reference mark and the second reference mark collaborating member aligns the connector on the card interface with the contact pads on the assay card.

108. The assay card reading system according to claim 107, wherein, in the second state, one or more connectors on the card interface are in contact with one or more contact pads of the assay card.

109. The assay card reading system according to claim 102, wherein the final alignment of the assay card along the x and y axes by the second reference mark and the second reference mark collaborating member aligns the optical detector and / or light source on the card interface with the optical aperture on the assay card.

110. The assay card reading system according to claim 89, wherein the assay card is received into the card bay through a slot to engage the first reference mark with the first reference mark cooperative member.

111. The assay card reading system according to claim 89, wherein the card interface is a platform suspended above the assay card bay.

112. The assay card reading system according to claim 111, wherein the platform is moved vertically between the first state and the second state by an actuator.

113. The assay card system according to claim 111, wherein the platform also includes one or more heating plates, and when the platform is in a second state, the heating plates control the temperature in the card bay.

114. The assay card reader according to claim 111, wherein in the second state, the compressive force of the platform on the assay card in the card bay seals one or more gaskets around the ports on the assay card, thereby transmitting pressure to or from the microfluidic flow cell on the assay card.

115. The assay card reading system according to claim 89, wherein the card reader includes at least one elastic member that biases the assay card upward or downward to keep it in place against the surface of the card bay, and when the card interface moves from the second state back to the first state, the second reference mark cooperating member on the card interface disengages from the second reference mark on the assay card so that the assay card can be removed from the card bay.

116. The assay card reader according to claim 115, wherein the elastic member biases the assay card upward or downward so that the alignment pins on the card interface disengage from the alignment holes on the assay card.

117. The assay card reader according to claim 115, wherein the elastic member is a leaf spring.

118. The assay card reader according to claim 89, wherein the at least one card interface comprises two or more card interfaces, and each of the card interfaces has an independent second reference mark cooperative member for aligning with the assay card independently of the other card interfaces.

119. The assay card reader according to claim 118, wherein the assay card has a second reference mark corresponding to a specific one of the card interfaces and an oversized through-hole corresponding to all other of the card interfaces, so that only one of the card interfaces is aligned with the assay card.

120. Assay card reading system, An assay card having sides and at least one first component of an interlocking mechanism along one of the sides, at least, A card bay for accepting assay cards, A second component of the interaction mechanism for engaging with the first component to hold the assay card in a predetermined position within the card bay, wherein the assay is positioned in the predetermined position by the interaction mechanism and not positioned by the register surface, A card reader including, Assay card reading system, including [specific component].

121. The assay card reading system according to claim 120, wherein the mutual engagement mechanism is at least one of a latch / catch, a cantilever snap-fit, a detent, a key and slot, or a magnetic mutual engagement.

122. The assay card reading system according to claim 121, wherein the mutual engagement mechanism is a latch / catch.

123. The assay card reading system according to claim 122, wherein the first component is a catch and the second component is a latch.

124. The assay card reading system according to claim 123, wherein the latch is a leaf spring that engages with the catch.

125. The assay card reading system according to claim 124, wherein the catch is a notch.

126. The assay card reading system according to claim 122, wherein the first component is a hole, and the second component is a spring-biased pin that engages with the hole.

127. The assay card reading system according to claim 120, wherein the assay card is received into the card bay by a sliding motion.

128. The assay card reading system according to claim 120, wherein the assay card is one of a group of assay cards of various lengths, and each assay card in the group has a first component on its side surface, and the predetermined position is defined by the mutual engagement of the first and second components.

129. The assay card reading system according to claim 128, wherein each assay card in the group includes at least one alignment reference mark, and the alignment reference mark is at the same distance from the first component along the insertion axis for each assay card in the group.

130. The assay card reader according to claim 129, wherein the group of assay cards has a length of 20 to 150 mm, 40 to 100 mm, or 50 to 75 mm and is held in the same predetermined position relative to the position of the first component.

131. The assay card reading system according to claim 120, wherein the mutual engagement of the first and second components provides the initial alignment of the assay card in the card bay.

132. The assay card reading system according to claim 120, wherein the second component is operated to engage with the first component.

133. The assay card reading system according to claim 132, wherein the operation is monitored by a sensor to detect the insertion or removal of the assay card.

134. The assay card reading system according to claim 133, wherein the lock prevents the operation of the second component.

