WO2013039738A1

WO2013039738A1 - Apparatus for temperature controlled label free assays

Info

Publication number: WO2013039738A1
Application number: PCT/US2012/053618
Authority: WO
Inventors: Joel Patrick Carberry; Qi Wu
Original assignee: Corning Incorporated
Priority date: 2011-09-12
Filing date: 2012-09-04
Publication date: 2013-03-21

Abstract

An apparatus for label-free assay, including: a housing; a circulator; a reader module; and a sensor module comprising a microplate holder, the holder receives and holds at least one microplate article having at least one sensor, the sensor module being spatially separated from the reader module by a cavity, the cavity provides a conduit for ambient temperature gas flow from the circulator to between the sensor module and the reader module within the housing, as defined and disclosed herein. Methods for interrogating a sensor in a microplate using the apparatus, as defined and disclosed herein.

Description

APPARATUS FOR TEMPERATURE CONTROLLED LABEL

FREE ASSAYS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of prioirity under 35 U.S.C. § 119 of U.S. Provisional Application Serial No. 61/533,438, filed on September 12, 2011, the content of which is relied upon and incorporated herein by reference in its entirety.

[0002] The entire disclosure of any publication or patent document mentioned herein is incorporated by reference.

BACKGROUND

[0003] The disclosure generally relates to an apparatus and method for imaging a sensor.

SUMMARY

[0003] The disclosure provides an apparatus and method for imaging a sensor, for example, as used in a microplate optical reader for label-independent detection.

BRIEF DESCRIPTION OF THE DRAWING(S)

[0004] In embodiments of the disclosure:

[0005] Fig. 1 shows a schematic of an exemplary air flow apparatus or system for maintaining the microplate at ambient temperature throughout a label free assay.

[0006] Figs. 2A and 2B, respectively, show a comparison of the wavelength shift of a 384 well plate, which was continuously monitored immediately after the plate is placed on a comparative reader having a simple plate holder (Fig. 2A), and the present disclosed reader having the air flow system (Fig. 2B).

[0007] Figs. 3 A and 3B, respectively, show a comparison of the wavelength shift time traces of a 384 well plate center- we 11 (dashed lines) and edge-well (solid lines) having a simple plate holder (Fig. 3A), and the disclosed reader having the air flow system (Fig. 3B).

[0008] Figs. 4A and 4B, respectively, show images of microplates illustrating the wavelength shifts, or in effective the thermal deviation or relative uniformity of the wells of the plate, of a 384 well sensor plate after it was placed on the comparative reader for 20 minutes having a simple plate holder (Fig. 4A) and when placed on the disclosed reader having the air flow system (Fig. 4B). [0009] Figs. 5A and 5B, respectively, show histograms of the wavelength shifts when a 384 well sensor plate was placed on the comparative reader for 20 minutes having a simple plate holder (Fig. 5 A) and on the disclosed reader having the air flow chamber under the plate holder (Fig. 5B).

[0010] Fig. 6 shows a schematic of an alternative microplate temperature control apparatus and method of use in a label- free detection reader system.

DETAILED DESCRIPTION

[0011] Various embodiments of the disclosure are described in detail with reference to the drawings. Reference to various embodiments does not limit the scope of the invention, which is limited only by the scope of the claims. Additionally, any examples set forth in this specification are not limiting and merely set forth some of the many possible embodiments for the claimed invention. Any aspect, feature, or embodiment of the disclosure can be used in any combination or permutation with any one or more other aspect, feature, or embodiment recited in the appended claims.

Definitions

[0012] "Biosensor," "sensor," or like term refers to an article, that in combination with appropriate apparatus, can detect a desired analyte or condition. A biosensor combines a biological component with a physico chemical detector component. A biosensor can typically consist of three parts: a biological component or element (such as tissue, microorganism, pathogen, cells, cell component, a receptor, and like entities, or combinations thereof); a detector element (operating in a physicochemical way such as optical, piezoelectric, electrochemical, thermometric, magnetic, or like manner); and a transducer associated with both components. In embodiments, the biosensor can convert a molecular recognition, molecular interaction, molecular stimulation, or like event occurring in a surface bound cell component or cell, such as a protein or receptor, into a detectable and quantifiable signal. A biosensor as used herein can include liquid handling systems which are static, dynamic, or a combination thereof. In embodiments of the disclosure, one or more biosensor can be incorporated into a micro-article. Biosensors are useful tools and some exemplary uses and configurations are disclosed, for example, in PCT Application No. PCT/US2006/013539 (Pub. No. WO 2006/108183), published Dec. 10, 2006, to Fang, Y., et al, entitled "Label- Free Biosensors and Cells," and U.S. Patent No. 7,175,980. Biosensor-based cell assays having penetration depths, detection zones, or sensing volumes have been described, see for example, Fang, Y., et al. "Resonant waveguide grating biosensor for living cell sensing," Biophys. J., 91, 1925-1940 (2006). Micro fluidic articles are also useful tools and some exemplary uses, configurations, and methods of manufacture are disclosed, for example, in U.S. Patent Nos. 6,677,131, and 7,007,709. U.S. Patent Publication 2007/0141231 and U.S. Patent No. 7,175,980, disclose a microplate assembly and method. These documents are hereby incorporated by reference in their entirety.

