Assay devices, methods and reagents including devices and methods for reducing crosstalk between CCD measurements

Abstract

The present disclosure relates to assay devices, methods, and reagents including devices and methods for reducing crosstalk between CCD measurements. Devices, systems, methods, reagents, and kits for performing assays and methods for their preparation are described. They are particularly suited for automated analysis in the form of porous plate assays. Systems, devices, and methods provided herein are also disclosed for generating measurements with reduced crosstalk between measurements of multiple spatially separated objects using a CCD camera-based system. The CCD camera-based system provided herein uses an improved reference level clamping circuit configured to completely reset the reference level during CCD readout, thereby reducing the level of crosstalk between measurements.

Description

[0001] This application is a divisional application of invention patent application 202080031975.4, filed on April 22, 2020, and entitled "Assay devices, methods and reagents including devices and methods for reducing crosstalk between CCD measurements."

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] The provisional patent application is related to U.S. Patent Application No. 14 / 147,216, entitled “Assay Apparatuses, Methods and Reagents,” filed on January 3, 2014, which claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 61 / 749,097, entitled “Assay Apparatus, Methods and Reagents,” filed on January 4, 2013. The disclosures of these patent applications are incorporated by reference in their entirety.

[0004] Reference is also made to commonly owned International Patent Application Publication No. WO 2014 / 107576, entitled “Assay Apparatuses, Methods and Reagents,” published on July 10, 2014, which shares the same specification and drawings as U.S. Patent Application No. 14 / 147,216. This International Patent Publication is incorporated herein by reference in its entirety.

[0005] Reference is also made to U.S. Application Publication Nos. 2011 / 0143947, 2012 / 0195800, 2007 / 0231217, 2009 / 0263904, and 2011 / 025663. The disclosures of each of these applications are incorporated herein by reference in their entirety. Field of the Invention

[0006] The present invention relates to devices, systems, methods, reagents and kits for performing assays. Certain embodiments of the devices, systems, methods, reagents and kits of the present invention can be used for automated sampling, sample preparation and / or sample analysis in a multi-well plate assay format. Background Art

[0007] Many methods and systems have been developed to perform chemical, biochemical, and / or biological assays. These methods and systems are essential in a variety of applications, including medical diagnostics, food and beverage testing, environmental monitoring, manufacturing quality control, drug discovery, and basic scientific research.

[0008] Multi-well assay plates (also referred to as microtiter plates or microplates) have become a standard format for processing and analyzing multiple samples. Multi-well assay plates can be used in a variety of forms, sizes, and shapes. For convenience, there are some standards for instruments for processing samples for high-throughput assays. Multi-well assay plates are usually made in standard sizes and shapes and have standard well arrangements. Well arrangements include those found in 96-well plates (12x 8-well arrays), 384-well plates (24x 16-well arrays), and 1536-well plates (48x 32-well arrays). ANSI / SLAS has published recommended microplate specifications. (See www.slas.org / SLAS / assets / File / ANSI_SLAS_1-2004_FootprintDimensions.pdf.)

[0009] A variety of plate readers can be used to measure measurements in multi-well plates, including readers that measure absorbance changes, luminescence (e.g., fluorescence, phosphorescence, chemiluminescence, and electrochemiluminescence), radiation emission, light scattering changes, and magnetic field changes. U.S. Patent Application Publication 2004 / 0022677 and U.S. Patent No. 7,842,246 by Wohlstadter et al. describe schemes that can be used for single and multiple ECL determinations in multi-well plate form, respectively. They include a plate that includes a plate top with through holes that form a hole wall and a plate bottom that is sealed against the plate top to form a hole bottom. The plate bottom has a patterned conductive layer that provides an electrode surface for the hole, and the electrode surface is used as a solid support for the binding reaction and an electrode for inducing electrochemiluminescence (ECL). The conductive layer may also include an electrical contact for applying electrical energy to the electrode surface.

[0010] Despite such known methods and systems for performing assays, there remains a need for improved devices, systems, methods, reagents, and kits for automated sampling, sample preparation, and / or sample analysis in a multi-well plate assay format.

[0011] In addition, plate reading systems such as CCD (charge coupled device) camera systems are often used to measure the characteristics of analytes in multi-well assay plates. Conventional CCD camera systems may suffer from excessive crosstalk between analyte measurements. The measurement of one analyte may affect the measurement of another analyte that is subsequently measured. The devices, systems, and methods described herein can reduce or eliminate the amount of crosstalk in CCD camera systems used to measure analytes in multi-well assay plates. Summary of the invention

[0012] Embodiments of the present invention include a system configured to measure multiple analytes in an assay plate with reduced crosstalk between analytes. The system includes a CCD camera comprising a CCD, an analog front end circuit, and an analog-to-digital circuit, the CCD camera being configured to measure multiple analytes and coupled to a focusing system and a plate carriage system, the plate carriage system being configured to position the assay plate to facilitate the measurement of multiple analytes. The analog front end circuit is configured to receive a readout signal from a serial register of the CCD and transmit the readout signal to the analog-to-digital circuit to generate multiple image data points, the analog front end circuit including a reference level clamp circuit configured with a time constant, the time constant being selected to completely reset the reference level between consecutive image data point readings, thereby reducing crosstalk between a first measurement of a first of a plurality of analytes and a second measurement of a second of the plurality of analytes.

[0013] In another embodiment, a method for reducing crosstalk between measurements of multiple analytes in an assay plate performed by a CCD camera. The method includes measuring multiple analytes with a CCD camera, the CCD camera including a CCD, an analog front end circuit, and an analog-to-digital circuit, and coupled to a focusing system and a plate carrier system, the plate carrier system being configured to position the assay plate to facilitate the measurement of the multiple analytes; receiving a readout signal from a serial register of the CCD by the analog front end circuit. Receiving the readout signal includes: transmitting the readout signal from the serial register to the analog-to-digital circuit to generate a first image data point, completely resetting a reference level of the analog front end circuit by a reference level clamp circuit having a time constant, transmitting the readout signal from the serial register to the analog-to-digital circuit to generate a second image data point, wherein the time constant is selected to reduce crosstalk between a first measurement of a first of the multiple analytes and a second measurement of a second of the multiple analytes.

[0014] The present invention relates to an apparatus comprising

[0015] a contact platform, wherein the contact platform comprises at least one pair of electrical contact probes, wherein the at least one pair of electrical contact probes comprises upstanding, spring-loaded pins;

[0016] a controller operably connected to the voltage source to conduct voltage to the at least one pair of electrical contact probes,

[0017] a plate carrier frame adapted to transport a single-well addressable multiwell plate and to position the multiwell plate relative to the contact platform so that a voltage can be applied to one or more wells on the plate,

[0018] and an optical sensor positioned above the contact platform, and a first alignment mechanism including a light source projected from the contact platform toward the optical sensor to align the contact platform relative to the optical sensor.

[0019] The apparatus may also include a second alignment mechanism including a plurality of holes on the plate carrier frame, and wherein a light source from the platform shines through the holes to further align the plate carrier frame with the contact platform. The plate carrier frame may include a rectangular opening sized and dimensioned to support a skirt on the periphery of the porous plate. The plurality of holes are located on at least two sides of the rectangular opening. The plate carrier frame may include a latch mechanism to hold the porous plate to the plate carrier frame.

[0020] The apparatus may further include a focusing mechanism disposed on the board carrier frame, which allows the optical sensor to be focused relative to the focusing mechanism. The apparatus may further include a third alignment mechanism, the third alignment mechanism including a conductive surface located on the board carrier frame, so that when the electrical contacts on the contact platform contact the conductive surface, current flows between the electrical contacts on the contact platform to indicate a predetermined distance between the electrical contacts and the board carrier frame.

[0021] In one embodiment, the present invention includes an apparatus for performing a luminescence assay in a multi-well plate. The apparatus includes a light detection subsystem and a plate handling subsystem, wherein the plate handling subsystem includes:

[0022] (a) a light-tight housing comprising an outer shell and a removable drawer, wherein

[0023] (x) a housing comprising a housing top, a housing front, one or more board introduction holes, an inspection hole, a sliding light-tight door for sealing the board introduction holes, and a plurality of alignment features, wherein the housing is adapted to receive the removable drawer, and

[0024] (y) The removable drawer comprises:

[0025] (i) an xy subframe including a plurality of associated alignment features configured to mate and engage with the plurality of alignment features to align a removable drawer within the housing relative to the optical detection subsystem, wherein the weight of the removable drawer is supported by the housing top;

[0026] (ii) one or more panel elevators having a panel elevator platform that can be raised or lowered, wherein the one or more panel elevators are located below the panel introduction hole;

[0027] (iii) a plate translation stage for translating the plate in one or more horizontal directions, wherein the stage includes a plate carrier for supporting the plate, the plate carrier having an opening to allow a plate elevator located below the plate carrier to access and lift the plate, and the plate translation stage is configured to position the plate below the inspection aperture and to position the plate above the plate elevator; and

[0028] (b) one or more board stackers mounted on top of the housing above the board introduction aperture, wherein the board stackers are configured to receive boards or transfer boards to the board elevator; and

[0029] The light detection subsystem includes a light detector mounted on the top of the housing and coupled to the detection hole through a light-tight seal.

[0030] The apparatus can be used to perform luminescence assays in multi-well plates and includes a plate handling subsystem including a plate carrier for supporting the multi-well plate, wherein the plate carrier includes a frame and a plate latch mechanism. The plate latch mechanism includes:

[0031] (a) Plate bracket ledge;

[0032] (b) a plate clamp arm perpendicular to the ledge and comprising a proximal end and a distal end relative to the ledge, wherein the arm is attached to the frame at the proximal end and the arm is rotatable in the xy plane at the distal end, and the arm further comprises an upper clamp including an inclined surface configured to engage a plate;

[0033] (c) a board positioning element comprising a rod, a pedal and a spring, wherein the rod is substantially perpendicular to the arm, substantially parallel to the ledge and attached to a distal end of the arm by the spring, and the pedal is attached to the rod at an angle; and

[0034] (d) a plate wall substantially parallel to the arm and substantially perpendicular to and disposed between the positioning element and the ledge, the wall comprising (i) a lower plate clamp configured to engage with the perforated plate skirt, and (ii) a lower plate clamp ramp configured to drive the lower plate clamp toward the skirt.

[0035] The present invention also relates to a method for joining a multi-well plate in the apparatus just discussed above. The method comprises the following steps:

[0036] (a) placing the plate on the frame;

[0037] (b) compressing the spring of the panel positioning element, thereby urging the pedal against the panel toward the ledge and rotating the arm toward the panel in the xy plane;

[0038] (c) contacting the upper clamp with the plate, thereby pushing the plate toward the bracket wall;

[0039] (d) Bring the lower plate clamp into contact with the skirt, thereby locking the plate in the bracket.

[0040] In addition, the present invention provides an apparatus for performing luminescence assays in multiwell plates and includes a plate handling subsystem including a plate holder for supporting the multiwell plate and a plate latch mechanism,

[0041] wherein the perforated plate has at least a first, a second, a third and a fourth side, and wherein the first and the third side are substantially parallel to each other and the second and the fourth side are substantially parallel to each other,

[0042] Wherein the plate holder defines a hole having a shape substantially the same as the porous plate and having a size smaller than the porous plate to support a ledge positioned around the periphery of the porous plate, wherein the plate holder further includes first (501) and second (513) stop members corresponding to the first and second edges of the porous plate, respectively.

[0043] wherein the plate latch mechanism is movable from an open configuration to receive a multiwell plate to a clamped configuration to latch the multiwell plate to the plate holder,

[0044] wherein the plate latch mechanism comprises a first latch member (509) biased to a clamping position and having a pedal (511) adapted to push a first edge of the multiwell plate toward a first stop, and a plate clamp arm (502) biased to a clamping position and having a bracket (503) pivotally connected to the plate clamp arm (502) and adapted to push a second edge toward a second stop (513), wherein the first latch mechanism (509) is connected to the plate clamp arm (502), and

[0045] The panel latch mechanism includes at least one biased clamp (515) positioned adjacent the second stop (513) to clamp the skirt of the perforated tray to the panel carrier.

[0046] Furthermore, the present invention provides a system comprising

[0047] (i) a multi-well assay plate selected from a single-well addressable plate or a multi-well addressable plate; and

[0048] (ii) A device configured to measure electrochemiluminescence (ECL) from a single well of a single-well addressable plate and from a group of wells of a multi-well addressable plate.

[0049] The present invention also includes a device for measuring luminescence of a multi-well plate of a plate type selected from a single-well addressable plate or a multi-well addressable plate, the device comprising:

[0050] (i) board type recognition interface, used to identify the board type;

[0051] (ii) a plate translation stage for holding and translating the multiwell plate in the xy plane;

[0052] (iii) a plate contact mechanism including a plurality of contact probes and located beneath a plate translation stage and within the range of motion of said stage, wherein said mechanism is mounted on a contact mechanism elevator which can raise and lower said mechanism to bring the probes into and out of contact with a bottom contact surface of a plate when placed on the translation stage;

[0053] (iv) a voltage source for applying a potential to the plate via the contact probe; and

[0054] (v) an imaging system positioned above the plate translation stage and vertically aligned with the plate contact mechanism, wherein

[0055] (a) an imaging system configured to image a P x Q matrix of wells, a plate contact mechanism configured to contact a bottom contact surface associated with the matrix, and a plate translation stage configured to translate the plate to position the matrix in alignment with the imaging system and the plate contact mechanism;

[0056] (b) the apparatus is configured to sequentially apply a voltage to each well in a matrix of a single well addressable plate and to image the matrix; and

[0057] (c) The device is configured to simultaneously apply a voltage to each well in a matrix of a multi-well addressable plate and to image the matrix.

[0058] Also provided is a method of measuring luminescence of a single-well addressable plate or a multi-well addressable plate, wherein the method comprises:

[0059] (a) Loading a plate on a plate translation stage;

[0060] (b) identifying the plate as a single-well or multi-well addressable plate;

[0061] (c) moving the plate translation stage to align a first P x Q matrix of wells with the plate contact mechanism and imaging system;

[0062] (d) raising the plate contact mechanism so that a contact probe on the contact mechanism contacts a bottom contact surface associated with the P x ​​Q matrix of holes;

[0063] (e) if the plate is a single-well addressable plate, generating and imaging luminescence in a P x Q matrix by sequentially applying a voltage to each well in the group while imaging the group;

[0064] (f) if the plate is a multi-well addressable plate, generating and imaging luminescence in a P x Q matrix by simultaneously applying a voltage to each well in the matrix while imaging the matrix; and

[0065] (g) Repeat steps (c) to (f) for the other P x Q matrices in the board.

[0066] The present invention also relates to an apparatus comprising a light detection system having a CCD sensor and an optical lens system. The light detection system is positioned over a single well in a multi-well plate at a time to perform electrochemiluminescence analysis on the single well. The area of ​​the CCD sensor is substantially the same as the area of ​​the single well. The light detection system also includes a cooling device sized and dimensioned to cool the CCD sensor. A heat removal system is provided that includes at least one fan oriented at an angle to draw hot air exhausted from the cooling device into a flow plenum and out of the apparatus.