135. The assay card reading system according to claim 134, wherein the assay card reader also includes a card interface above or below the card bay coupled to the lock, the card interface being configured to move perpendicularly to the card bay, and the vertical position of the card reader determines whether the lock engages with or disengages from the second component.

136. The assay card reading system according to claim 131, wherein the at least one first component comprises a first component on each side of the assay card, and the at least one second component comprises two second components, each located on a different side of the assay card in the card bay.

137. The assay card reading system according to claim 120, wherein the card reader includes at least one elastic member that biases the assay card upward or downward to position the assay card in a specific vertical position within the card bay.

138. The assay card reader according to claim 137, wherein the elastic member is a leaf spring.

139. The assay card reading system according to claim 120, wherein the assay card has a polarized front edge that allows the assay card to be inserted into the card bay in only one direction.

140. The assay card reading system according to claim 139, wherein the assay card reader has an obstacle that prevents the front edge from being inserted into the card bay.

141. The assay card reading system according to claim 140, wherein the front edge has a shape that clears the obstacle, and the rear edge has a different shape that does not clear the obstacle.

142. The assay card reading system according to claim 140, wherein the shape of the front edge is asymmetrical, with one side enabling the clearance of an obstacle but the other side not.

143. The assay card reading system according to claim 142, wherein each corner of the front edge has a taper of a different size for clearing obstacles of different sizes.

144. The assay card reading system according to claim 140, wherein the obstacle is a fixed obstacle or a displaceable obstacle.

145. The assay card reading system according to claim 144, wherein the displaceable obstacle is the second component.

146. The assay card reading system according to claim 145, wherein during the insertion of the assay card, the second component is contacted by the tapered corner of the assay card and displaced away from the card bay to allow clearance for the front edge of the assay card, and then acts back toward the card bay to engage with the catch when the assay card is fully inserted.

147. The assay card reading system according to claim 146, wherein the lock prevents displacement of the second component.

148. The assay card reading system according to claim 147, wherein the assay card reader also includes a card interface above or below the card bay coupled to the lock, the card interface being configured to move perpendicularly to the card bay, and the vertical position of the card reader determines whether the lock engages with or disengages from the second component.

149. The assay card reading system according to claim 120, wherein the card reader further includes a slot through which the assay card is inserted into the card reader.

150. The assay card reading system according to claim 120, wherein the assay card includes at least one alignment reference mark, the alignment reference mark is at a specific distance from the first component along the insertion axis.

151. It is an assay card reader, A card bay for accepting assay cards, A card interface positioned above or below the card bay, wherein the card interface has a plurality of elastic connectors, and the card interface has at least two states, in a first state, the card interface is spaced apart from the card bay, the elastic connectors are fully extended, allowing the assay card to be received in the card bay without contact with the card interface, and in a second state, the elastic connectors are in contact with the assay card and are at least partially compressed. Assay card reader, including...

152. The assay card reader according to claim 151, wherein the elastic connector is a spring connector.

153. The assay card reader according to claim 152, wherein the spring connector is at least partially compressed when the card interface is operated to the second state.

154. The assay card reader according to claim 153, wherein the spring connector is compressed from the fully extended length when the card interface is in the first state to the contact length when the card interface is in the second state, and the degree of compression of the spring connector varies to accommodate various assay card thicknesses.

155. The assay card reader according to claim 154, wherein the card reader is configured to accept assay cards having a thickness of 0.1 to 5 mm, or 0.2 to 4 mm, or 0.3 to 3 mm.

156. The assay card reader system according to claim 154, wherein the card reader is configured to accept assay cards whose thickness varies in a ratio of at least 1:50, 1:40, 1:30, 1:20, or 1:10 from the thinnest to the thickest.

157. The assay card reader according to claim 154, wherein, due to a compressive force from the card interface on the assay card, the assay card cannot be removed when the card reader is in the second state.

158. The assay card reader according to claim 154, wherein the spring connector is a spring biasing pin.

159. The assay card reader according to claim 151, wherein the card reader includes at least one elastic member that biases the assay card upward or downward when the assay card is inserted, thereby keeping it aligned within the card bay.

160. The assay card reader according to claim 159, wherein the elastic member is a leaf spring.

161. The assay card reader according to claim 160, wherein the elastic member is configured to be pushed upward by the front edge of the assay card during insertion, allowing the assay card to pass through the elastic member and be received in the card bay.

162. The assay card reader according to claim 161, wherein the leaf spring is fully compressed so that it becomes flush with the card interface, allowing assay cards of various thicknesses to be inserted.