[0013] "Ambient" refers, for example, to an open lab condition, or an enclosed space, either of which contains the reader instrument and the liquid handling system. A liquid handing system is preferably thermally isolated from the assay plate during compound addition (discussed further below). If there is residual temperature rise in the assay plate after compound addition, the plate can be first exposed to strong direct air flow, which rapidly brings the temperature to ambient, before being returned into the reader.

[0014] The apparatus and methods of the disclosure are particularly well suited for biosensors based on label-independent detection (LID), such as for example an Epic^® system or those based on surface plasmon resonance (SPR). The articles and methods of the disclosure are also compatible with an alternative LID sensor, such as Dual Polarized

Intereferometry (DPI). In embodiments, the biosensor system can comprise, for example, a swept wavelength optical interrogation imaging system for a resonant waveguide grating biosensor, an angular interrogation system for a resonant waveguide grating biosensor, a spatially scanned wavelength interrogation system, surface plasmon resonance imaging, and like systems and applications, or a combination thereof.

[0015] Commonly owned and assigned copending U.S. Patent Applications USSN 13/021,945 and 12/939,606 disclose systems and methods for optically reading microplates. These documents and their corresponding provisional applications are hereby incorporated by reference in their entirety.

[0016] "About" modifying, for example, the quantity, dimension, process temperature, process time, and like values, and ranges thereof, employed in describing the embodiments of the disclosure, refers to variation in the numerical quantity that can occur, for example:

through typical measuring and handling procedures used; through inadvertent error in these procedures; through differences in the manufacture, source, or quality of components, and like considerations. The term "about" also encompasses amounts that differ due to aging of or environmental effects on components. The claims appended hereto include equivalents of these "about" quantities.

[0017] "Optional," "optionally," or like terms refer to the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, the phrase "optional component" or like phrase means that the component can or can not be present and that the disclosure includes both embodiments including and excluding the component.

[0018] "Consisting essentially of in embodiments refers, for example, to optical readers and associated components, to an assay, to method of using the assay to screen compounds, and to articles, devices, or any apparatus of the disclosure, and can include the components or steps listed in the claim, plus other components or steps that do not materially affect the basic and novel properties of the articles, apparatus, or methods of making and use of the disclosure, such as particular components, a particular light source or wavelength, a particular surface modifier or condition, or like structure, material, or process variable selected. Items that may materially affect the basic properties of the components or steps of the disclosure or that may impart undesirable characteristics to aspects of the disclosure include, for example, having inadequate gas transport to maintain an "ambient" temperature equilibrium among system components, or having gas turbulence which can produce disfavored localized heating or cooling within the system.

[0019] The indefinite article "a" or "an" and its corresponding definite article "the" as used herein means at least one, or one or more, unless specified otherwise.

[0020] Abbreviations, which are well known to one of ordinary skill in the art, may be used (e.g., "h" or "hr" for hour or hours, and "rt" for room temperature, "nm" for nanometers, and like abbreviations).

[0021] Specific and preferred values disclosed for components, times, operations, and like aspects, and ranges thereof, are for illustration only; they do not exclude other defined values or other values within defined ranges. The article, apparatus, and methods of the disclosure include those embodiments having any value or any combination of the values, specific values, more specific values, and preferred values described herein.

[0022] The Corning, Inc., Epic^® system is a high throughput label free detection technology platform for studying bio-molecular interactions and live cells. A commercially available Epic^® instrument can detect the average response of each biosensor in a microplate. Label free imaging methods continue to evolve and can now provide spatially resolved high content label free responses within each sensor (see for example, commonly owned and assigned U.S. Patent 7,599,055, to Gollier et al, entitled "Swept wavelength imaging optical interrogation system and method for using same"). This document is incorporated by reference in its entirety.

[0023] In embodiments, the disclosure provides an improved microplate optical reader for optical reader imaging of biochemical, live-cell, and like label-independent-detection (LID) assays.