[0067] Preferably, the optical lens system comprises a plurality of lenses and the plurality of lenses have both spherical surfaces and aspherical surfaces. The area of ​​the plurality of lenses is larger than the area of ​​the single hole and larger than the area of ​​the CCD sensor.

[0068] Preferably, the light detection system is mounted on the top surface of the housing in a substantially vertical orientation, and the heat removal system is also mounted on the top surface of the housing.

[0069] Preferably, the at least one fan is housed in a flow plenum, and the flow plenum also houses at least one printed circuit board (PCB) and includes at least one opening to allow electrical connection between the at least one PCB and electrical components outside the plenum. Preferably, a flow baffle is positioned within the plenum to minimize air recirculation within the apparatus. Preferably, hot air flows over the at least one PCB away from the camera and board before exiting the apparatus.

[0070] Also provided is an apparatus comprising a contact platform, wherein the contact platform comprises at least one pair of electrical contact probes, the electrical contact probes preferably being upright, spring-loaded pins; a controller operably connected to a voltage source to conduct voltage to the at least one pair of electrical contact probes; a plate carrier frame adapted to transport a single-well addressable multi-well plate and position the multi-well plate relative to the contact platform so that voltage can be applied to one or more wells on the plate; and a vertical lift system. The vertical lift system preferably moves the contact platform to contact corresponding electrode contacts located at the bottom of the multi-well plate. The vertical lift system preferably comprises a worm gear mated to the gear portion of the lead screw so that rotation of the worm gear rotates the lead screw. The lead screw is threadedly connected to a support base supporting the contact platform so that rotation of the lead screw raises or lowers the support base and the contact platform.

[0071] Preferably, a motor is used to rotate the worm gear, the motor being controlled by the controller. The vertical lift system may include a guide shaft sized and dimensioned to slide within a corresponding hole in the support base to allow the support base to slide along the guide shaft. Preferably, the worm gear is oriented in a substantially horizontal direction and the lead screw is oriented in a substantially vertical direction. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] The foregoing and other features and advantages of the present invention will become apparent from the following description of the embodiments of the present invention as shown in the accompanying drawings. The accompanying drawings, which are incorporated herein and form a part of the specification, further serve to explain the principles of the invention and enable those skilled in the relevant art to make and use the invention. The accompanying drawings are not drawn to scale.

[0073] 1(a)-(b) show front and back views, respectively, of the device 100 with a stylized cover, and FIGS. 1(c)-(d) show corresponding front and back views, respectively, of the device without the cover.

[0074] Figure 2(a)-(c) show detailed views of the board handling subsystem and the light detection subsystem.

[0075] Figure 3 A view of a removable drawer of a plate handling subsystem within the apparatus 100 is shown.

[0076] 4(a)-(f) show various detailed views of the removable drawer 240 and the subcomponents located within the drawer.

[0077] 5(a)-(o) show detailed views of the board bracket and board latch mechanism.

[0078] Figures 6(a)-(b) show two alternative embodiments of optical focusing mechanisms that may be incorporated into the device.

[0079] Figure 7(a)-(l) show detailed views of the plate contact mechanism.

[0080] Figure 8(a)-(c) show the various components of the light detection subsystem.

[0081] Fig. 9 Non-limiting embodiments of lens configurations that may be used in a light detection subsystem are shown.

[0082] Figures 10(a) and (b) are perspective and side views, respectively, of a device 1000 having a stylized cover; Figure 10(c) is a perspective view of the device 1000 with the stylized cover partially cut away; and Figure 10(d) is a perspective view of the device 1000 with the stylized cover partially cut away.

[0083] Figures 11(a) and (b) show detailed views of the plate contact mechanism of device 1000; Figure 11(c) is an enlarged view of the contact platform; Figure 11(d) is a diagram showing the overlap of electrical contact pins and electrical contacts on a single-well addressable multi-well plate.

[0084] FIG. 12( a ) is a perspective view of the device 1000 without a cover; FIG. 12( b ) is an enlarged partial perspective view showing the heat removal system.

[0085] Fig.13 This is a partial enlarged view of another heat removal system.

[0086] Fig.14 is a cross-sectional view of the light detection system 1010 .

[0087] Fig.15 is a schematic diagram of a lens in the light detection system 1010 .

[0088] Fig.16 is a schematic diagram of a CCD camera device consistent with the implementation scheme of this article.

[0089] Fig.17 A comparison is shown between test measurements completed with a conventional CCD camera system and an improved CCD camera system consistent with embodiments herein.

[0090] Fig.18 Results of analyte measurements performed with a conventional CCD camera system are illustrated.

[0091] Fig.19 is a schematic diagram of an example analog front end circuit optimized to reduce analyte measurement crosstalk.

[0092] Fig. 20 is a process flow diagram depicting a process flow for measuring analytes with reduced crosstalk. DETAILED DESCRIPTION

[0093] The detailed description section provides a description of certain embodiments of the present invention, which should not be considered as limiting, but are intended to illustrate certain creative aspects. Unless otherwise defined herein, scientific and technical terms related to the present invention should have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context requires otherwise, singular terms should include pluralities, and plural terms should include singularities. The articles "one" and "an" are used herein to refer to one or more than one (i.e., at least one) grammatical object of the article. For example, "an element" refers to one element or more than one element. In addition, claims that refer to "including" allow other elements to be included within the scope of the claim; the present invention is also described by referring to the transition phrase "essentially composed of..." (i.e., if other elements do not have a substantial impact on the operation of the present invention, they are allowed to be included within the scope of the claim) or "composed of..." (i.e., only the elements listed in the claim are allowed, rather than auxiliary elements or insignificant activities that are usually associated with the present invention) instead of such claims of the term "including". Any of these three transition words can be used to claim protection for the present invention.

[0094] Specific embodiments of the present invention are now described with reference to the accompanying drawings. The following detailed description is merely exemplary in nature and is not intended to limit the present invention or the application and use of the present invention. In addition, it is not intended to be bound by any express or implied theory presented in the foregoing technical field, background technology, summary of the invention or the following detailed description.

[0095] Described herein is a device for performing assays in a multiwell plate format having one or more of the following desirable attributes: (i) high sensitivity, (ii) large dynamic range, (iii) small size and weight, (iv) array-based multiplexing capability, (v) automated operation; and (vi) the ability to process multiple plates. We also describe components and subsystems used in such a device and methods of using the device and subsystems. The device and method can be used with a variety of assay detection techniques, including but not limited to techniques for measuring one or more detectable signals. Some of them are suitable for electrochemiluminescence measurements, particularly embodiments of multiwell plates (and assay methods using these plates) with integrated electrodes, such as those described in U.S. Publication 2004 / 0022677 and U.S. Patent 7,842,246 to Wohlstadter et al. and U.S. Patent 7,807,448 to Glezer et al., respectively.

[0096] In a preferred embodiment, a device for performing luminescence determination in a multi-well plate is provided. One embodiment includes a light detection subsystem and a plate handling subsystem, wherein the plate handling subsystem includes a light-tight housing that provides a lightless environment in which luminescence measurements can be performed. The housing includes a housing and a removable drawer placed in the housing. The housing also includes a housing top having one or more plate introduction holes, through which the plate can be lowered onto a plate translation stage in the drawer or removed (manually or mechanically) from the plate translation stage. Before performing the luminescence measurement, a sliding light-tight door in the housing is used to seal the plate introduction hole to protect it from ambient light. The housing also includes a detection hole coupled to a light detector mounted on the top of the housing, and one or more plate stackers mounted above the plate introduction hole on the top of the housing, wherein the plate stacker is configured to receive the plate or transfer the plate to a plate elevator in the removable drawer. The removable drawer includes a plate translation stage for horizontally translating the plate in the drawer to an area within the device in which a specific determination process and / or detection step is performed. The removable drawer also includes one or more board elevators having a board lift platform that can be raised and lowered within the drawer, wherein the board elevator is located below the one or more board introduction holes. The board translation stage is configured to position the board below the inspection hole and to position the board above the board elevator on the board lift platform.

[0097] The device also includes a photodetector (e.g., through a light-tight connector or baffle) mounted to the detection hole at the top of the housing. In certain embodiments, the photodetector is an imaging photodetector, such as a CCD camera and may also include a lens. The photodetector may be a conventional photodetector, such as a photodiode, an avalanche photodiode, a photomultiplier tube, etc. Suitable photodetectors also include arrays of such photodetectors. Available photodetectors also include imaging systems, such as CCD and CMOS cameras. The photodetector may also include lenses, light guides, etc., for guiding, focusing and / or imaging light on the detector. In certain specific embodiments, the imaging system is used to image the luminescence of the binding domain array in one or more holes of the assay plate, and the assay device reports the luminescence value of the luminescence emitted from each element of the array. The photodetector is mounted on the top of the housing with a light-tight seal. Additional components of the device include plate contacts for making electrical contact with the plate and providing electrical energy (e.g., for inducing ECL) to the electrodes in the holes below the photodetector.

[0098] Specific embodiments of the device of the present invention are shown in the drawings. Figures 1(a)-(b) show front and rear views of the device 100 with a stylized cover, respectively, and Figures 1(c)-(d) show corresponding front and rear views of the device without a cover, respectively. As shown, for example in Figure 1(c), the device includes a light detection subsystem 110 and a board handling subsystem 120. More detailed views are provided in Figures 2(a)-(b). The board handling subsystem 120 includes a light-tight housing 130, which includes a housing 231 having a housing top 232, a bottom 233, a front 234 and a rear 235. The housing also includes a plurality of alignment features and the housing is adapted to receive a removable drawer 240, which includes a removable drawer front and is formed of an integral cast element. The walls of the removable drawer define a rigid xy subframe, 415 in Figure 4(d), which includes a plurality of accompanying alignment features. When the drawer is properly placed within the housing, the alignment and accompanying alignment features mate and engage, thereby aligning the drawer and its components with the components of the light detection subsystem. When the alignment / accompanying alignment features are engaged, the weight of the removable drawer is supported by the top of the housing. The removable drawer 240 in the device 100 depicted in Figures 1(a)-(b) is best shown in Figure 3 4(a) , which carries various internal subsystems described in detail below, and is mounted within the housing 231 in FIG. 4(b) , wherein the housing rear 235 and housing sides are omitted for clarity. FIG. 4(c) shows the housing 231 with openings and alignment features 405, 406, and 407 positioned and sized to receive the removable drawer 240.

[0099] In one embodiment, the board handling subsystem further includes a board sensor configured to detect a board in the subsystem. Suitable board sensors include, but are not limited to, capacitive sensors, contact switches, ultrasonic sensors, weight sensors, or optical sensors, or combinations thereof.

[0100] Referring to FIG. 2( a), the housing top 232 also includes one or more plate introduction (and discharge) holes 236 and 237, respectively, through which the plate is lowered onto or removed from the plate translation stage (manually or mechanically). Before the luminescence measurement is performed, a sliding light-tight door (shown as 239 in FIG. 2( c)) is used to seal the plate introduction holes 236, 237 from ambient light. In addition, the housing top also includes an identifier controller to read and process data stored in the identifier on the plate. In one embodiment, the identifier controller is a barcode reader (238) mounted above the hole in the housing top through a light-tight seal, wherein the barcode reader is configured to read a barcode on a plate placed on the plate translation stage within the housing. In a preferred embodiment, the barcode on the plate is read once the plate is lowered into the drawer. In an alternative or additional embodiment, the plate includes an EEPROM or RFID and the housing top and / or the drawer includes an identifier controller suitable for communicating with each of these identifiers. In a further additional embodiment, the identifier controller can be provided separately from the device. In this embodiment, information stored in an identifier attached to or associated with a board or a group of boards is transmitted to the device via a computer and / or a network connected thereto and / or manually input via a user interface of the computer and / or network. In this regard, reference is made to U.S. Application Publication No. US2011 / 0022331 and U.S. Patent No. 8,770,471, the disclosures of which are incorporated herein by reference.

[0101] The board handling subsystem further includes one or more board stackers mounted on the housing top 232 above the board introduction openings 236, 237, wherein the board stackers are configured to receive boards or transfer boards to the board elevator. The board handling subsystem optionally includes a heating and / or cooling mechanism (e.g., a resistive heater, a fan, a heat sink, or a thermoelectric heater / cooler) to maintain the temperature of the subsystem at desired conditions. It may also include a humidity control mechanism (e.g., a humidifier and / or dehumidifier, or a desiccant chamber to maintain the humidity of the subsystem at desired conditions).

[0102] FIG4 shows a detailed view of a removable drawer of a plate handling subsystem. Referring to FIG4(a), the drawer includes (i) a plate lifting mechanism 400 having elevating plate lifting platforms 401 and 402; and (ii) a plate translation stage 403 for translating a plate in one or more horizontal directions, wherein the stage includes a plate bracket 404 for supporting the plate. The plate bracket 404 preferably has an opening 420 to allow a plate elevator 400 positioned below the plate bracket 404 to access and lift the plate, and the plate translation stage 403 is configured to position the plate below the detection hole on the housing top 232 and below the light detector in the light detection system 110, and to position the plate above the plate elevator 400. The plate lifting platforms 401, 402 of the plate elevator 400 preferably include a non-slip surface to prevent movement of the plate on the plate lifting platforms during movement in the device. The plate translation stage 403 has horizontal motion, e.g., motion in a substantially horizontal plane or in the X-direction and the Y-direction, for horizontally translating plates in a drawer to one or more areas within the apparatus where specific assay processing and / or detection steps are performed. In one non-limiting example, as shown in FIG. 4( e), the plate translation stage 403 is movable in one horizontal direction along tracks 422, and the plate carriage 404 is movable in an orthogonal horizontal direction on tracks 424 on the plate translation stage 403. In a preferred embodiment, the plate translation stage has two axes of motion, x and y, and motors coupled to the axes of motion allow for automated movement of the plates on the stage.

[0103] The inclusion of a removable drawer 240 in the light-tight housing 130 enhances the operability and manufacturability of the device. To ensure proper alignment of the drawer 240 within the housing 231 and thus proper alignment of the subsystem within the drawer 240 with the light detection subsystem 110, the housing includes a plurality of alignment features and the xy subframe of the drawer includes a plurality of accompanying alignment features configured to mate and engage with the alignment features of the housing. A cross-sectional view of the drawer 240 placed within the housing 231 is shown in FIG. 4( b ), wherein the housing rear 235 and housing sides are omitted for clarity and properly aligned with the light detection subsystem 110.