163. The assay card reader according to claim 160, wherein the solid length of the leaf spring is configured to accept assay cards having a thickness of 0.1 to 5 mm, or 0.2 to 4 mm, or 0.3 to 3 mm.

164. The assay card reader according to claim 160, wherein the assay card is inserted by a sliding motion.

165. The assay card reader according to claim 164, wherein the sliding motion occurs through a slot in the card reader.

166. The assay card reader according to claim 159, wherein at least one elastic member is configured as a shutter positioned adjacent to the slot.

167. The assay card reader according to claim 166, wherein the shutter blocks light from entering the slot at least partially.

168. The assay card reader according to claim 166, wherein the shape of the shutter is configured to be pushed upward by the front edge of the assay card inserted through the slot in the card reader.

169. The assay card reader according to claim 151, wherein the card interface moves from the second state to the first state so that the assay card can be removed from the card bay.

170. The assay card system according to claim 151, wherein the card interface includes a heating element, and when the card interface is in a second state, the heating element controls the temperature in the card bay.

171. The assay card reader system according to claim 151, wherein in the second state, the compressive force of the card interface on the assay card in the card bay seals one or more gaskets around the ports on the assay card, thereby transmitting pressure to or from the microfluidic flow cell on the assay card.

172. The assay card reader system according to claim 151, further comprising one or more latching members for capturing the assay card while it is being inserted into the card bay.

173. The assay card reader system according to claim 172, wherein the latching member on at least one side of the card bay provides initial alignment between the assay card and the card bay.

174. The assay card reader system according to claim 173, wherein no corners or walls of the card bay are required to capture the assay card or to provide initial alignment of the assay card within the card bay.

175. The assay card reader system according to claim 151, wherein the edge connector does not make an electronic connection to the assay card while it is being inserted into the card bay.

176. The assay card reader system according to claim 173, wherein two or more alignment pins on the card interface are received by two or more alignment holes on the assay card when the card interface moves from the first state to the second state, providing a final alignment between the electronic contacts on the assay card and the electronic connector on the assembly.

177. The assay card reader system according to claim 151, wherein the card interface includes a heating element, and the elastic connector is sufficiently compressed in the second state so that the heating element contacts the assay card.

178. A card reader, and at least, A card bay for accepting assay cards, A card interface located above or below the card bay and configured to move vertically, wherein the card interface has at least two states, in a first state the card interface is separated from the card bay, allowing an assay card to be received into the card bay, and in a second state the card interface is in contact with the assay card, A card interface having at least one discrete heating zone, wherein the heating zone is in contact with the assay card and controls the temperature of the assay card when the card interface is in the second state, A card reader, including...

179. The card reader according to claim 178, wherein the card interface includes at least two heating zones.

180. The card reader according to claim 179, wherein the heating zone is thermally isolated.

181. The card reader according to claim 180, wherein thermal isolation is achieved by constructing a card interface with a support between the heating zones made of a material having low thermal conductivity.

182. The card reader according to claim 181, wherein the support is made of a polymer material such as rubber or plastic.

183. The card reader according to claim 179, wherein the heating zones are arranged such that a short assay card is exposed only to the first heating zone, and an assay card of increasing length can be exposed to one or more additional heating zones.

184. The card reader according to claim 178, wherein the heating zone comprises a metal heating plate and a closed-loop temperature control system.

185. The card reader according to claim 184, wherein the heating plate is made of aluminum.

186. The card reader according to claim 178, wherein the heating zone comprises an upper heating plate on the card interface and a lower heating plate below the card bay.

187. The card reader according to claim 178, wherein the compressive force of the card interface on the assay card in the second state prevents the assay card from being removed while it is heated.

188. The card reader according to claim 187, wherein the reader is configured to activate the card interface to the first state, thereby releasing the compressive force on the card interface on the assay card and allowing the assay card to be removed.

189. The card reader according to claim 188, wherein the reader is configured to activate the card interface to a third state, in which the upper heating plate is separated from the lower heating plate to maximize the airflow between the heating plates for faster cooling.

190. The card reader according to claim 189, further comprising an airflow system having a high-flow fan configured to move air between an upper heating plate and a lower heating plate in order to maximize cooling.

191. The card reader according to claim 189, wherein one or more obstruction elements partially block entry into the card bay, and the obstruction elements are locked during a cooling phase to prevent insertion of the assay card until the heating zone has cooled to a set temperature.