[0024] Swept wavelength imaging system for interrogating a resonant waveguide grating (RWG) based Epic® biosensor has been described and demonstrated, see for example, the aforementioned US 7,599,055. The optical system described in US Patent 7,599,055, was designed to interrogate an entire SBS format microplate. The technology recently evolved from using a narrow band tunable laser to a tunable light source with optimum temporal and spatial coherence (see commonly owned and assigned copending USSN 12939606, entitled "Tunable light source for label independent optical reader," first filed Nov. 10, 2009). The tunable light source is much lower in cost compared to tunable lasers and commercially available tunable filters. The tunable light source eliminated optical speckles and simplified data processing.

[0025] Label- free optical biosensors such as Surface Plasmon Resonance (SPR) and resonant waveguide grating (RWG) can detect minute changes in refractive indices as a result of molecular interactions or changes in mass distributions inside live cells. The sensor readout can also be affected by temperature because the optical index of a buffer solution is thermally sensitive. For example, a RWG biosensor has a temperature coefficient of about 27 picometers (pm)/°C. For this reason, the majority of commercially available SPR instruments, such as Biacore, precisely control the sensor temperature in the instrument. RWG based Epic® sensors are made in microplate format. Although temperature controlled microplate readers for non-label free modalities are known (see for example, WO2004EP2705 A, entitled "Autonomous device with active temperature regulation," and US5459300A entitled

"Microplate heater for providing uniform heating regardless of the geometry of the

microplates"), temperature control of biosensors on such a scale and to such precision can be more challenging. In addition to the temperature sensitivity of the buffer, the plastic microplate may expand or contract, and warp as temperature changes. It is therefore important that the microplate be maintained at an equilibrated temperature during

measurement. In practice, the optical reader instrument contains electronics that can cause a higher temperature than ambient. Active temperature control often implies operating at elevated temperatures greater than ambient to avoid the use of refrigeration. Such a temperature control scheme has been used in certain commercial Epic® systems, where the plate temperate is typically held at 26 °C with a precision of ±0.1°C. Other commercial Epic ® systems do not actively control the plate temperature, so the heat generated by the instrument still brings the plate temperature above ambient by about 2 to 3 °C. Still other Epic® label free imaging reader concepts (see for example, the aforementioned U.S. Patent 7,599,055, and USSN 12939606 entitled "Tunable light source for label independent optical reader"), are likely to have comparable amounts of heat dissipation and hence a similar degree of temperature elevation.

[0026] When the reader temperature is different from ambient, it can take, for example, about 30 minutes for a microplate coming from an ambient environment to thermally equilibrate inside the instrument prior to measurement. This dwell time can be inconvenient and inefficient for users especially for high through put applications. Furthermore, compound addition is often handled by external liquid handling system at ambient temperature conditions, which has a different temperature from the reader. The microplate experiences a temperature excursion when it is transported outside of and back inside the instrument. Temperature re-equilibration can take an additional 20 to 30 minutes. Although in-well referencing can eliminate the second wait time for biochemical assays, the technique is not applicable to cell assays. Kinetic responses of cell assays with small responses or low compound doses can be skewed by the thermal disturbances during the assays. Moreover, there are cell lines that respond to sudden temperature changes, which can further deteriorate the data quality of those assays.

[0027] A superior instrument control and further enhanced assay capabilities are desirable. In the present disclosure rather than operating the microplate at an elevated temperature, an excellent solution is to maintain the plate uniformly at ambient temperature throughout the assay, thereby minimizing the temperature excursions. The disclosure provides a method and apparatus to achieve this feature.

[0028] In embodiments, the disclosure provides an apparatus that maintains a microplate in a reader instrument or reader apparatus at ambient temperature even though the reader instrument may be at a higher temperature. The apparatus includes an air flow system that is integrated within the reader. The air flow system thermally insulates the plate from the instrument body and rapidly removes or minimizes the thermal gradient. Experiments have demonstrated a temperature excursion of less than 0.1 °C when the plate was transferred from ambient conditions to the disclosed apparatus. Additionally, the plate equilibration time was reduced to, for example, less than about 3 minutes.

[0029] An advantage of the disclosed system includes, for example, a more cost effective solution compared to actively controlling the microplate temperature. The disclosed apparatus eliminates the temperature difference between the plate holder area of the reader and ambient, enabling, for example, the use of an external liquid handling system for compound addition without creating a temperature mismatch, which mismatch can adversely affect the assays. The disclosed apparatus improves the accuracy of small signal cell assays. The disclosed apparatus also enables small signal biochemical assays without in-well referencing, which can further lower the plate cost.