[0104] In a preferred embodiment, the alignment features of the drawer 240 include a plurality of holes, and the corresponding alignment features on the housing 231 include a plurality of pins sized to fit within the holes. As shown in FIG. 4( c), the housing 231 preferably includes at least three alignment pins, pins 405 and 406 positioned on the housing front 234, and pin 407 positioned on the opposite end of the housing. Additional alignment features may be included in the housing and the drawer as desired. Preferably, the alignment features are positioned or calibrated relative to the housing top so that the weight of the drawer 240 is supported by the housing top 232. The accompanying alignment features on the drawer configured to mate and engage with the alignment pins 405, 406, and 407 are shown in FIG. 4( d) as holes 408, 409, 410 (in the embodiment in FIG. 4( d), the alignment pin 405 mates and engages with the hole 408, the pin 406 mates and engages with the hole 409, and the pin 407 mates and engages with the hole 410). In addition, the drawer also includes alignment latches 416 and 417 (as shown in Figure 4(a)), which mate and engage with accompanying alignment locks 418 and 419 (Figure 4(c)) to lock / unlock the drawer within the housing.

[0105] Because the alignment features 405-407 and 408-410 are positioned or aligned to the housing top 232, when the removable drawer 240 is inserted into the housing 231 guided by the XY frame 415, after the removable drawer 240 is fully inserted into the housing 231, the weight of the drawer 240 and the components thereon are supported by the housing top 232. An advantage of this feature is that, since the light detection system 110 is also mounted on the housing top 232, any calibration or alignment of the subsystems on the drawer 240 with the light detection system 110 can be performed directly with respect to the light detection system 110 without having to take into account any gaps or spacing between the drawer 240 and the housing top 232.

[0106] One or more additional engagement / locking features may be included in the housing and / or drawer, for example, as shown in FIG4(e), where a spring-loaded pin 411 is mounted to the drawer 240 and is configured to mate and engage with a hole 412 located in the board bracket 403. In one embodiment, a solenoid is used to actuate a spring-loaded pin, such as pin 411. In the embodiment shown in FIG4(f), when the board bracket and the board translation stage are aligned, the alignment feature, i.e., pin 411, in the board translation stage mates and engages with a corresponding locking feature, i.e., element 412, in the board bracket, as shown in FIG4(f). These alignment and / or engagement features lock the board bracket in place to protect the subassembly from damage, such as during shipping and / or installation.

[0107] In a further preferred embodiment, as shown in Figures 4(c)-(d), the top of the housing includes an electrical connection contact mechanism 413, and the front of the drawer includes an accompanying electrical connection, i.e., element 414, wherein the electrical connection and its accompanying elements are configured to mate and engage with each other after the drawer is properly inserted and aligned in the housing.

[0108] Referring to FIG. 4( a ), in a preferred embodiment, the plate carrier includes a carrier platform 404 and a plate latch mechanism configured to receive and engage an exemplary plate, hereinafter labeled 426, placed on the carrier platform 404 as shown in FIG. 5( a )-( b ) ( FIG. 5( a ) shows a view of the plate carrier with plate 426 locked in place, and FIG. 5( b ) shows the same view with components of the plate latch mechanism visible and engaged with the plate in the locked position). As shown in FIG. 5( b ), the outer edge of the plate follows standard design conventions for multiwell plates and includes a skirt 522 that surrounds the plate wall and is lower in height than the plate wall (an enlarged view is shown in FIG. 5( o )). In other words, the skirt 522 is positioned proximate to the bottom of the plate 426. The plate latch mechanism is designed to push the outer edges of the skirt on two orthogonal sides of the plate against two corresponding physical stops in the plate carrier to provide a clear and reproducible positioning of the plate in the carrier. The panel latch mechanism is also designed to apply a downward physical force at a defined location on the top of the panel skirt to repeatably and securely retain the panel in the vertical dimension.

[0109] A view of a plate carrier 404 and a plate latch mechanism with a plate 426 is shown in Figures 5(a)-(b). A sequence illustrating the operation of the plate latch mechanism is shown in Figures 5(c)-5(f) and discussed below. In a particular embodiment, the plate carrier 404 supports a porous plate 426 (or a consumable having the same footprint and external physical geometry as a porous / microtiter plate configured for use in an apparatus as described herein) having at least a first side, a second side, a third side, and a fourth side, wherein the first side and the third side are substantially parallel to each other and the second side and the fourth side are substantially parallel to each other. The plate carrier 404 defines a hole 420 having a shape substantially the same as the porous plate 426 and a size smaller than the porous plate to support a skirt or ledge 522 positioned around the periphery of the porous plate 426. The plate carrier also includes first (501) and second (513) stop surfaces that define the horizontal position of the skirt 522 on the first side and the second side of the porous plate, respectively, when the plate 426 is fully locked. The plate latch mechanism is movable from an open configuration, e.g. Figure 5(i) and 5(j) As best shown, to receive the plate 426 into a clamping configuration to lock the plate to the plate bracket, as Figure 5(a) and 5(b) Best shown.

[0110] The plate latch mechanism includes (i) a first latch member (509) biased to a clamped position and consisting of a pedal 511, an actuating rod 510 and a spring 512, which provides a biasing force and preferably has a high spring force. The pedal (511) is used to push the first edge of the porous plate 426 toward the first stop 501 and the plate clamp arm (502) which is also biased to a clamped position by the spring 512, wherein the first latch mechanism (509) is connected to the plate clamp arm (502). The plate latch mechanism also includes (ii) a bracket (503) which is pivotally connected to the plate clamp arm (502) and is suitable for pushing the second edge of the plate 426 toward the second stop (513). The plate latch mechanism also includes (iii) at least one biasing clamp (515) which is positioned near the second stop (513) to clamp the skirt 522 of the porous plate 426 to the plate bracket 404, thereby preventing vertical movement. The clamp 515 engages with the plate skirt and applies a downward force on the skirt of the plate. The support (503) preferably includes at least two legs (504, 506) and both are in contact with the fourth side of the porous plate. At least one leg (504, 506) includes a ramp (507, 508) to apply a lateral force towards the second stopper and a downward force to the skirt of the porous plate (as shown in Figures 5 (e)-(i)).

[0111] The first latch member 509 includes an actuating rod (510) which is biased to a clamped position by a spring (512) and extends past one edge of the board carrier in the clamped position (as shown in FIG. 5( c)). During loading and unloading of boards, as the board carrier 404 moves into alignment with the board elevator, an extension 510a of the actuating rod (510) is pushed against a physical stop in the housing, such as the rear wall of the drawer 240 or the rear portion 235 of the housing, which pushes the extension 510a of the rod (510) into the carrier, as best shown in FIG. 5( d) where the rod 510 is not yet engaged and in FIG. 5( e) where the rod 510 is pushed. It should be noted that as the board carrier 404 moves against the physical stop, the rod 510 and the two biasing clamps 515 are pushed, Figure 5(d) and 5(i)For clarity only the retraction of the rod 510 is shown. The movement of the rod (510) forces the pedal 511 to retract toward the rod 510 to make room for the plate 426. As shown in FIG. 5( c), the pedal 511 is a cantilevered arm connected to the rod 510 and has the ability to bend like a spring. When the rod 510 is pushed inward, the fulcrum 524 fixedly connected to the plate bracket 404 forces the pedal 511 to retract or move in the direction of the arrow shown in FIG. 5( d). The fulcrum 526 can also be located on a sheath 526 covering the first latch member 509, as best shown in FIG. 5( a). The plate clamp arm 502 is preferably pivotally connected to the rod 510 at one end 528 and is preferably pivotally connected to the plate bracket 404 at the opposite end 530. The bracket 503 is pivotally connected to the plate clamp arm 502 at a pivot point 531. As best shown in FIG. 5( d ), as the rod 510 is pushed inwardly, the pedal 511 and the plate clamp arm 502 with the bracket 503 are retracted or moved away from the opening 420 .

[0112] An advantage of pivotally connecting the bracket 503 to the plate clamp arm 502 is that the bracket 503 can be rotated, preferably slightly relative to the plate clamp arm 502, so that the legs 504 and 506 of the bracket 503 can contact the plate 426 during the latching process.

[0113] As described above, when the plate carrier 404 moves against the physical stop, the rod 510 and the two biasing clamps 515 are pushed. When the extension 515a of the biasing clamp 515 is pushed inward, this action lifts the biasing end 515b upward against the force of the spring 532. When the biasing end 515b is lifted to the open position, it is sized and dimensioned to receive the skirt 522 of the plate 426, and when the biasing clamp 515 is released, the spring 532 forces the biasing end 515b downward and clamps on the skirt 522 to hold the plate 426 against upward movement.

[0114] The device also includes an ejector (516) for releasing the plate 426 from the latch mechanism. The ejector 516 has an extended actuating element (521) and a similar actuating rod (510) is also pushed against a stop in the apparatus when the plate carriage is aligned with the plate elevator, so that the ejector moves the porous plate 426 away from the second stop 513. The ejector 516 is preferably spring loaded by a spring 514 and it optionally includes an over-travel preventer 534. The ejector 516 pushes the plate 426 away from the stop 513 when activated, and when the ejector 516 is activated, the rod 510 and the biasing clamp 515 also move to the open position so that the plate 426 can be pushed away from the stop 513 and the biasing end 515b. The over-travel preventer 534 can be elastically deformed to absorb some of the movement of the ejector. Movement of the carriage plate 404 away from the plate loading / unloading position (ie, aligned with the plate elevator) reverses the movement of their rods (510) and ejectors (516) and resets the latch mechanism to the latched configuration.

[0115] The engagement of the perforated plate 426 with the plate latch mechanism to lock the plate 426 in the plate carrier 404 is shown in Figures 5(i)-(m). Figure 5(i) is similar to Figure 5(d), showing the first latch member 509 with the pedal 511 retracted and the arm 502 / bracket 503 in the open position. In Figure 5(j), the latch mechanism remains unengaged and in the open position, allowing the perforated plate 426 to be placed on the opening 420 in the plate carrier 404. In the open configuration depicted in Figure 5(j), the pedal 511, the clamp arm 502, the bracket 503 and the biasing clamp 515 are biased away from the opening 420 to allow the plate 426 to be loaded into the plate carrier 404. As shown in Figure 5(j), both the extensions 510a and 515a are pushed inward by the movement of the plate carrier 404 against the rear stop (e.g., the rear side of the drawer 240 or the rear portion 235 of the housing).

[0116] When the plate 426 is placed into the plate carrier 404 and the plate carrier 404 moves away from the rear stop as shown in FIG5(k), the pedal 511 moves away from the fulcrum 524 and outward to push and bias the plate 426 against the first stop 501. The plate clamp arm 502 also moves with the rod 510, thereby allowing the bracket 503 to push the plate 426 against the second stop 513. As shown in FIG5(k), only the leg 504 is in contact with the plate 426; however, due to the pivot connection at the pivot point 531, the second leg 506 will automatically and quickly contact the plate 426 when the bracket 503 rotates about the pivot 531. The biasing clamp 515, preferably spring loaded by the spring 532, engages the plate skirt 522 of the perforated plate 426 on the second edge of the plate as shown in FIG5(l), and the bracket 503 also engages and pushes down on the plate skirt 522. As described above, the legs 504 and 506 of the bracket 503 have ramps 507, 508 and are angled as shown. When the legs 504 and 506 push the plate 426, the ramps 507, 508 contact the skirt 522 and push the plate 426 in two directions: toward the second stop 513 and downward. As shown in FIG. 5(m), the biasing clamp 515 engages with the plate skirt 522.

[0117] In a preferred embodiment, the plate holder 404 also includes an optical focusing mechanism used by an optical sensor in the device, such as a light detector within the above-mentioned light detection system 110, to measure contrast and focus. The optical focusing mechanism includes at least two, or preferably at least three patterned surfaces at different heights relative to the plate holder, and therefore relative to the target surface, for focusing (i.e., the bottom of the wells of the 96-well plate 426 is held in the plate holder 404). The present invention includes a method for imaging multiple surfaces and calculating the magnitude and direction of the image adjustment required to focus the target surface based on the image. In one embodiment, the contrast value of the image of each surface is calculated, and the focus height is determined as the height at which the contrast changes minimally with height changes, or, alternatively, the height below a predetermined threshold.

[0118] In one embodiment, the plate carrier includes at least three patterned surfaces, each surface being at a different height relative to the plate carrier. Two alternative embodiments of the optical focusing mechanism are shown in Figures 6(a)-(b). In certain preferred embodiments, the surface has a pattern of different transparencies (e.g., a pattern etched or cut into an opaque substrate or a patterned opaque ink or film printed on a transparent surface), so that the pattern can be imaged using light transmitted through the substrate. In alternative embodiments, the surface / pattern is not transparent, and the pattern is imaged using a light source that reflects light off the surface.

[0119] The focusing mechanism includes at least high, medium and low patterned surfaces spaced from the optical sensor, wherein the medium patterned surface and the target surface are aligned to substantially the same plane level, wherein a first distance between the high and medium patterned surfaces and a second distance between the medium surface and the low patterned surface are substantially equal, and wherein the optical sensor and the patterned surface are moved relative to each other until a difference between a first pair of contrast values ​​between the high and medium patterns and a second pair of contrast values ​​between the medium pattern and the low pattern is less than a predetermined value of about ±2.0 dimensionless units, as described below. The difference can be ±3.0 or ±4.0, or as low as ±1.0. The higher the contrast difference, the easier but less accurate the focusing, and the lower the contrast difference, the more difficult but more accurate the focusing.

[0120] As shown in Fig. 6 (a)-(b), the mechanism preferably includes a plurality of patterned surfaces, such as at least two and optionally three patterned surfaces (601-603), and the patterned surface includes substantially the same pattern, such as a grid pattern. The patterned surfaces are preferably adjacent to each other in a group. In the embodiment shown in Fig. 6 (a), the mechanism also includes an unpatterned surface 604. Preferably, each patterned surface is located on a parallel plane. In a preferred embodiment, the height of the middle patterned surface is effectively equal to the focal position of the hole in the porous plate 426 filled with a predetermined amount of fluid. The low patterned surface is at a height of about 0.25mm below the middle patterned surface, and the upper patterned surface is at a height of about 0.25mm above the middle patterned surface. In one embodiment, the low patterned surface is located above the plate bracket (that is, above the support platform on which the plate rests) at a height of about 4-4.75mm. Preferably, the low patterned surface is at a height of about 4.5-4.7mm above the plate carriage, and most preferably, the low patterned surface is at a height of about 4.6-4.7mm above the plate carriage. The medium patterned surface is at a height of about 4.5-5.0mm above the plate carriage, preferably, at about 4.7-4.9mm above the plate carriage, and most preferably, at about 4.7-4.8mm above the plate carriage. And the high patterned surface is at a height of about 4.75-5.10mm above the plate carriage, preferably at about 4.8-5.0mm above the carriage platform, and most preferably at about 4.85-4.95mm above the plate carriage. It is worth noting that any one of the surfaces 601, 602 and 603 can be a medium patterned surface, a high patterned surface or a low patterned surface. In a preferred embodiment, the optical focusing mechanism is adjacent to the plate carriage.