192. The card reader according to claim 191, further comprising a locking mechanism for locking the obstruction element in a predetermined position, wherein the card interface is coupled to the locking mechanism, and a specific vertical position of the card interface engages or disengages the locking mechanism.

193. The card reader according to claim 192, wherein in the third state, the card interface moves vertically to the specific vertical position further away from the card bay than in the first state, engaging the locking mechanism and locking the obstruction element in place during cooling.

194. The card reader according to claim 193, wherein, after cooling, the card interface moves to return to a vertical position to the first state, thereby releasing the locking mechanism from the obstructing element and enabling insertion of the assay card.

195. The card reader according to claim 193, wherein the card interface is coupled to the locking mechanism by a spring, and the spring releases the locking mechanism when the card interface moves vertically from the third state back to the first state.

196. It is an assay card, Bass and, A microfluidic channel defined within or on the base, wherein the channel is configured to guide fluid from a port, A capacitive element comprising two or more capacitive elements arranged continuously along the channel, wherein the capacitive elements are electrically connected by conductive traces outside the channel, and a discrete increase in capacitance can be detected when the fluid flows from one capacitive element to a successive capacitive element, Assay card, including

197. The assay card according to claim 196, wherein the capacitive element is arranged adjacent to at least one reference element at a set voltage.

198. The assay card according to claim 197, wherein the capacitive element and the adjacent reference element are comb-shaped.

199. The assay card according to claim 198, wherein a series of capacitive elements share a reference element.

200. The assay card according to claim 196, wherein the capacitive element includes a conductive element covered with a dielectric in the channel.

201. The assay card according to claim 200, wherein the conductive element is silver.

202. The assay card according to claim 201, wherein the conductive element is screen-printed silver ink.

203. The assay card according to claim 201, wherein the silver conductive element is at least partially covered with carbon.

204. The assay card according to claim 200, wherein the dielectric is a screen-printed dielectric ink.

205. The assay card according to claim 200, wherein the channel includes at least one exposed conductive element not covered with a dielectric, and contact between the exposed conductive element and the fluid sample results in a short circuit.

206. The assay card according to claim 205, wherein the exposed conductive element is arranged continuously downstream from the capacitive element, and the capacitive element and at least one exposed conductive element are also electrically connected by a conductive trace outside the flow cell.

207. The assay card according to claim 196, wherein the two or more capacitive elements include more than three consecutive capacitive elements.

208. The assay card according to claim 196, wherein the distance between consecutive capacitive elements is greater than the size of the meniscus of the fluid in the channel.

209. The assay card according to claim 205, wherein the ratio of the distance between consecutive capacitive elements to the thickness of each element is 3 to 50, or 5 to 30, or 10 to 20.

210. It is an assay card, A first and a second layer, wherein the first layer covers the second layer and defines a space between them, The space includes a microfluidic channel, The second layer and the channel are formed within a unified body. Assay card.

211. The assay card according to claim 210, wherein the unified body is a pre-molded body.

212. The assay card according to claim 211, wherein the uniform body is embossed.

213. The assay card according to claim 210, wherein the aforementioned unified body is printed on it.

214. The assay card according to claim 210, wherein the aforementioned unified body is integrally molded.

215. The assay card according to claim 214, wherein the unified body is injection molded.

216. The assay card according to claim 210, further comprising at least one of a cell wall defining a cell in the space, a lateral flow promoter along the channel, or an edge structure along the channel, wherein the cell wall, the lateral flow promoter, or the edge structure is formed within the unified body.

217. The assay card according to claim 215, further comprising the cell wall, wherein the cell wall is formed within the unified body.

218. The assay card according to claim 216, further comprising the edge structure, wherein the edge structure is formed within the unified body.

219. The assay card according to claim 217, further comprising the lateral flow promoter, wherein the lateral flow promoter is formed within the unified body.

220. The assay card according to claim 219, wherein the aforementioned unified body is printed on it.

221. The assay card according to claim 219, wherein the uniform body is embossed.

222. The assay card according to claim 219, wherein the aforementioned unified body is integrally molded.

223. The assay card according to claim 222, wherein the unified body is injection molded.

224. The assay card according to claim 210, wherein a lateral flow promoter is printed or otherwise deposited on the unified body along the channel.

225. The assay card according to claim 224, wherein the transverse flow promoter is a surface treatment.

226. The assay card according to claim 210, wherein the first and second layers are adhesively joined together.

227. The assay card according to claim 210, wherein the first and second layers are welded together.

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