[0030] In embodiments, the presently disclosed method and apparatus provide the following benefits. The present disclosure provides assays having reduced assay time. Plates from ambient temperature do not need the long wait time for thermal equilibration. The present disclosure enables low cost plates. With a consistent and low temperature gradient, temperature change can be accurately referenced by only a few wells. This result enables high sensitivity biochemical assays without self-referencing sensors. The present disclosure enables new cell assays. Because of minimal temperature excursion and effective

temperature referencing, small cell kinetics can be detected. The apparatus and method also enables assays on cells that respond to temperature changes. The apparatus and method of the present disclosure are low cost. The disclosed system does not require active temperature control. Hardware costs can be minimized.

[0031] The disclosed apparatus and method can be used with optical biosensors in any format. The disclosed apparatus and method can be used with all generations of Epic® readers, such as where the microplate is heretofore measured at higher temperatures than ambient. In presently disclosed systems, the air flow chamber can reduce the time to reach thermal equilibrium and remove the thermal gradient that is otherwise trapped beneath the bottom of the microplate.

[0032] In embodiments, the disclosure provides an apparatus for label-free assay, with reference to Fig. 1, comprising:

a housing;

a circulator (310);

a reader module (500); and a sensor module comprising a microplate holder (400), the holder receives and holds at least one microplate article (200) having at least one sensor, the sensor module being spatially separated from the reader module by a cavity (i.e., a flow chamber), the cavity provides a conduit for ambient temperature gas flow from the circulator to between the sensor module and the reader module within the housing.

[0033] In embodiments, a housing can be, for example, a box, a container, and like isolation or shield means to prevent or minimize excursions in "ambient" temperature of the apparatus components and the sensor work piece components, such as resonant waveguide sensors, chemical or biological samples, reagents, reactants, buffers, ligand candidates, chemical compounds, and like materials . In embodiments, where ambient temperature control within the housing is good to excellent the housing can be, for example, a container, a box, a room, a laboratory, a building, and like structures, having a small, a large, or intermediate size sufficient to accommodate the apparatus. In embodiments, a circulator can be, for example, a blower, a propeller, a turbine, a compressor, a compressed gas source, such as a fan as shown, a vacuum pump source or exhaust, and like adiabatic gas circulator or gas propulsion device for creating relative motion of the gas within the apparatus;

[0034] The apparatus can further comprise a compound module having a source of at least one compound, the compound module being in fluid communication (not shown) with the sensor module. For example, the compound module can include a means for transferring at least one compound solid, compound solution, compound suspension, compound aerosol, or like compound formulation, from the compound module to the sensor module, such as a tube, a pipettor, a pipette, or like conduit for liquid transfer.

[0035] The apparatus can further comprise a mixer manifold (320) situated between the circulator and the cavity.

[0036] The cross-section of the mixer manifold (320) can have, for example, dimensions of the width and length within greater than or less than about 30% to about 50% cross-section area of a microplate situated in the plate holder.

[0037] The apparatus is free of an active temperature source, i.e., no active heating or cooling, for example, the apparatus does not employ a heater, a cooler, or a combination thereof, or any associated active temperature control equipment, such as a thermostat, a temperature sensor, or like devices. [0038] The gas flow between the circulator and the sensor module can be, for example, laminar, turbulent, or a combination thereof, and preferably substantially laminar or entirely laminar.

[0039] The circulator can provide a laminar gas flow through the cavity at a rate of about 1 to about 50 cubic feet per minute (CFM), preferably about 5 to about 20 CFM and more preferably about 8 to about 15 CFM, and an output cross section of about 250 mm² or an output cross section of, for example, of about 50x50 mm² and like dimensions.

[0040] The reader module further comprises a leak-proof window (520) having a tilt angle of about 1 to 5° (e.g., 3°). The window prevents back reflections into the reader and is optional if the reader is otherwise leak-proof to liquids or gases.

[0041] The microplate has edge wells and interior wells. The edge and interior wells of a microplate used in the disclosed apparatus and method can have substantially the same temperature profile over time for wavelength change or wavelength stability.

[0042] In embodiments, the disclosure provides a label-free assay method comprising: placing a label- free sensor article at ambient temperature into a sensor module, such as the above described apparatus, and providing a compound module having a source of at least one compound, the compound module being in fluid communication with the sensor module;

maintaining the sensor article at ambient temperature under gas circulation between the sensor article and the reader module with the circulator;

optionally measuring the baseline label-free signal of the sensor article;

transferring at least one compound from the compound module to the sensor article; and

measuring the label-free signal from the sensor article in the reader before and after the transfer of at least one compound and comparing the respective signals.