[0121] Therefore, the present invention provides a method for focusing an optical sensor to a target surface, comprising the following steps: (a) providing at least high, medium and low patterned surfaces 601-603, wherein the medium patterned surface and the target surface are located at the same focal height, and wherein a first distance between the high patterned surface and the medium patterned surface is substantially equal to a second distance between the medium surface and the low patterned surface; (b) obtaining a first contrast value difference between the high and medium patterned surfaces using an optical sensor; (c) obtaining a second contrast value difference between the medium and low patterned surfaces using an optical sensor; (d) comparing the first and second contrast value differences and determining whether the target surface is in focus and / or determining the magnitude and direction of focus adjustment required to bring the target surface into focus.

[0122] During operation, the plate translation stage 403 translates the plate carriage 404 to position the optical focusing mechanism at Figure 7(a)-7(c)(1)7( c )(1) . The light outlets 725-728 may be connected to a single light emitting diode (LED) or each light outlet may have its own LED or other light source. The light source is illuminated and the light beam is irradiated on the underside of the optical focusing mechanism, more specifically under the surfaces 601-603. Preferably, the light outlets 725-728 provide uniform illumination for the surfaces 601-603. Thus, the optical sensor or camera in the light detection subsystem 110 images the optical focusing mechanism, calculates the difference in the above contrast values, and determines whether the target is in focus and / or determines the magnitude and direction of the focus adjustment required to put the target surface in focus. Based on the calculation, the focus of the optical sensor is adjusted manually or automatically accordingly, for example by using a motorized focus adjustment. Preferably, the method further comprises the steps of adjusting the distance between the optical sensor and the target surface, and repeating the steps of obtaining the first and second contrast values ​​and comparing these contrast values ​​until the difference between the first and second contrast values ​​is less than a predetermined value. A suitable calculation for determining the contrast value is to obtain a region of interest (ROI) of the image covered by a dot pattern of a focused object, such as surface 601, 602 or 603 or a portion thereof. The mean and standard deviation of all pixels within the ROI are measured. The mean (AVG) and standard deviation (StDEV) are measured or determined to calculate the contrast value (%CV) for the ROI.

[0123] %CV = (StDEV / AVG) x 100

[0124] The %CV for each ROI (high and low) is then subtracted to create a difference value reported to the operator. The %CV shown above is a unitless or dimensionless value.

[0125] The preferred predetermined value for the difference in %CV contrast values ​​is experimentally determined to be ±2.0 by comparing the ECL values ​​as a function of defocus from nominal. The magnitude of this difference may vary depending on the contrast function. A certain amount of defocus is acceptable without affecting the ECL. The preferred value of ±2 is within this range. Smaller values, such as ±1.5 or ±1.0 would be more accurate but also more difficult to achieve during focusing operations. Larger values, such as ±3.0 or ±4.0 would be less accurate but easier to achieve. A person of ordinary skill in the art can balance accuracy and operational difficulty based on the teachings of the present invention. Differences in contrast values ​​between ±1.0 and ±4.0 are within the scope of the present invention.

[0126] Other methods of calculating or determining contrast values ​​may be used, such as those discussed by Eli Peli in "Contrast in Complex Images," Journal of the Optical Society of America, Vol. 10, October 1990, pp. 2032-2040. This reference is incorporated herein by reference in its entirety.

[0127] In addition, the plate bracket 404 includes a plurality of reference elements. One reference element includes a conductive bottom surface 536 disposed on the bottom surface of the plate bracket 404, as shown in FIG. 5( n), which is used to train the positioning of a contact mechanism during the setup of the device, the contact mechanism being used to contact the bottom of the plate 426 held in the plate bracket 404. The contact mechanism described in more detail below includes a series of spring-loaded contact members and can be raised to contact the bottom surface of the plate 426, for example to start an ECL measurement. As shown in FIG. 5( n), the conductive bottom surface 536 is located on the lower side of the plate bracket 404, and when the plate 426 is locked in the plate bracket 404, it is configured to be at the same height as the bottom of the plate. During the setup or adjustment of the device, the contact mechanism is raised until it reaches a height at which the contact member contacts the surface 536, as detected by electrically measuring the drop resistance between the contact members, indicating that the contact member has properly contacted the conductive surface 536 and will properly contact the bottom of the plate during the ECL measurement. The measured height is used to set the contact mechanism height of the contact plate 426 held in the plate bracket 404.

[0128] Further, the plate holder 404 includes another reference element (depicted in FIG. 5( c) as semicircular holes cut into the plate holder 404, i.e., elements 517-520). A light source in the contact mechanism, such as a light outlet or LED 722, is projected through each hole 517-522. The plate translation stage 403, which moves in the horizontal plane discussed above, positions each hole 517-522 above the light outlet 722 shown in FIG. 7( c)(1). The light projected through each hole is imaged by a light detector in the light detection system 110 to reference the position of the plate holder 404 in the xy space of the horizontal plane relative to the other components of the device. In a preferred embodiment, the reference element includes one or more notches or cutouts, for example, on the edge of the plate platform, for example, as shown in FIG. 5( c), at both ends of the reference surface / stop (501) and (503). Advantageously, the element can also be imaged to confirm whether the plate is in the correct orientation.

[0129] Light outlet 722 and light outlet 725-728 are preferably illuminated by a single LED. Suitable LED can be connected to a light pipe or waveguide to the light outlet. Depending on the voltage applied, suitable LED can have different intensity outputs. In an example, as shown in Fig. 7 (h), LED 739 is connected to multiplexer 738. Microprocessor 729 can instruct multiplexer 738 to apply a first voltage to LED 739 to activate light outlet 722 and apply a second voltage to LED 739 to activate light outlet 725-728. Or, multiple LEDs can be used for light outlet.

[0130] The board handling subassembly also includes one or more transport locks for locking the board tray in place during transport, as described above and best illustrated in Figure 4(e). In a preferred embodiment, the transport lock includes a solenoid-driven pin 411 on the removable drawer 240, which is received in a hole 412 on the board translation stage 403. The board tray 404 rides on rails 422, 424 and preferably includes a clamp to lock the tray in place. Further, the board tray 404 includes a board orientation sensor, such as an accelerometer or electronic level commonly used in smartphones, to ensure that the porous plate 426 placed on the board tray 404 is in the correct orientation. Alternatively, a reflective optical sensor can be attached or adhered to the board tray and sensed by the camera. Optionally, ultrasonic sensors, contact switches, and capacitive sensors can also be used.

[0131] The plate handling subassembly 120 also includes a plate contact mechanism, which includes an electrical contact probe mounted on a plate contact elevator, which is used to raise the probe to contact the electrical contacts on the bottom of the porous plate 426 discussed above, which are in turn connected to electrodes in the holes of the plate. The contact probe is used to apply an electrical potential to the electrodes in one or more holes of the porous plate 426. The plate contact mechanism and the imaging device are aligned so that electrical contact is formed, wherein the hole or hole group is located directly below and in the imaging area of ​​the imaging device. The contact mechanism is shown in Figures 7 (a)-(b) and includes a contact mechanism platform 701, which includes four interrogation zones 702-705, each of which includes a pair of electrical contact probes to conduct voltage potential to the interrogation zone. Preferably, the interrogation zones 702-705 are arranged in quadrants or a 2×2 matrix. However, the interrogation zones can be arranged in a linear manner or in any PxQ matrix, where P and Q are integers and can be different from each other. As discussed in more detail below, the multiwell plates 426 useful in the apparatus 100 of the present invention can be arranged in an MxN matrix, where an MxN matrix is ​​larger than a PxQ matrix. As described above, a PxQ matrix can be 12x8, 24x16, 48x32 wells, or any number of wells.

[0132] The device also includes a controller operably connected to a voltage source, wherein the voltage source is connectable to one or more pairs of electrical contact probes, and a multiplexer connected to the controller and the voltage source for selectively connecting the voltage source to a pair of electrical contact probes at a single interrogation zone or to the pair of electrical contact probes at more than one interrogation zone. As used herein, "voltage source" includes both voltage sources and current sources. A block diagram showing the components of the controller is shown in FIG7(h), which includes a microprocessor 729 connected to a power supply 730 and a digital-to-analog converter 731, which is connected to low pass filters 732 and 733, a current monitor 734, another optional power supply 737 and an analog-to-digital converter 736, and a multiplexer 738. The controller is also operably connected to an LED 739, which is a component of the contact mechanism described above.

[0133] The multiplexer 737 controlled by the processor 729 guides the application of the potential identified as above based on the type of plate used in the apparatus. If the porous plate 426 is configured to analyze one hole at a time, referred to herein as a single-hole addressable plate, wherein the hole of the plate corresponds to the area of ​​the contact mechanism platform, the multiplexer 737 will guide the selective application of the potential by electrically isolating each area and selectively applying the potential only in the first area. On the other hand, if the porous plate is configured to analyze two or more holes at a time, referred to herein as a porous addressable plate, the multiplexer 737 will guide the selective application of the potential by electrically connecting two or more areas and selectively applying the potential in these two or more areas. In one embodiment, the plate includes a bar code, and the bar code includes plate configuration information and the device 100 includes a bar code reader 238, which reads the plate configuration information and identifies the type of the plate placed in the stacker.

[0134] In a preferred embodiment, the device includes a plurality of interrogation zones 702-705 arranged in a P x Q matrix. Preferably, the P x ​​Q matrix is ​​a 2 x 2 matrix. The pair of electrical contact probes on the plate contact mechanism platform 701 preferably include upright pins, such as spring-loaded pins. Further, the device preferably also includes an optical sensor, such as a light detector in the light detection system 110 located above the platform 701, and the platform 701 includes a first alignment mechanism, which includes a light source, such as a light outlet 722 protruding from the platform toward the optical sensor, to align the platform 701 relative to the optical sensor. In one embodiment, the light source (e.g., an LED or other type of bulb) is located below a hole in the contact mechanism (e.g., a through hole (722) centered on the platform (701) as shown in Figure 7(c)(1)) and emits light. The device also preferably includes a second alignment mechanism, which includes a plurality of holes located on the board carrier frame (e.g., elements 517-520 shown in Figure 5 (c)) and a light source 722 from the platform 701 can be illuminated through these holes and detected by the optical sensor to further align the board carrier frame with the platform 701. The plurality of holes can be positioned on at least two sides of the board carrier frame (see the description above). In addition, the device also preferably includes a third alignment mechanism, which includes a conductive surface located on the board carrier frame (e.g., surface 536 in Figure 5 (n)) so that when the electrical contacts on the platform contact the conductive surface, current flows between the electrical contacts on the platform to indicate a predetermined distance between the electrical contacts and the board carrier frame. The device preferably includes a fourth alignment / focusing mechanism, which includes a patterned focusing target (e.g., surfaces 601-603 in Figures 6 (a) and 6 (b)), and the contact mechanism platform includes one or more light sources for passing light through the pattern to achieve imaging of the pattern discussed above. The light source can be a light source below the hole (722) as described above. Optionally, multiple light sources (e.g., LEDs or other types of bulbs) can be used to produce a wider and more uniform light field, for example, four LEDs (725-728) embedded in the plate contact mechanism platform as shown in Figure 7(c)(1).

[0135] In a preferred embodiment, the device is adapted to interrogate a sample contained in a multiwell plate, wherein the multiwell plate comprises a plurality of wells arranged in an M×N matrix, and the device comprises a carriage frame configured to support the multiwell plate, wherein the carriage frame is movable relative to a contact mechanism platform comprising a plurality of interrogation zones, wherein each interrogation zone comprises at least one pair of electrical contact probes to apply a voltage potential to at least one well. The device further comprises a controller operably connected to the motor to move the carriage frame relative to the platform and operably connected to a voltage source, wherein the voltage source is connectable to one or more pairs of electrical contacts, and a multiplexer connected to the controller and the voltage source for selectively connecting the voltage source to a pair of electrical contact probes of a single interrogation zone or to connecting the voltage source to at least one pair of electrical contact probes of more than one interrogation zone. Preferably, the interrogation zones are arranged in a P×Q matrix and the M×N matrix is ​​larger than the P×Q matrix, and the P×Q matrix may be a 2×2 matrix. Preferably, each interrogation zone is sized and dimensioned to interrogate one well on the multiwell plate 426.

[0136] Preferably, the electrical contact probe on the contact mechanism platform includes a plurality of working electrode contact probes, and the plurality of working electrode contact probes are selectively connected to a voltage source by a controller to determine the number of holes to be queried. In one embodiment, the working electrode probe is connected to the working electrode in a hole, or alternatively, a working electrode probe is connected to the working electrode in multiple holes. The unconnected working end electrode probe can be electrically isolated in a multiplexer when not in use, thereby allowing the use of multiple working electrode probes (e.g., 4 probes) to apply potentials to multiple working electrodes in multiple holes, one hole at a time (e.g., applying potentials to a group of 4 holes, one hole at a time). The electrical contacts on the platform can also include a plurality of counter electrode probes, which are electrically connected to at least one electrical circuit or an electrical path, or alternatively at least one electrical grounding. In one embodiment, the bottom electrical contacts of the porous plate connected to the counter electrode probes for multiple holes on the platform are electrically connected. Alternatively, the bottom electrical contacts of the porous plate connected to the counter electrode probes for all holes on the platform are electrically connected. Further, the bottom electrical contacts of the porous plate connected to the counter electrode probes for at least one hole on the platform can be electrically isolated. The controller can interrogate P x ​​Q or fewer holes simultaneously.

[0137] Referring to Figures 7(c)(2)-(g), the contact mechanism platform 701 includes a plurality of working contact probes 706-713 and counter contact probes 714-721. As shown in Figure 7(c)(2), if the controller 709 is configured to electrically connect two or more interrogation zones, the instrument 100 selectively applies a potential within two or more zones (e.g., zones 703 and 704), thereby applying a potential between the working electrode contact probes 706 and 710 and 709 and 713, respectively, and connecting the counter electrode contact probes 714-717 and 718-721. The connection of the counter electrodes at the platform 701 and the plate 426 is discussed below. As also described below, only one working contact electrode and one counter contact electrode are required. Every two connections provide redundancy for the system so that an ECL signal is generated even when one electrode fails.

[0138] Alternatively, if the switch mechanism is configured to electrically isolate each region, the instrument selectively applies a potential within a first region, e.g., as in FIG. 7( d ), where region 703 is isolated and a potential is applied across working electrode contact probes 706 and 710. In one embodiment, all counter electrode contact probes 714-717 and 718-721 connected to ground are electrically connected at platform 701. As discussed below in conjunction with FIG. 7( k ), the counter electrode contact probes for each well are isolated by the counter electrode at the bottom of plate 426. In the example shown in FIG. 7( d ), the well directly above region 703 has counter electrodes connected to counter electrode contact probes 718 and 719, but is isolated from the other counter electrode contact probes on platform 701. Alternatively, the counter electrodes for each interrogation zone may be isolated at platform 701.