[0043] In embodiments, the ambient temperature can be maintained, for example, at less than 0.2 °C variation throughout the assay.

[0044] In embodiments, the temperature gradient can be, for example, less than 0.1 °C throughout the apparatus, such as 0.01 to 0.09°C.

[0045] In embodiments, the illumination side of the sensor article can be exposed to uniform laminar air flow having ambient temperature. The ambient air traverses the illumination side or face of the sensor article as schematically shown in Fig. 1, that is, for example, the air flows parallel to and in close proximity to the face of the sensor article being illuminated.

[0046] In embodiments, the plate illumination surface, such as the bottom of the plate, can be, for example, exposed to uniform direct air flow in ambient temperature, such as at 0° to about 45° angle relative to the plate illumination surface.

[0047] In embodiments, the mixer manifold can have an input cross section that approximately or exactly matches the inlet of the circulator, and the manifold output cross section can approximately or exactly match the inlet of the cavity.

[0048] In embodiments, the mixer manifold can have a gas velocity or gas speed circulation that is consistent, i.e., uniform or constant and without turbulence, across the mixer manifold outlet of within greater than or less than from about 20% of the average gas velocity.

[0049] In embodiments, the cavity of the apparatus can have surfaces that are smooth and the cross section along the chamber is uniform or consistent, i.e., constant and without turbulence. The apparatus can further comprise having the sensor module optionally having one or more of, for example, a plate positioning mechanism, an air inlet having a width equal to the width or length of the plate, an air outlet, one or more side walls, a bottom plate and like options, or combinations thereof.

[0050] In embodiments, the disclosure provides a method of reading a microplate in, for example, the aforementioned apparatus, the method comprising:

forming a microplate assembly by engaging a microplate holder in a reader apparatus with a microplate having at least one well and the well having at least one sensor;

maintaining the reader apparatus including a reader module and the microplate assembly under constant ambient air flow conditions through an internal cavity between the microplate assembly and the reader module for a time;

illuminating the microplate;

collecting the reflected light from the illuminated microplate;

forming an image of the reflected light on an image sensor; and

optionally analyzing the formed image;

a collimated tunable light source; a broadband light source;

a light source focused on a single spot at a time;

or a combination thereof.

[0051] In embodiments, the disclosure provides an apparatus suitable for use with a variety of reader systems including, for example, a full-plate or microplate swept wavelength imaging system, or a non- wave length swept system, including for example:

a broadband source or a tunable light source having a spectral width substantially similar to the resonance width of at least one sensor of a full-plate, for example, from 0.3 time to 3 times the width of the resonance;

a lens ensemble for collimating the tunable light source on the full-plate and for transmitting any reflected light from the full-plate;

a beam splitter for diverting a portion of the transmitted light;

an imaging lens for collecting diverted light and producing an optical image of the at least one sensor of the full-plate; and

an image sensor for receiving the optical image of the at least one sensor of the full-plate,

wherein the apparatus housing the imaging system is adapted to maintain a microplate in a reader instrument or reader apparatus at ambient temperature even though the reader instrument can be at a higher temperature.

[0052] In embodiments, the disclosure provides a method for interrogating a resonance waveguide (RWG) sensor of a microplate with any of the abovementioned systems or apparatus, comprising:

emitting an optical beam from the tunable source light source having a spectral width substantially similar to the resonance width of at least one sensor of the microplate;

converting the optical beam into one or more interrogation beams with the lens ensemble;

illuminating one or more sensors of the microplate with the one or more of the interrogation beams;

collecting the reflected light of the illuminated one or more plate sensors; and forming an image on the image sensor,

wherein the method maintains a microplate in a reader instrument or reader apparatus at ambient temperature even though the reader instrument can be at a higher temperature. [0053] The apparatus can further comprise, for example, a microplate, a well plate, a microscope slide, a chip format, or like analyte container, support member, or sample presentation article, and optionally including, for example, microfluidic flow facility.

[0054] In embodiments, the apparatus can have at least one microplate, having at least one well, the well having the at least one optical sensor therein, and the sensor having a signal region and an optional reference region. The microplate can be an array of wells such as commerically available from Corning, Inc.

[0055] In embodiments, the disclosure provides a method of reading an evanescent wave sensor in the abovementioned apparatus having a user-provided, engaged microplate having at least one sensor.

[0056] In embodiments, a received or provided microplate (work-piece) can have a base or substrate thickness, for example, of from about 10 micrometers to about 10,000

micrometers, about 50 micrometers to about 10,000 micrometers, and 100 micrometers to about 1,000 micrometers, including any intermediate values and ranges. A specific example of a microplate base thickness is, for example, of from about 0.1 millimeters to about 10 millimeters, such as 0.3 millimeters to about 1.0 millimeters. A thinner microplate base can, for example, reduce distortion and can improve image quality. A thin microplate base can be, for example, glass, plastic, and like materials, or combinations thereof, having a thickness of about 0.7 mm to 1.0 mm, and can be representative of the thicknesses found in certain commercial products. Glass, plastic, or like materials, having a thickness of less than about 0.4 mm is operatively a thin base plate material.

[0057] In embodiments, the incident beam can contact at least one optical sensor in, for example, at least one of: a single well, two or more wells, a plurality of wells, or preferably all wells of the received microplate.

[0058] The evanescent wave sensor can be, for example, a resonant waveguide biosensor, a surface plasmon resonance (SPR) sensor, and like sensors, or a combination of such sensors.

[0059] In embodiments, the sensor can include on its surface, for example, at least one of a live-cell, a bioentity, a chemical compound, a coating, and like entities, or a combination thereof.

[0060] In embodiments, the method can, for example, further comprise simultaneously or sequentially contacting the optical sensor with a fluorescence inducing incident beam and recording the received fluorescent image with a suitable recorder. That is, to accomplish, for example, cellular or sub-cellular fluorescence imaging (see, for example, commonly owned and assigned copending application US SN 12/151,175, entitled "SYSTEM AND METHOD FOR DUAL-DETECTION OF A CELLULAR RESPONSE").

[0061] Although the sensor can be interrogated using a swept wavelength imaging technique, a simpler intensity imaging technique, which is commonly used in SPR imaging, can be employed because of the wide resonance width. This method can be facilitated by the use of a low coherence light source, which removes the parasitic interference fringes (see for example commonly owned and assigned copending copending USSN 61/259802)

[0062] In embodiments, the disclosed imaging technique can be applied to compact Epic^® configurations and applications by, for example, redesigning the field of view to cover 4x3 wellplate configurations, or like configurations. In embodiments, the disclosed system provides high spatial resolution and at reduced cost because the disclosed system can avoid a precision swept wavelength mechanism. The system's overall simplicity provides an optical reader having lower overall cost. However, in this operating mode the readout can be more sensitive to defects on the sensor surface.

EXAMPLES

[0063] The following examples serve to more fully describe the manner of using the above-described disclosure, as well as to further set forth the best modes contemplated for carrying out various aspects of the disclosure. It is understood that these examples do not limit the scope of this disclosure, but rather are presented for illustrative purposes. The working example(s) further describe(s) how to make and use the apparatus and practice the methods of the disclosure.

Example 1

[0064] Reader Apparatus having Ambient Temperature Control The disclosed apparatus and method, in embodiments, provide a uniform and steady flow of ambient air across a major surface of a microplate, such as the irradiated or the bottom side of the microplate opposite the wells. In particular, the disclosure provides an apparatus having an air flow system integrated into a label free reader instrument.

[0065] Referring to the Figures, Fig. 1 illustrates a SBS standard microplate (200) with a label- free biosensor embedded in the bottom of the wells is placed on a reader (100).

Because reader instrument contains electronics that dissipate heat, the instrument can have a higher temperature than ambient by, for example, 2 °C to 4 °C. To exacerbate the problem, the outer skirt of a microplate can form a trap for the rising hot air from the reader electronics. Even if the microplate holder is thermally insulated from the instrument, the hot air eventually heats the microplate and brings the plate's temperature above ambient. Because of the lack of air flow in the bottom of the microplate, the thermal gradient can vary over time as the ambient temperature changes. Both temperature and temperature gradient variations can adversely affect assay results.

[0066] Fig. 2A illustrates the thermal effect on the microplate when measured by a reader with a conventional plate holder. The microplate is filled with water and first equilibrated in ambient temperature. The plate is then placed on a reader which is at 2°C higher than ambient temperature. Continuous measurement and recording begins immediately after placing the 384 well microplate on the reader. Wavelength shifts appear to stabilize after about 25 minutes, and the microplate temperature is increased by about 1 °C. Wavelength shifts of peripheral wells or edge wells are significantly different from other wells because the edge wells have faster kinetics than wells situated in the interior and the center of the plate. Two example traces are compared in Fig. 3A. As a result, ambient temperature change affects the edge wells differently from center wells, causing a time-varying

temperature gradient. Wavelength shift across the plate at 20 minutes is shown in Fig. 4 A. The spatial distribution also varies considerably in different time points. A histogram of the wavelength shift at 20 minutes is shown in Fig. 5A. The standard deviation of the

wavelength shift is 2.8 picometers (pm). The wells around the edges of the microplate are more susceptible to ambient temperature change than the inner wells. Zee prime or Zed prime (Ζ') is a well known metric of the statistical goodness of an assay.