[0139] Similarly, Figures 7(e)-(g) show how the contact mechanism is configured to apply a potential within first regions 702 (Figure 7(e)), 705 (Figure 7(f)) and 704 (Figure 7g), and to apply a potential at working contact probes 707 and 712 (in Figure 7(e)), 708 and 711 (in Figure 7(f)) or 709 and 713 (in Figure 7(g)), respectively, although counter-contact probes 714-717 and 718-721 are electrically connected at platform 701, but the counter-contact probes of each interrogation zone are isolated by counter electrodes on holes in plate 426 directly above each interrogation zone. Preferably, the contact probes are each an independent spring-loaded contact member, such as a contact pin.

[0140] In a preferred embodiment, the multi-well plate 426 includes a bottom electrical contact for each well located on the bottom surface of the plate, wherein the bottom electrical contact is configured to contact a pair of electrical contact probes on the platform 701. The bottom electrical contacts include a counter electrode contact connected to the counter electrode in the well of the plate and a working electrode contact connected to the working electrode in the well of the plate. Each well includes at least one working electrode and one counter electrode, which, depending on the plate format, can be electrically connected (bused) or electrically independent of the working electrodes and counter electrodes in other wells of the plate.

[0141] A non-limiting set of exemplary bottom electrical contact patterns are shown in Figures 7(i)-(l), where Figure 7(i) shows a pin contact configuration of platform 701 substantially similar to Figure 7(c)(2). Figure 7(k) shows the overlap of bottom electrical contacts under exemplary four wells covering interrogation zones 702-705. Each well has a bottom counter electrode 740 and two working electrodes 742 and 744 in an exemplary "Z-shape". The bottom counter electrodes 740 are not electrically connected to each other, so the counter electrodes for each well or each interrogation zone are separated or isolated at plate 426.

[0142] For region 703, a Z-shaped bottom counter electrode 740 is connected to counter electrodes 718 and 719. Bottom working electrodes 742 and 744 are connected to working electrodes 710 and 706, respectively.

[0143] For region 705, Z-shaped bottom counter electrode 740 is connected to counter electrodes 720 and 721. Bottom working electrodes 742 and 744 are connected to working electrodes 711 and 708, respectively. Regions 702 and 704 are similarly connected.

[0144] The next electrical connection is to the interior of the well itself. As shown in FIG. 7( l ), ​​each well in this example has a well working electrode 750 and well counter electrodes 752 and 754. Here, the well working electrode 750 has a Z shape and is connected to the bottom working electrodes 742 and 744, and the well counter electrodes 752 and 754 are connected to the bottom counter electrode 740.

[0145] For region 705, working electrodes 711 and 708 on platform 701 are connected to bottom electrodes 742 and 744 of each well and well working electrode 750. Counter electrodes 720 and 721 on platform 701 are connected to bottom counter electrodes 740 and well counter electrodes 752 and 754 of each well. The Z-shape of bottom electrode 740 and well electrode 750 is designed to bear sufficient electrical contact. Any shape can be used and the present invention is not limited to any particular shape.

[0146] As shown in the above discussion, each well and each interrogation zone has two working electrodes, such as 708 and 711 for region 705, and two counter electrodes, such as 720 and 721 for region 705. As shown above, both the working and counter electrodes are electrically connected to the well. Only one pair of working and counter electrodes is required to conduct the ECL potential into the well. The other pair is used for redundancy in case one or more electrodes fail.

[0147] It should also be noted that in the examples discussed above in conjunction with Figures 7(i), 7(k) and 7(l) where each well can be interrogated individually, the working electrode of each interrogation zone and well is isolated at platform 701 and multiplexer 738, and the counter electrode of each interrogation zone and well is isolated at plate 426 and its bottom electrode and well electrode.

[0148] FIG7( j) illustrates an example where four wells covering interrogation zones 702-705 can be interrogated simultaneously using contact pins or electrodes from the same platform 701. As shown, the multi-well plate 426 has a bottom working electrode 760 covering working electrodes 707, 708, and 709. The plate 426 also has a bottom counter electrode 762 covering at least counter electrodes 719, 720, 715, and 716. The bottom working electrode 760 and the bottom counter electrode 762 are electrically connected upward to all four wells. Activating one or more working electrodes 707, 708, and 709 and one or more counter electrodes 719, 720, 715, and 716 will provide ECL potentials for all four wells. Multiple available working electrodes and counter electrodes also provide redundancy.

[0149] According to an embodiment of the present invention, the bottom of the plate includes an internal electrical contact conduit connected to the bottom electrical contact to conduct the voltage potential into the hole. In one embodiment, the bottom electrical contact of at least one hole is electrically isolated from the bottom electrical contact of the adjacent hole, and optionally, the internal electrical contact conduit of at least one hole can be electrically isolated from the bottom electrical contact of the adjacent hole. Reference is made to U.S. Patent 7842246 and U.S. Application 20040022677 (both filed on June 28, 2002, entitled "AssayPlates, Reader Systems and Methods for Luminescence Test Measurements", which are incorporated herein by reference), which discloses other embodiments of the bottom of the plate that can be interrogated by the contact mechanism disclosed herein.

[0150] Thus, the present invention provides a method for interrogating a sample contained in a multiwell plate having an M x N well matrix, comprising the steps of: (a) providing a plate contact mechanism platform having a plurality of interrogation zones, (b) providing at least one pair of electrical contact probes (e.g., a working electrode contact probe and a counter electrode contact probe) for each interrogation zone, wherein each interrogation zone is adapted to interrogate a single well, (c) selectively applying a voltage potential to: (i) one interrogation zone to interrogate one or more wells simultaneously or (ii) multiple interrogation zones to interrogate multiple wells, and (d) moving the multiwell plate relative to the platform to interrogate additional wells. A single well may be interrogated, or M x N wells (where M x N is greater than a P x Q matrix) may be interrogated. The method may further comprise the step of: (e) controlling the application of the voltage potential in step (c) by selecting at least one positive active contact probe (e.g., a working electrode probe) of the pair of electrical contact probes on the platform to be connected to the voltage potential. Step (e) may further comprise the step of electrically isolating at least one positive active contact probe that is not connected to the voltage potential. The method may also include the steps of: (f) providing a bottom electrical contact on the bottom surface of the multiwell plate, and optionally, (g) electrically isolating at least one electrical loop or optionally at least one ground contact probe (e.g., a counter electrode probe) from the bottom electrical contact. Optionally, all electrical loops or ground contact probes from the bottom electrical contact are isolated from each other.

[0151] As described above, the device can be used to measure the luminescence from two alternative types of porous plates, single-hole addressable plates (i.e., plates interrogated one hole at a time by the device) and / or multi-hole addressable plates (i.e., plates interrogated one sector at a time by the device, wherein a sector is a group of adjacent holes). Various types of porous plates are described in U.S. Patent 7842246 and U.S. Application 20040022677, including single-hole and multi-hole addressable plates (both filed on June 28, 2002, entitled "Assay Plates, Reader Systems and Methods for Luminescence Test Measurements", incorporated herein by reference). The plate of the present invention includes several elements, including but not limited to the top of the plate, the bottom of the plate, a plurality of holes, a working electrode, a counter electrode, a reference electrode, a dielectric material, an electrical connection, a conductive through hole and an assay reagent. The hole of the plate is defined by the hole / opening at the top of the plate, the bottom of the plate can be fixed to the top of the plate directly or in combination with other components, and the bottom of the plate can be used as the bottom of the hole. One or more assay reagents can be included in the hole and / or assay domain of the plate. These reagents can be fixed or placed on one or more surfaces of the hole, preferably on the electrode surface, most preferably on the working electrode surface. The determination reagent can be contained or positioned by the feature in the hole, for example, the dielectric material of patterning can limit or position the fluid. The top of the plate preferably includes an integral molded structure made of rigid thermoplastic materials such as polystyrene, polyethylene or polypropylene. The bottom of the plate preferably includes an electrode (for example, working and / or counter electrode), which includes carbon, preferably a carbon layer, more preferably a screen printing layer of carbon ink. In another preferred embodiment, the bottom of the plate includes an electrode consisting of a screen printing conductive ink deposited on the substrate.

[0152] The single-well addressable plate comprises a plate top having a plate top opening and a plate bottom mating with the plate top to define a well of the single-well addressable plate, the plate bottom comprising a substrate having a top surface with patterned electrodes thereon and a bottom surface with patterned electrical contacts thereon, wherein the electrodes and contacts are patterned to define a plurality of well bottoms of the single-well addressable plate, wherein the pattern within the well bottoms comprises: (a) a working electrode on the top surface of the substrate, wherein the working electrode is electrically connected to the electrical contact; and (b) a counter electrode on the top surface of the substrate, wherein the counter electrode is electrically connected to the electrical contact but is not electrically connected to an additional counter electrode in an additional well of the single-well addressable plate. Preferably, the electrodes and contacts of the single-well addressable plate are individually addressable.

[0153] The multi-well addressable plate includes a plate top having a plate top opening and a plate bottom that is mated with the plate top to define the holes of the multi-well addressable plate, the plate bottom including a substrate having a top surface with patterned electrodes thereon and a bottom surface with patterned electrical contacts thereon, wherein the electrodes and contacts are patterned to define two or more independently addressable sectors of two or more jointly addressable assay wells, each sector including two or more wells, which have: jointly addressable working electrodes on the top surface of the substrate, wherein each working electrode is electrically connected to each other and connected to at least a first electrical contact; and (b) jointly addressable counter electrodes on the top surface of the substrate, wherein each counter electrode is electrically connected to each other, but not to the working electrode, and connected to at least a second electrical contact. In one embodiment, the independently addressable sectors include less than 50% of the holes of the multi-well addressable plate, more preferably less than 20% of the holes of the multi-well addressable plate. The independently addressable sectors may include a 4x4 array of holes or a 2x3 array of independently addressable sectors. Alternatively, the independently addressable sectors may comprise one or more rows or one or more columns of apertures.

[0154] The single-hole or multi-hole addressable plate can be a 4-hole plate, a 6-hole plate, a 24-hole plate, a 96-hole plate, a 384-hole plate, a 1536-hole plate, a 6144-hole plate, or a 9600-hole plate. The electrodes in any plate form include carbon particles and they may further include a printed conductive material, wherein one or more electrodes include a plurality of measurement domains formed thereon. The plurality of measurement domains may include at least four measurement domains, preferably seven measurement domains, and more preferably at least ten measurement domains, and the plurality of measurement domains may be defined by openings in one or more dielectric layers supported on the working electrode. Plates useful for the device are available from Meso Scale Discovery (Rockville, MD; www.mesoscale.com) and include, but are not limited to, the following multi-well addressable plates (Meso Scale Discovery catalog numbers): L15XA-3, L15XB-3, L15AA-1, L15AB-1, L15SA-1, L15SB-1, L15GB-1, L45XA-3, L45XB-3, N45153A-2, N45153B-2, N45154A-2, and N45154B-2; and the following single-well addressable plates (Meso Scale Discovery catalog numbers): L55AB-1, L55SA-1, L55XA-1, and L55XB-1.

[0155] Thus, the device measures luminescence from a multiwell plate by first detecting the type of plate in the device, such as by reading a barcode on the multiwell plate that includes plate configuration information, aligning the contact mechanism and the imaging device so that the interrogation zone or zones are directly below and within the imaging field of the imaging device, and guiding the selective application of the potential by (a) electrically isolating each interrogation zone of the contact mechanism and selectively applying a potential only within the first zone (for a single-well addressable plate); or (b) electrically connecting two or more zones and selectively applying a potential within the two or more zones (for a multiwell addressable plate).

[0156] If a multi-well addressable plate is used in the apparatus, the imaging system and contact mechanism are aligned with an interrogation zone corresponding to a group or sector of adjacent wells (e.g., a group of four adjacent wells), and the apparatus selectively applies a voltage to all wells of that sector. The apparatus then moves the plate via the plate translation stage to reposition the contact mechanism and imaging system with an additional interrogation zone corresponding to an additional sector or group of wells, and selectively applies a voltage to the wells of that additional sector.

[0157] If a single-well addressable plate is used in the apparatus, the imaging system and contact mechanism are aligned with an interrogation zone corresponding to a group or sector of adjacent wells (e.g., a group of four adjacent wells), and the apparatus selectively applies a voltage to each well of the sector one at a time. Likewise, the plate is moved by the plate translation stage to reposition the contact mechanism and imaging system with additional interrogation zones corresponding to additional sectors of wells to interrogate each well of the additional sector one at a time.

[0158] According to another embodiment of the present invention, another device, preferably another ECL reader, is specifically configured to interrogate or read a single hole addressable multi-well plate. Device 1000 is shown in Figures 10 (a) and (b). The reader can have a substantially same horizontal footprint as the device 100 shown and discussed above, although with a different stylized cover 1001. Figure 10(c) and 10(d) The internal mechanisms or subsystems of the devices 100 and 1000 are compared and shown to be comparable except as described below. As shown in Figures 10(c) and (d), both devices have a light-tight housing 130 with a removable drawer 240 for accessing the light-tight housing. Both devices have a housing top 232 having intake and exhaust ports 236 and 237, and a bar code reader 238 mounted thereon. Also mounted on the housing top 232 of the device 100 is the light detection subsystem 100. Mounted on the housing top 232 of the device 1000 is a different light detection system 1010, as described below.

[0159] Since the device or reader 1000 is designed to read a single-hole addressable multi-well plate specifically, its plate contact mechanism is simplified, as shown in Figure 11 (a)-(c). The worm gear mechanism 723 is driven by a motor 723a to rotate the mating gear bottom 724a of the lead screw 724. The lead screw 724 has a threaded spindle 723b, which is screwed into the corresponding threaded hole in the support base 700. When the worm gear 723 rotates, it rotates the mating portion 724a of the lead screw 724, which rotates in the threaded hole of the support base 700. These rotational movements lift or lower the support base 700 to adjust the vertical height of the plate contact mechanism, as discussed herein, to contact the conductive bottom of the multi-well plate. A guide shaft 700a, which is preferably not threaded and is suitable for sliding in the corresponding hole in the support base 700, is included to guide the lifting and lowering of the support base 700.

[0160] The contact platform 1701 is sized and dimensioned to electrically contact a single hole on the plate 426 at one time, which in this embodiment is a single-hole addressable plate. The contact platform 1701 includes at least one working electrode contact probe and one counter electrode contact probe to conduct current to the working electrode and counter electrode in the hole where the ECL analysis is performed. Preferably, a set of spare or redundant working and counter electrode probes are included. In this example, four contact probes 1703 including two working electrode contact probes and two counter electrode contact probes are illustrated. Preferably, the contact probes 1703 are upright, spring-loaded needles.

[0161] FIG. 11( d) shows the overlap of contact probes 1703 on the electrical contacts on the bottom of a single-hole addressable multi-well plate. Plate 426 has at least one working electrode contact 1705 and at least one counter electrode contact 1707 below each hole. A contact probe 1703 that transmits a positive charge will contact at least one working electrode contact 1705. As described above, a second contact probe 1703 that contacts another working electrode contact 1705 is a standby. At least one contact probe 1703 that is preferably connected to an electrical loop or alternatively grounded contacts a counter electrode contact 1707. Another contact probe 1703 that contacts a counter electrode contact 1707 is a standby.

[0162] Contact platform 1701 may be smaller in size than contact platform 701 since it only needs to contact a single hole at a time. Preferably, contact platform 1701 also includes light outlet 722 for positioning purposes and light outlets 725-728 for illuminating a focusing mechanism, such as patterned surfaces 601-603, as discussed herein.

[0163] The device 100, 1000 also has an improved heat removal system 1200, which includes an angled oriented fan 1202 and a cover manifold 1204, which separates the fan and the electronics (e.g., a printed circuit board (PCB) including a control board) from the rest of the interior of the device 1000, such as Figure 12(a) and 12(b) 10 (d) as a translucent piece. As described below, the CCD sensor within the optical detection system 110 or 1010 is cooled, and the heat generated by this cooling is removed from the optical detection 110, 1010 by the cooling fan 1208. The removed heat typically rises to the top of the device 1000 (best shown in FIG. 10 (d)) or the cover 1001 of the device 100. The fan 1202 is directed toward the top of the optical detection system 110, 1010 to draw the generated heat, as shown by arrows 1210, from the interior of the device 100, 1000 and more specifically from the top of the optical detection 110, 1010 into the cover manifold 1204 and out of the device 1000 through the exhaust port 1212 as shown by arrows 1214. As the hot air leaves, fresh ambient air is drawn into the device.

[0164] The cover manifold 1204 acts as a flow plenum, where heated air is pulled into the cover manifold and forced out through exhaust ports 1212 to minimize flow recirculation within the decorative or stylized cover 1001, which would reduce heat removal efficiency. The PCBs within the cover manifold 1204 can also generate heat, which is also removed by the airflow 1210, 1214 as it passes over the PCBs before exiting the device 1000.

[0165] The cover manifold 1204 has one or more openings 1216 for electrical connections 1220 between the PCB and the electrical and electronic components on the housing top 232. To minimize flow recirculation that may occur through the openings 1216, baffles 1218 are provided within the cover manifold 1204. As shown in FIG. 12( b), the baffles 1218 are generally vertical and preferably extend downwardly toward the housing top 232 behind the electrical connections 1220.

[0166] Fig.13 FIG. 1 is an enlarged view of a heat removal system 200 for device 100, which includes two fans 202 and a cover manifold 204. Fans 202 are located near exhaust port 1212. Heat removal system 200 can also be seen in FIGS. 1(c), 1(d), 2(c), 4(b), and 4(c). Each fan 202 is smaller in size and has a lower flow rate than fan 1202. In one non-limiting example, fans 202 each have a flow rate of approximately 11.3 ft 3 / min flow rate and has a footprint of 40 mm x 40 mm. Fan 202 operates at approximately 9,500 rpm. A single fan 1202 can have a flow rate of approximately 36.3 ft 3 / min flow rate and has a footprint of 70 mm x 70 mm. Fan 1202 rotates at approximately 3,900 rpm. Fan 1202 has a significantly higher effective flow rate and lower rotation speed than fan 202. As shown in the following test results, heat removal system 1200 cools device 100 or 1000 more effectively and at a lower noise level.

[0167]

[0168]

[0169] Additionally, the average temperature of the four corner wells in the 96-well plate and the center well was approximately 1.2°C for the dual-fan configuration and a lower 0.5°C for the larger single-fan configuration.

[0170] In a specific embodiment, the device can measure luminescence from a single well addressable plate or a multi-well addressable plate, wherein the device comprises:

[0171] (i) board type recognition interface, used to identify the board type;

[0172] (ii) a plate translation stage for holding and translating the multiwell plate in the xy plane;

[0173] (iii) a plate contact mechanism including a plurality of contact probes and located beneath a plate translation stage and within the range of motion of said stage, wherein said mechanism is mounted on a contact mechanism elevator which can raise and lower said mechanism to bring the probes into and out of contact with a bottom contact surface of a plate when placed on the translation stage;

[0174] (iv) a voltage source for applying a potential to the plate via the contact probe; and

[0175] (v) an imaging system positioned above the plate translation stage and vertically aligned with the plate contact mechanism, wherein

[0176] (a) an imaging system configured to image a P x Q matrix of wells, a plate contact mechanism configured to contact a bottom contact surface associated with the matrix, and a plate translation stage configured to translate the plate to position the matrix in alignment with the imaging system and the plate contact mechanism;

[0177] (b) the apparatus is configured to sequentially apply a voltage to each well in a matrix of a single well addressable plate and to image the matrix; and

[0178] (c) The device is configured to simultaneously apply a voltage to each well in a matrix of a multi-well addressable plate and to image the matrix.

[0179] Preferably, the P×Q matrix is ​​a 2×2 array of wells for an exemplary multi-well addressable plate. The imaging system can collect a separate image in the matrix of a single well addressable plate for each sequential application of voltage to a single well, where the P×Q matrix is ​​a 1×1 array of wells. The plate type identification interface can include a barcode reader, an EPROM reader, an EEPROM reader, or an RFID reader, or alternatively, the plate type identification interface includes a graphical user interface configured to enable a user to input plate type identification information.

[0180] Thus, a method of measuring luminescence from a single-well addressable plate or a multi-well addressable plate using such a device comprises:

[0181] (a) Loading a plate on a plate translation stage;

[0182] (b) identifying the plate as a single-well or multi-well addressable plate;

[0183] (c) moving the plate translation stage to align a first P x Q matrix of wells with the plate contact mechanism and imaging system;

[0184] (d) raising the plate contact mechanism so that a contact probe on the contact mechanism contacts a bottom contact surface associated with the P x ​​Q matrix of holes;

[0185] (e) if the plate is a single-well addressable plate, generating and imaging luminescence in a P x Q matrix by sequentially applying a voltage to each well in the group while imaging the group;

[0186] (f) if the plate is a multi-well addressable plate, generating and imaging luminescence in a P x Q matrix by simultaneously applying a voltage to each well in the matrix while imaging the matrix; and

[0187] (g) Repeat steps (c) to (f) for the other P x Q matrices in the board.

[0188] The removable drawer may include a light source (e.g., LED) located below the detection aperture and below the height of the plate translation stage. In one embodiment, the light source or light sources are part of the plate contact mechanism. As described above with reference to the optical focusing mechanism, the light source in the contact mechanism is used in conjunction with the optical focusing mechanism to adjust the contrast and focus of the light detector relative to the plate.

[0189] A method for measuring luminescence from a single-well addressable plate, as shown in FIGS. 10-12 and its subsections, may include the following steps:

[0190] (a) Loading a plate on a plate translation stage;

[0191] (b) optionally confirming that the plate is a single-well addressable plate;

[0192] (c) moving the plate translation stage to align the first hole with the plate contact mechanism and the imaging system;

[0193] (d) raising the plate contact mechanism so that a contact probe on the contact mechanism contacts a bottom contact surface associated with the first hole;

[0194] (e) generating and imaging luminescence in the P x ​​Q matrix by applying a voltage to the first aperture while imaging the pair;

[0195] (g) Repeat steps (c) to (e) for the remaining wells in the plate.

[0196] In other embodiments, one or more light sources may also be used in conjunction with the fiducial holes or windows to correct errors in plate alignment. Light from the light source passes through the fiducial and is imaged on an imaging device to determine the correctness of the alignment of the plate. Advantageously, a board formed of a board bottom mated to a board top (e.g., a board having a screen-printed board bottom mated to an injection molded board top as described in U.S. Pat. Nos. 7,842,246 and 6,977,722) includes a reference patterned (e.g., screen printed) or cut into the board bottom to correct for misalignment of the board bottom relative to the board top. In a particular embodiment, the board top on such a board includes a hole (e.g., in an outer frame of the board top) that is aligned with the reference on the board bottom to allow imaging of the reference. Thus, imaging of light generated under the board can be used to communicate the exact position of the board to image processing software and also to provide a camera focus check. The board can then be realigned using a two-axis positioning device. Thus, the device can process a board by a board positioning method comprising: (1) providing a board having an optical path opening; (2) illuminating the board from the bottom; (3) detecting light passing through the optical path opening; and (4) optionally, realigning the board.

[0197] In a preferred embodiment, the contact mechanism platform includes a first alignment feature 722 and the optical detection subsystem includes a camera positioned above the platform, the camera being adjustable relative to the first alignment feature. Preferably, the first alignment feature is a light source, such as an LED. The camera in the optical detection subsystem is adjustable relative to the alignment feature in the xy plane. The platform may also include a plurality of additional alignment features, such as at least one additional alignment feature in each quadrant, and the camera position is adjustable relative to each additional alignment feature. The additional alignment features may include a light source, such as an LED. Thus, as described above, the device may use an optical focusing mechanism to confirm the correct alignment of the contact mechanism and the detection hole by the following steps: (1) illuminating the contact mechanism alignment feature; (2) detecting light from the alignment feature; and (4) optionally, realigning the plate translation stage, the light detector and / or the contact mechanism. In a preferred embodiment, the device confirms the correct alignment of the contact mechanism before contacting the plate, and then confirms the plate position by detecting light from the light path opening in the plate and realigning the plate as needed.

[0198] As shown in Fig. 7 (a)-(b), the height of the contact mechanism platform is adjustable because the platform further includes a worm gear 723 driving a lead screw mechanism 724 and a support base 700, as described above. In one embodiment, the gear mechanism includes a worm gear. In a preferred embodiment, the platform includes a plate surface area that is sized to accommodate a microtiter plate, such as a multiwell plate, and the platform also includes an overflow collection area surrounding the plate surface area to protect the components of the drawer from accidental overflow of fluid that may be contained in the multiwell plate.

[0199] The light detection subsystem 110 of the device 100 includes a light detector, which can be mounted to the detection hole at the top of the housing through a light-tight connector or baffle. In some embodiments, the light detector is an imaging light detector, such as a CCD camera, and it also includes a lens. An exemplary light detection subsystem 110 is shown in Figure 8 (a). The subsystem includes a light detector housing 801, which surrounds the light detector (not shown) and is attached to the top of the housing via a casting component 802, which is bolted to the top of the housing through the detection hole. Above the casting component is a buckle or clamp 803, which includes an adjustment mechanism consisting of a screw 804 and a gear 805, as shown in Figure 8 (b). The camera focusing mechanism is also configured to focus the camera in the x, y and z directions as needed, manually, by an electric element or both. The light detection subsystem also includes one or more light-tight elements to prevent light leakage in the light detection subsystem or at the joint between the light detection subsystem and the top of the housing. For example, molded rubber or other compressible materials can be clamped between the connected components to prevent light leakage. In addition, the light detector housing includes one or more vents and / or cooling elements to cool the light detector within the housing. In one embodiment, the housing includes an air inlet and an exhaust port, each located at opposite ends of the housing. Additional vents may be located in the housing. In a preferred embodiment, the air inlet is sized to match a cooling fan positioned within the housing.

[0200] A lens coupled to the camera is used to provide a focused image of the luminescence generated by the plate in the light-tight housing. The diaphragm sealed to the lens and the detection hole at the top of the housing allow the imaging system to image the light from the housing while keeping the housing in a light-tight environment that is not affected by ambient light. Suitable cameras for the imaging system include, but are not limited to, conventional cameras such as film cameras, CCD cameras, CMOS cameras, etc. The CCD camera can be cooled to reduce electronic noise. Preferably, the lens is a high numerical aperture lens that can be made of glass or injection molded plastic. The imaging system can be used to image one or more holes of the plate at a time. Because the size of the CCD chip is more closely matched to the imaged area, the light collection efficiency for imaging the light from a single hole is higher than for imaging a group of holes. The reduction in the size of the imaging area and the increase in the collection efficiency allow the use of small, inexpensive CCD cameras and lenses while maintaining high sensitivity for detection.

[0201] If high resolution is not required, the sensitivity of the measurement can be improved by using hardware binning on the CCD during image acquisition. Binning is the process of combining the accumulated charges in adjacent pixels in the CCD to form superpixels, which effectively reduces the electronic read noise per unit area. The preferred binning depends on the field of view, the reduction ratio, and the size of the CCD pixels. In a preferred embodiment, the light detector 110 of the device 100 includes a camera with a CCD having 512×512 pixels, each pixel having a size of 24×24 microns, a total area of ​​12.3×12.3 mm, and a lens with an image reduction ratio of 1.45X. For such a detector and lens combination, a 4x 4 binning readout (i.e., creating a superpixel by combining 16 pixels in a 4x 4 pixel group) is preferred, resulting in a superpixel size of approximately 100x100 microns, which translates to a resolution of approximately 150 microns in the object plane at the ECL electrode. Due to their low cost and size, it is particularly advantageous to use uncooled cameras or cameras with minimal cooling (preferably about -20°C, about -10°C, about 0°C, or higher). In a preferred embodiment, the light detection subsystem includes a lens assembly consisting of a series of lens elements (904 and 905) designed to produce a telecentric view of the imaging aperture and an optical bandpass filter (903) in the optical path within the lens assembly so that light passing through the filter is at substantially normal incidence relative to the filter. Fig. 9 In the embodiment shown, the camera is provided with a telecentric view of the imaging aperture (901).

[0202] The optical detection system 1010 of the device 1000, as described above, is suitable for reading single-hole addressable multi-well plates and can have a smaller size or footprint on the top 232 of the housing than the optical detection system 110 of the device 100, as shown in Figures 10 (c) and (d). As taught in co-owned U.S. patent application 14 / 147,216, because the size of a typical CCD chip matches the imaged area, i.e., the area of ​​a single hole in a multi-well plate, more closely, the light collection efficiency for imaging light from a single hole is higher than for imaging a group of holes. In addition, imaging light from a single hole at a time avoids the need to correct for optical crosstalk between holes. The reduction in the size of the imaging area and the increase in collection efficiency allow the use of smaller, inexpensive CCD cameras and lenses while maintaining high sensitivity for detection. The size of the CCD camera of the device 1000 is preferably smaller than that of the CCD camera of the device 100.

[0203] In one example, the CCD camera of the light detection system 1010 has 1392 pixels x 1040 pixels, and each pixel is 6.45 x 6.45 μm. The total area of ​​the CCD is about 8.98 mm x 6.7 mm, which is smaller than the CCD camera of the light detection system 110. A suitable CCD camera for the light detection system 1010 is the Sony ICX 825CCD. The lens that can be used with the light detection system 1010 has a 1:1 ratio without any magnification or reduction. A 4x 4 binning readout technique is also preferred.

[0204] Fig.14 1900. A camera 1012 is located at the top of the detection system. A CCD sensor is located at one end of the lens system 1900, as described below. In an exemplary system, the camera window may include an optical bandpass filter located in the optical path. A camera-to-lens adapter 1014 is provided to connect the lens to the camera 1012. A clamping mechanism 1016 secures the light detection system to the housing top 232. An optical bandpass 1018 may be included in the optical path to limit the wavelength of light passing through the lens system 1900. Preferably, the range of wavelengths passing through the optical bandpass 1018 is discussed below.