[0067] The disclosed apparatus and method, in embodiments, directs smooth and uniform air flow on the reading side (e.g., underneath) of the microplate bottom to effectively maintain ambient temperature in the microplate. The air flow also eliminates temperature gradients. The air flow chamber is designed as part of the plate holder, with the reading side (e.g., bottom) of the microplate forming the top of the chamber. In embodiments, a laminar air flow can be created around or about the plate. The flow chamber can include the plate mounting mechanisms that are designed to ensure smooth flow through the microplate without turbulence or dead zones.

[0068] As illustrated in Fig. 1, air is generated from a circulator (310), such as a blower or a fan, which can have, for example, a flow rate of 10 cubic feet per minute (CFM) and an output cross section of about 50x50 mm². An air filter can optionally be placed in the intake of the blower to remove dust particles. The air flow passes through a mixer (manifold) (320) and into the inlet of the flow chamber, the width of which can be equal to the width or length of the microplate. The height of the inlet can be, for example, 6 mm, and the back pressure is preferably minimal. The mixer smoothly guides the air flow into the cavity or flow chamber situated between the reader module and the sensor module and plate holder (400), with a uniform flow rate across the inlet. The flow chamber is formed between the sensor module including the plate holder (400), and the microplate (200), and the reader module plate (510), which can be part of the instrument (500). The plate holder can have smooth features (330), which avoid the generation of air turbulence. A "spill-proof or "leak-proof reader window (520) can optionally be incorporated into the bottom plate of the flow channel. The window (520) can be tilted at an angle of about 3° to prevent back reflections into the reader. The window is optional if there is no air leakage into the reader. The bottom surface of the flow chamber can be formed by reader module plate (510) and reader window (520) and can be further designed to conform to the top surface. The top surface of the chamber corresponds, in part, to the base or bottom of the microplate(210), so that the cross sectional area of the flow chamber is nearly constant throughout the flow chamber. Having a constant cross section along the length of the chamber facilitates maintaining a consistent gas flow rate in the chamber.

[0069] The gap (430) between the bottom of the microplate skirt and the surface of the plate holder is preferably less than, for example, 0.25 mm to minimize air leakage. The microplate can be supported, for example, on the insert surface by three balls (410) for angular repositioning accuracy. The locations of the mounting balls are designed to minimize air obstruction. Lateral positions of the microplate are typically defined by three dowel pins (420). It can be preferable that the air flow direction is against the pins which serve as natural stops.

[0070] In embodiments, the separation or gap between the plate holder (400) and the reader module (510), that is the height of the cavity or flow chamber (405), in a single plate configuration, can be, for example, about 0.2 cm to about 4 cm, such as about 0.6 cm.

Spacing can vary depending, for example, on the number of plates in the configuration and gas flow rates. The plate holder (400) is preferably thermally insulated from the reader (100). The flow rate required to maintain ambient temperature is lower in this instance. In practice, a larger flow rate can reduce the time to reach thermal equilibrium. The flow rate can also be variable. For example, the flow rate can be set to high when a microplate has just been placed on the instrument, and the gas flow rate can be systematically reduced after

equilibrium.

[0071] As a comparison, the same experiment was repeated on the disclosed reader apparatus having the disclosed an air flow chamber. The flow rate was about 10 cubic feet per minute (CFM). Fig. 2B shows an average wavelength shift of -1.6 pm or -0.06°C temperature drop on the microplate when it is placed from ambient onto the reader, even though the reader temperature is 2°C higher than ambient. Wavelength shift traces appear well-synchronized after three minutes, suggesting that the temperature gradient has stabilized. With a constant thermal gradient, only a small number of wells are sufficient to reference out the temperature variation. Wavelength shifts of two wells are compared in Fig. 3B.

Wavelength shift across the plate at 20 minutes is shown in Fig. 4B. The distribution is consistent at different time points independent of ambient temperature variation. A

histogram of the wavelength shift at 20 minutes is depicted in Fig. 5B. The standard deviation of wavelength shift is 0.8 pm. This value is more than three times less than that of the reader with a simple plate holder. Improving the flow chamber design can further reduce the variation. Equilibration time was reduced from about 25 minutes to about 3 minutes.