[0205] Fig.15 The suitable exemplary lens system 1900 shown in is located between a single hole on a multi-well plate and a CCD sensor. In an exemplary embodiment, the lens system is designed for spectral bands of about 550nm to about 750nm and about 570nm to about 670nm at half power. Preferably, the lens system 1900 is larger in size than the area of ​​a single hole or CCD. Without being bound by any particular theory, a larger lens can maintain high light collection efficiency by capturing a wider cone angle of light. The lens system 1900 has caps 1902, 1904 located at its ends, which protect the optical elements during transportation and are removed before installation. Two exemplary dual elements 1906 are arranged in a housing 1908 in opposite orientations to each other. Each dual element 1906 includes an outer lens 1910 and an inner lens 1912 that contact each other at a spherical surface 1914. The outer surface 1916 and the inner surface 1918 are aspherical. The surface profile of an aspherical lens is not a part of a sphere or a cylinder. Aspheric lenses are used in optical systems for many reasons, including but not limited to reducing optical aberrations such as astigmatism and simplifying more complex optical systems. Aspheric lenses can also be used to reduce the thickness of the lens. Other suitable lens systems are disclosed in commonly owned U.S. Patent Application Publication US2012 / 0195800 and International Application Publication WO 2009 / 126303, the entire contents of which are incorporated herein by reference. The lenses discussed herein can be made of glass or plastic.

[0206] The top of the housing of the plate handling system also includes a plate stacker installed on the top of the housing, above the plate introduction hole, wherein the plate stacker is configured to receive the plate or transfer the plate to the plate elevator. The plate stacker may include a removable stacking slot that is configured to accommodate multiple plates and prevent the plate from moving on the apparatus, thereby coordinating each plate in the stacking slot to be correctly introduced onto the plate elevator. In one embodiment, the stacking slot can accommodate at least 5 plates, preferably at least 10 plates, and the stacking slot can accommodate a plate nesting extension element that is configured to further expand the stacking slot capacity. The plate elevator includes a plate detection sensor, such as a capacitive sensor, and the stacker may also include a plate detection sensor, such as a capacitance, weight or optical sensor.

[0207] A method for measuring in a multi-well plate using the device is provided. The plate can be a conventional multi-well plate. The measurement techniques that can be used include, but are not limited to, techniques known in the art, such as cell culture-based assays, binding assays (including agglutination tests, immunoassays, nucleic acid hybridization assays, etc.), enzymatic assays, colorimetric assays, and the like. Other suitable techniques will be apparent to those of ordinary skill in the art.

[0208] The method for measuring the amount of analyte also includes the technology of measuring analyte by detecting the label that can be directly or indirectly (for example, by using the label binding partner of analyte) connected to analyte. Suitable labeling includes labeling that can be directly visualized (for example, particles that can be seen visually and labels that produce measurable signals such as light scattering, light absorption, fluorescence, chemiluminescence, electrochemiluminescence, radioactivity, magnetic field, etc.). Useable labeling also includes enzyme or other chemically active substances, which have the chemical activity that causes measurable signals such as light scattering, absorbance, fluorescence, etc. The formation of product can be detectable measurable properties such as absorbance, luminescence, chemiluminescence, light scattering, etc. relative to substrate, for example, due to differences. Some (but not all) measuring methods that can be used together with solid phase binding methods according to the present invention can benefit from or need washing steps to remove unbound components (such as labels) from solid phase.

[0209] In one embodiment, the measurement performed with the device of the present invention can be performed in the form of an electrochemiluminescence-based assay, such as an electrochemiluminescence-based immunoassay. The high sensitivity, wide dynamic range, and selectivity of ECL are important factors in medical diagnosis. Commercially available ECL instruments have shown excellent performance and are widely used for reasons such as their excellent sensitivity, dynamic range, precision, and tolerance to complex sample matrices. Substances that can be induced to emit ECL (ECL active substances) have been used as ECL markers, such as (i) organometallic compounds, wherein the metal is from, for example, Group VIII noble metals, including Ru- and Os-containing organometallic compounds, such as terpyridyl ruthenium (RuBpy) moieties, and (ii) luminol and related compounds. Substances with ECL labels in the ECL process are referred to herein as ECL co-reactants. Commonly used co-reactants include tertiary amines (e.g., see U.S. Patent No. 5,846,485), oxalates, and persulfates for ECL from RuBpy and hydrogen peroxide for ECL from luminol (see, e.g., U.S. Patent No. 5,240,863). The light generated by the ECL label can be used as a reporter signal in diagnostic procedures (Bard et al., U.S. Pat. No. 5,238,808, incorporated herein by reference). For example, the ECL label can be covalently coupled to a binding agent, such as an antibody, a nucleic acid probe, a receptor, or a ligand; the participation of the binding agent in the binding interaction can be monitored by measuring the ECL emitted from the ECL label. Alternatively, the ECL signal from the ECL-active compound can indicate the chemical environment (see, for example, U.S. Pat. No. 5,641,623, which describes an ECL assay for monitoring the formation or destruction of an ECL co-reactant). For more background on ECL, ECL labeling, ECL detection, and apparatus for performing ECL assays, see U.S. Patent Nos. 5,093,268; 5,147,806; 5,324,457; 5,591,581; 5,597,910; 5,641,623; 5,643,713; 5,679,519; 5,705,402; 5,846,485; 5,866,434; 5,786,141; 5,731,147; 6,066,44 8; 6,136,268; 5,776,672; 5,308,754; 5,240,863; 6,207,369; 6,214,552 and 5,589,136 and published PCT Nos. WO99 / 63347; WO00 / 03233; WO99 / 58962; WO99 / 32662; WO99 / 14599; WO98 / 12539; WO97 / 36931 and WO98 / 57154, all of which are incorporated herein by reference.

[0210] In certain embodiments, as described in U.S. Patent No. 7842246, a plate suitable for electrochemiluminescence (ECL) assay is used. The device of the present invention can use a plate configured to detect ECL from one hole at a time or more than one hole at a time. As described above, the plate configured to detect ECL from one hole at a time or more than one hole at a time includes electrodes and electrode contacts that are specially patterned to allow electrical energy to be applied to electrodes in only one hole at a time or more than one hole at a time. The device may be particularly suitable for assaying in a plate containing dry reagents and / or sealed holes, for example, as described in U.S. Patent No. 7,807,448 to Glezer et al.

[0211] In one embodiment, the method includes: (a) introducing a plate into a plate stacker, (b) opening a light-tight door, (c) lowering the plate from the plate stacker to a lift platform on a plate translation stage, (d) sealing the light-tight door, (e) translating the plate to position one or more wells below a photodetector, (f) detecting luminescence from the one or more wells, (g) opening the light-tight door, (h) translating the plate to a position below the plate stacker, and (i) lifting the plate to the plate stacker. In a preferred embodiment, the method also includes reading a plate identifier on the plate and identifying the plate configuration, translating the plate to position one or more wells below the photodetector, optionally imaging one or more alignment features on a contact mechanism and adjusting the position of the photodetector relative to the contact mechanism, and selectively applying an electrical potential within one or more interrogation zones based on the plate configuration. The method may also include translating a plate carrier to position one or more additional wells below the photodetector and detecting luminescence from the one or more additional wells. The method may also optionally include applying electrical energy to electrodes in one or more wells (e.g., to induce electrochemical luminescence).

[0212] ECL-based multipath testing is described in the following documents: U.S. Publication 2004 / 0022677 and 2004 / 0052646 for U.S. Patent Nos. 7,842,246 and 6,977,722, respectively; U.S. Publication 2003 / 0207290 for U.S. Patent No. 7,063,946; U.S. Publication 2003 / 0113713 for U.S. Patent No. 7,858,321; U.S. Publication 2004 / 0189311 for U.S. Patent No. 7,497,997; and U.S. Publication 2005 / 0142033 for U.S. Patent No. 7,981,362.

[0213] A method for measuring a biological agent using a device as described herein is also provided. In one embodiment, the method is a binding assay. In another embodiment, the method is a solid phase binding assay (in one example, a solid phase immunoassay) and includes contacting the assay composition with one or more binding surfaces that bind to an analyte of interest (or its binding competitor) present in the assay composition. The method may also include contacting the assay composition with one or more detection agents that can specifically bind to the analyte of interest. The multiple binding assay method according to the preferred embodiment may involve a variety of formats available in the art. Suitable assay methods include sandwich or competitive binding assay formats. Examples of sandwich immunoassays are described in U.S. Patents 4,168,146 and 4,366,241. Examples of competitive immunoassays include those disclosed in U.S. Patents 4,235,601, 4,442,204, and 5,208,535 of Buechler et al. In one example, small molecule toxins such as marine toxins and fungal toxins can be advantageously measured in a competitive immunoassay format.

[0214] In one example, the device 100 described above is an ECL reader suitable for performing ECL testing on (i) a multi-well addressable multi-well plate, such as a four-well addressable 96-well plate or (ii) a single-well addressable multi-well plate, such as a single-well addressable 96-well plate. The wells can be 1 point or 1 point small point, 4, 7, 10 points. Depending on the multi-well addressable mode or the single-well addressable mode, the device 100 can read the plate in about 1:29 minutes or 2:42 minutes. For a properly calibrated ECL reader, the dark noise of 10 points is about 13 / 14 ECL counts, and the saturation of 10 points is about 1.9x10 6 / 2.2x10 6 ECL counts, depending on the mode, yielding approximately 1.4x10 5 To about 1.5x10 5 The effective dynamic range (saturation value / dark noise) of the apparatus 100 is calibrated to provide a nominal signal of 15,000 counts for an ECL generated in an MSD QUICKPLEX plate containing an MSD Free Tag ECL 15,000 solution available from Meso Scale Diagnostics of Rockville, Maryland. The apparatus 100 is compatible with V-PLEX, U-PLEX, and R-PLEX assay kits, which are also available from Meso Scale Diagnostics. V-PLEX, U-PLEX, and other assay kits are described in co-owned International Published Patent Applications WO 2018 / 017156A1 and WO 2017 / 015636A1, which are incorporated herein by reference in their entirety.

[0215] In another example, the device 1000 described above is an ECL reader designed to perform ECL testing on a single-well addressable multi-well plate, such as a single-well addressable 96-well plate 1 spot or a single-well addressable 96-well plate 1 spot small spot. The device 100 can also be used with 4-spot, 7-spot or 10-spot plates. When properly calibrated as described above, the device 1000 can read one such plate in about 2:37 minutes, where the 1-spot dark noise is about 8 ECL counts and the 1-spot saturation is about 1.3x10 6 ECL counts, yielding approximately 1.6x10 5 The effective dynamic range (saturation value / dark noise) of the apparatus 1000 is 1.5 saturation value / dark noise. The apparatus 1000 is compatible with U-PLEX and R-PLEX assay kits, which are available from Meso Scale Diagnostics of Rockville, Maryland. The specifications of the non-limiting exemplary apparatus 1000 are as follows:

[0216]

[0217] Binding agents, binding components of binding surfaces and / or bridging agents that can be used as detection agents include, but are not limited to, antibodies, receptors, ligands, haptens, antigens, epitopes, mimetic sites, aptamers, hybridization partners and intercalators. Suitable binding agent compositions include, but are not limited to, proteins, nucleic acids, drugs, steroids, hormones, lipids, polysaccharides and combinations thereof. The term "antibody" includes complete antibody molecules (including hybrid antibodies reassembled by antibody subunits in vitro), antibody fragments and recombinant protein constructs comprising the antigen-binding domain of an antibody (such as described in Porter & Weir, J. Cell Physiol., 67 (Suppl 1): 51-64, 1966; Hochman et al., Biochemistry 12: 1130-1135, 1973; incorporated herein by reference). The term also includes complete antibody molecules, antibody fragments and antibody constructs that have been chemically modified (e.g., by introducing a label).

[0218] As used herein, measurement is understood to include both quantitative and qualitative measurements, and encompasses measurements performed for a variety of purposes, including but not limited to detecting the presence of an analyte, quantifying the amount of an analyte, identifying a known analyte, and / or determining the identity of an unknown analyte in a sample. According to one embodiment, the amount of the first binding reagent and the second binding reagent bound to one or more binding surfaces can be expressed as a concentration value of the analyte in the sample, i.e., the amount of each analyte per volume of sample.

[0219] Analytes can be detected using an electrochemiluminescence-based assay format. Electrochemiluminescence measurements are preferably performed using binding reagents that are fixed or otherwise collected on the electrode surface. Particularly preferred electrodes include screen-printed carbon ink electrodes that can be patterned at the bottom of a specially designed ink cartridge and / or a multi-well plate (e.g., 24-, 96-, 384-, etc. well plate). As described in U.S. Patent Nos. 7,842,246 and 6,977,722 (both filed on June 28, 2002, entitled "Assay Plates, Reader Systems and Methods for Luminescence Test Measurements," incorporated herein by reference), an imaging plate reader is used to induce and measure electrochemiluminescence from ECL markers on the carbon electrode surface. Similar plates and readers are now available ( and Board and Devices, MesoScale Discovery, a division of Meso Scale Diagnostics, LLC, Rockville, MD).

[0220] In one embodiment, antibodies immobilized on electrodes within the plate can be used to detect selected biological agents in a sandwich immunoassay format. In another embodiment, an antibody microarray patterned on an integrated electrode within the plate will be used to detect a variety of selected biological agents in a sandwich immunoassay format. Thus, each well contains one or more capture antibodies, which are immobilized on the working electrode of the plate and optionally in dry form or as a separate component, such as in a kit; labeled detection antibodies and all additional reagents required for analyzing the sample and for performing positive and negative controls.

[0221] Preferably, an ECL reader such as the above-described devices 100 and 1000 is validated before first use or periodically. Preferably, the steps to validate the ECL reader should be completed together at the beginning of the validation process, because any assay run requires an operational ECL reader. ECL validation includes the steps of running the ECL reader using an electronic plate, which measures the current applied to the plate. This ensures that the applied current is sufficient and uniform. Another step (which may be the next step) is to run the ECL reader using an empty assay microplate (e.g., an MSD 96-well plate) to measure the level of electronic noise or background / dark noise within the ECL reader. Another step that can follow the other two steps is to fill the assay plate with a reagent consisting of unbound SULFO-TAG in Meso Scale Diagnostics Read Buffer (hereinafter referred to as "free label") to verify that the ECL reader is reading the expected counts. For example, a free label of 300,000 counts can be used as a detection reagent to generate an ECL signal. Therefore, the ECL reader should read approximately 300k counts from each well within a small predetermined range. 300k free tags are available from Meso Scale Diagnostics.

[0222] In one example, the electronic plate is similar to a standard 96-well plate with 8 rows (AH) and 12 columns (1-12), and the verification step can read the wells at example well positions A9, B10, C11, D12 and E4, F3, G2, H1 to determine whether the reading is at or above a predetermined amount, such as 2000 counts. Other well positions and different numbers of well positions can be selected. The electronic plate should be verified for each well configuration that the ECL reader is expected to read.