[0072] When conducting compound assays, the compound plates (600) can be placed on a holder (700), which can be in close proximity to the sensor plate (200). Air can be circulated on top of the sensor plate (200), and around the compound source plates (600), so that the plates are well equilibrated with ambient. When the assay plate is removed for compound addition, the replacement plate is placed on the reader to maintain consistent thermal condition in the reader.

Example 2

[0073] Reader apparatus having ambient temperature control An alternative implementation of the Reader apparatus having ambient temperature control is illustrated in Fig. 6, where the microplate directly deflects a uniform air flow without forming a flow chamber. This method may necessitate a larger spacing, for example, a spacing which is greater than about 1 inch (2.5 cm), between the plate holder (400) and the reader module (510). In embodiments, the larger separation or spacing gap (405) between the plate holder (400) and the reader module (510) in a single plate configuration, can be, for example, about 0.5 cm to about 8 cm, such as about 2 cm. [0074] The disclosure has been described with reference to various specific embodiments and techniques. However, it should be understood that many variations and modifications are possible while remaining within the scope of the disclosure.

Claims

What is claimed is:

1. An apparatus for label- free assay, comprising:

a housing;

a circulator;

a reader module; and

a sensor module comprising a microplate holder,

the holder receives and holds at least one microplate article having at least one sensor, the sensor module being spatially separated from the reader module by a cavity, the cavity provides a conduit for ambient temperature gas flow from the circulator to between the sensor module and the reader module within the housing.

2. The apparatus of claim 1 further comprising a compound module having a source of at least one compound, the compound module being in fluid communication with the sensor module.

3. The apparatus of claim 1 further comprising a mixer manifold situated between the circulator and the cavity.

4. The apparatus of claim 3 wherein the cross-section of the mixer manifold has dimensions of the width and length within greater than or less than about 30% to 50% of a microplate situated in the plate holder.

5. The apparatus of claim 1 wherein the apparatus is free of an active temperature source.

6. The apparatus of claim 1 wherein the gas flow between the circulator and the sensor module is substantially laminar.

7. The apparatus of claim 1 wherein the circulator provides a laminar gas flow through the cavity at a rate of about 1 to about 50 cubic feet per minute (CFM) and an output cross section of about 250 mm².

8. The apparatus of claim 1 wherein the reader module further comprises a leak- proof window having a tilt angle of about 1 to 5°.

9. The apparatus of claim 1 wherein the microplate has edge wells and interior wells, the edge and interior wells having substantially the same temperature profile over time for wavelength change or stability.

10. A label- free assay method comprising:

placing a label- free sensor article at ambient temperature into the sensor module of the apparatus of claim 2;

optionally measuring the baseline label-free signal of the sensor article;

measuring the label- free signal from the sensor article in the reader before and after the transfer of at least one compound and comparing the respective signals.

11. The method of claim 10, wherein the ambient temperature is maintained at less than 0.2 °C variation throughout the assay.

12. The method of claim 10, wherein the temperature gradient is less than 0.1 °C throughout the apparatus.

13. The method of claim 10, wherein the illumination side of the sensor article is exposed to uniform laminar air flow having ambient temperature, and the air flow traverses the illumination side of the sensor article.

14. The method of claim 10, wherein the plate bottom is exposed to uniform direct air flow in ambient temperature at 0° to about 45° angle relative to the plate bottom surface

15. The apparatus of claim 1 wherein the mixer manifold has an input cross section that matches the inlet of the circulator, and the manifold output cross section can match the inlet of the cavity.

16. The apparatus of claim 1 wherein the mixer manifold has a gas circulation velocity that is uniform across the mixer manifold outlet of from about 20% greater than or less than of the average velocity.

17. The apparatus of claim 1 wherein the cavity chamber has smooth surfaces and the cross sectional along the chamber is uniform.

18. The apparatus of claim 1 further comprising having the sensor module having a plate positioning mechanism, an air inlet having a width equal to the width or length of the plate, an air outlet, one or more side walls, and the illuminated reading surface of a microplate is received by and held by the apparatus.

19. A method of reading a microplate, comprising:

illuminating the microplate;

collecting the reflected light from the illuminated microplate;

forming an image of the reflected light on an image sensor; and

optionally analyzing the formed image. The method of claim 19 wherein illuminating the microplate is selected from at one of:

a coUimated tunable light source;

a broadband light source;

a light source focused on a single spot at a time;

or a combination thereof.