[0223] Patents, patent applications, publications and test methods cited in this disclosure are incorporated herein by reference in their entirety. The scope of the present invention is not limited by the specific embodiments described herein. In fact, various modifications to the present invention, in addition to those described herein, will become apparent to those skilled in the art based on the foregoing description and the accompanying drawings. Such modifications are intended to fall within the scope of the claims.

[0224] As described above, the measurement of one analyte may affect the measurement of another subsequently measured analyte. According to other embodiments of the present invention, the devices, systems and methods described herein can reduce or eliminate the amount of crosstalk in a CCD camera system used to measure analytes in a multi-well assay plate. The following discussion is preferably incorporated into the device 100 and can also be used in conjunction with the device 1000.

[0225] Fig.16is a schematic diagram of a CCD camera system 1300. The CCD camera system includes a CCD 1301, an analog front end circuit 1303 having a CCD clamp capacitor 1322, and an analog-to-digital circuit 1304. The CCD camera system 1300 provides an output to an output device 1321. The output device 1321 may include any suitable device for interpreting data output by the CCD camera system 1300, such as a display monitor or a computer system. The CCD camera system 1300 is configured to measure a plurality of spatially separated analytes in an assay plate and is coupled to a focusing system and a plate carrier system that is configured to position the assay plate to facilitate measurement of the plurality of analytes, as described above.

[0226] The CCD 1301 includes a plurality of pixels 1311 arranged in rows and columns. During exposure of the CCD 1301, the pixels 1311 of the CCD 1301 detect light striking the CCD and accumulate charge as a function of the detected light. After exposure, the charge value in each pixel 1311 is received by the analog front end circuit as a readout signal as follows. The charge of the bottom row of pixels 1311 is transferred to the serial register 1320. The analog front end circuit 1303 is used to transfer the readout signal of the CCD 1301 to the analog-to-digital circuit 1304 for conversion into a plurality of image data points. After conversion, the plurality of image data points can be output to any relevant system for analysis.

[0227] Between successive measurements of image data points, the clamp capacitor 1322 is reset to a reference voltage level via the reference level clamp circuit portion of the analog front end circuit 1303. After resetting, the clamp capacitor 1322 receives a data voltage from the next (or first) data element of the serial register 1320. The analog front end circuit 1303 operates by sampling the reference level and the data level from the clamp capacitor 1322 (e.g., by correlated double sampling or other suitable techniques). The difference between the reference level voltage amplitude and the data voltage amplitude is then converted to a digital signal by the analog-to-digital circuit 1304 to be output as an image data point. After reading each data voltage, the reference level clamp circuit restores the clamp capacitor 1322 to the reference level voltage and then transfers the data voltage of the next data element of the serial register 1320 to the clamp capacitor 1322. After all data elements of the serial register 1320 are read, the voltage from the next row of pixels 1311 is pulled down into the serial register 1320 to be sequentially read by the analog front end circuit 303. In further embodiments, pixels 1311 are read out together in groups, such as 2x2 groups or 4x4 groups of pixels, for averaging prior to readout.

[0228] In this way, the entire pixel 1311 can be read by the analog front end circuit 1303 and converted into an image data point by the analog to digital circuit 1304. In the analog front end circuit 1303, the reference level clamp circuit part operates to completely reset the reference level voltage to a predetermined value after each image data point is measured. That is, after each image data point is read, the reference level voltage returns to substantially the same voltage (e.g., within the resolution range of the analog to digital circuit 1304). The reference level clamp circuit has a time constant that is selected to return the reference level voltage to a predetermined value. In particular, the decay time constant of the reference level clamp circuit is selected to ensure that the reference level voltage is completely reset. Completely resetting the reference level voltage ensures that each consecutive image data point reading is not affected by the previous image data point reading. This in turn reduces crosstalk between measurements of different analytes on the assay plate.

[0229] This is in contrast to some conventional systems where the timing of the reference level clamp circuit portion fails to fully reset the reference level voltage on the clamp capacitor between each image data point read. In particular, this failure to fully reset the reference level voltage may be more common after a high voltage value is read from a data element of the serial register. Such high voltage reads may occur when the CCD is recording a bright image. The high voltage read of the bright image may affect the readout of several pixels recorded after the bright pixel, causing "smearing" or "banding" to appear in the image because the image data point recorded after the bright spot is artificially raised in value. The effect of this streaking is related to Fig.17 Shown.

[0230] Fig.17 Illustrated is the effect of tailing in a pinhole test setup. Fig.17 The pinhole CCD test results before and after the analog front-end circuit is optimized are shown. Pinhole CCD testing is performed using a pinhole test fixture that is configured to allow a single spot of light to reach the CCD. The light source is arranged behind an opaque material with a pinhole in it. At a clear point, the pinhole allows light to pass through the opaque material and reach the CCD. If the CCD is working properly, the output image data points will correspond only to those portions of the spotlight's light that fall on the CCD.

[0231] Images 1401, 1402, 1403, 1404, and 1405 were produced using a pinhole test fixture to increase the illumination level by 1 millisecond from image 1401 to 1405. Images 1401, 1402, 1403, 1404, and 1405 each illustrate pinhole spot illumination and the accompanying white streaking. In producing images 1401, 1402, 1403, 1404, and 1405, an analog front end circuit having a reference level circuit time constant that does not fully reset the clamp capacitor was employed. The streaking is an artifact produced by a reference level circuit having a non-resetting time constant, as described above. Bright spot illumination on the CCD results in a failure to reset the reference level between reading consecutive image data points. Because the reference level in the reference level clamp circuit is not properly reset, the measured values ​​of consecutive image data points are affected or influenced by the measured values ​​of previous image data points. Bright image data points illuminated by the spot cause consecutive image data points outside the spot to artificially increase in value. As can be seen in images 1401 through 1405, this artifact extends through many of the image data points to the right of the illumination point. Because each row of pixels is read from the serial register from left to right, bright pixels cause a streak that initially extends to the right from the illumination point. After the last pixel in a row is read, the first pixel in the next row is read. Therefore, the last pixel read of one row can affect the first pixel read of the next row, which causes the smearing effect to "wrap around" and appear in the next row, as shown in images 1402 through 1405.

[0232] Images 1411, 1412, 1413, 1414, and 1415 were generated using a pinhole test fixture with increasing illumination levels from image 1411 to 1415 for 1 millisecond. The illumination levels of images 1411 to 1415 correspond to the levels of images 1401-1405, respectively. In capturing images 1411 to 1415, an improved analog front end circuit 1303 as modified and disclosed herein was used. As described above, the analog front end circuit 1303 includes a reference level clamp circuit having a time constant that is selected to fully reset the clamp capacitor between data reads. The improved reference level clamp circuit allows the reference level to be fully reset after each image data point is read. Fully resetting the reference level clamp circuit after each image data point is read reduces or eliminates the effect of the first image data point read on the second consecutive image data point read.

[0233] When attempting to measure multiple analytes within a single image, relative to Fig.17 The artifact errors and images 1401 to 1405 depicted may be particularly troublesome. Fig.18The captured image of multiple analytes 1501 occupying multiple wells 1510 (here, four wells) of an assay plate 1500 is illustrated. Each well 1510 of the assay plate 1500 contains four spatially separated analytes 1501. A CCD camera is used to capture images of the four wells 1510 and all of the analytes 1501 contained therein. Analyte measurement problems caused by tailing can be seen in each analyte 1501. Using a CCD camera coupled to an analog front end circuit that cannot fully reset the clamping capacitor to capture the image can result in bright spot tailing, as described above. The image of the analyte 1501 is tailed into the image of the adjacent analyte 1501. Each analyte 1501 has a tail 1503 that extends right into the dot position of the next analyte 1501, which can result in erroneous measurements of these analytes 1501. When using a conventional analog front end circuit, similar tailing will occur for all analytes 1501 in the wells 1510. Eliminating tailing by using an analog front end circuit having a time constant selected to fully reset the clamping capacitor, consistent with an embodiment of the present invention, results in superior accuracy because the effect of the measurement of the first analyte 1501 on the second analyte 1501 is reduced or eliminated.

[0234] like Fig.18 As shown, crosstalk between analytes 1501, such as tailing, may occur between multiple analytes in a single well of an assay plate and / or between multiple analytes contained in different wells of an assay plate. A system based on a CCD camera of the present invention using an analog front end circuit 1303 consistent with an embodiment of the present invention can improve camera tailing crosstalk in two situations.

[0235] Fig.19 An example of an analog front end circuit 1600 consistent with an embodiment of the present invention is illustrated. The analog front end circuit 600 includes a reference level clamp circuit 1601, an analog-to-digital converter buffer circuit 1602, and a clamp capacitor 1603. The clamp capacitor 1603 is connected to the output of the CCD and the preamplifier 1604. The clamp capacitor 1603 converts the voltage level of the data element of the serial register from the CCD and the preamplifier 1604 to the voltage on the analog-to-digital converter buffer circuit 1602. Between readings of the data voltage on the clamp capacitor 1603, the reference level clamp circuit 1601 resets the voltage on the clamp capacitor 1603 to a reference level. The analog-to-digital conversion circuit (not shown) alternately samples the data voltage and the reference level voltage, and converts the voltage difference between the two into a digital signal to be output as an image data point.

[0236] The reference horizontal clamp circuit 1601 is optimized to completely reset the reference horizontal voltage between data voltage readings on the clamp capacitor 1603. In particular, the time constant of the reference horizontal clamp circuit 601 is selected so that a data voltage reading on the clamp capacitor 1603 does not affect a subsequent data voltage reading. That is, the reference horizontal clamp circuit 1601 completely resets the reference horizontal voltage at the clamp capacitor 1603 so that there is no memory between consecutive data voltage readings. This optimization serves to reduce or eliminate the streaking or tailing shown in images 1401-1405 caused by the combination of bright pinhole spots and a non-optimized reference horizontal clamp circuit 1601.

[0237] Fig. 20 1700 is a process flow chart illustrating an embodiment of an analyte measurement process 1700. The various processing operations and / or data flows depicted in FIG. 7 are described in more detail herein. The described operations can be completed using some or all of the system components described in detail above, and in some implementations, various operations can be performed in different orders and various operations can be omitted. Additional operations can be performed together with some or all of the operations shown in the depicted flow chart. One or more operations can be performed simultaneously. Therefore, the operations shown (and described in more detail below) are exemplary in nature and should not be considered restrictive.

[0238] In operation 1702, process 1700 includes using a CCD camera to measure multiple analytes in one or more imaging areas. The CCD camera includes a CCD, an analog front end circuit, and an analog-to-digital circuit. The CCD camera is coupled to a focusing system and a plate carriage system, which is configured to position an assay plate to facilitate the measurement of multiple analytes. The CCD camera measures multiple analytes by capturing or detecting light from multiple analytes during CCD exposure. In further embodiments, light from any other inspection object can be captured or detected during CCD exposure to generate CCD measurements.

[0239] At operation 1702 , process 1700 includes receiving, by an analog front end circuit, a readout signal from a serial register of a CCD.

[0240] In operation 1704, receiving the readout signal from the serial register includes transmitting the readout signal from the serial register to an analog-to-digital circuit to generate a first image data point.

[0241] In operation 1706, receiving the readout signal further includes resetting a reference level of the analog front end circuitry by a reference level clamp circuit having a time constant. The time constant is selected to reduce crosstalk between a first measurement of a first of the plurality of analytes and a second measurement of a second of the plurality of analytes.

[0242] In operation 1708, receiving the readout signal further includes transmitting the readout signal from the serial register to an analog-to-digital circuit to generate a second image data point.

[0243] Therefore, systems, devices and methods for reducing crosstalk between measurements of multiple analytes are provided. Although various embodiments according to the present invention have been described above, it should be understood that they are presented only by way of illustration and example, not limitation. The improved CCD camera-based system proposed herein can be used.

[0244] It will be apparent to those skilled in the relevant art that various changes may be made in form and detail without departing from the spirit and scope of the present invention. Therefore, the breadth and scope of the present invention should not be limited by any of the exemplary embodiments described above. Further embodiments and combinations are described in the following numbered paragraphs. It will also be understood that each feature of each embodiment discussed herein and each feature of each reference cited herein may be used in combination with the features of any other embodiment. Aspects of the above-mentioned methods for presenting tactile effects may be used in any combination with other methods described herein, or these methods may be used alone. All patents and publications discussed herein are incorporated herein by reference in their entirety.

Claims

1. A system configured to measure a plurality of analytes in an assay plate with reduced crosstalk between the analytes, the system include: a CCD camera comprising a CCD, analog front end circuitry, and analog-to-digital circuitry, the CCD camera being configured to measure a plurality of analytes and coupled to a focusing system and a plate carrier system configured to position the assay plate to facilitate measurement of the plurality of analytes, The analog front end circuit is configured to receive a readout signal from a serial register of the CCD and transmit the readout signal to the analog-to-digital circuit to generate a plurality of image data points, the analog front end circuit including a reference level clamp circuit configured with a time constant, the time constant being selected to completely reset the reference level between consecutive image data point readings, thereby reducing crosstalk between a first measurement of a first one of the plurality of analytes and a second measurement of a second one of the plurality of analytes.

2. The system of claim 1, wherein a first one of the plurality of analytes and a second one of the plurality of analytes are contained in a single well of the assay plate.

3. The system of claim 1, wherein a first one of the plurality of analytes and a second one of the plurality of analytes are contained in different wells of the assay plate.

4. The system of claim 1, wherein the reference level is completely reset between consecutive image data point readings such that the magnitude of the reference level returns to a predetermined value between consecutive image data point readings.

5. The system of claim 1, wherein completely resetting the reference level between consecutive image data point readings reduces the effect of a first image data point value on a second image data point value obtained consecutively after the first image data point value.

6. The system of claim 1, wherein the time constant is a decay time constant of the reference level clamp circuit.

7. The system of claim 1, wherein each of the plurality of image data points represents a pixel of the CCD.

8. The system of claim 1, wherein each of the plurality of image data points represents a plurality of binned readout pixels of the CCD.

9. The system of claim 1, wherein the plurality of analytes are spatially separated from one another.

10. A method of reducing crosstalk between measurements of multiple analytes in an assay plate performed by a CCD camera, the method include: measuring the plurality of analytes with the CCD camera, the CCD camera comprising a CCD, analog front end circuitry, and analog-to-digital circuitry, and coupled to a focusing system and a plate carrier system configured to position the assay plate to facilitate measurement of the plurality of analytes; Receiving a readout signal from a serial register of a CCD through the analog front-end circuit, wherein receiving the readout signal comprises: transmitting the readout signal from the serial register to the analog-to-digital circuit to generate a first image data point, The reference level of the analog front-end circuit is completely reset by a reference level clamp circuit having a time constant, transmitting the readout signal from the serial register to the analog-to-digital circuit to generate a second image data point, Wherein the time constant is selected to reduce crosstalk between a first measurement of a first one of the plurality of analytes and a second measurement of a second one of the plurality of analytes.

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