Lambda theta reflectometry for biosensing applications

Abstract

Determination of the presence and / or concentration of proteins contained in biological serum can be accomplished by optically monitoring a surface reflectivity of a thin film on a chip. Lambda theta reflectometry can be used to measure thin film layer thickness by identifying a surface reflectivity null as a function of incident light wavelength and angle of incidence. Lambda theta reflectometry generates a plurality of images while the chip, illuminated by a light source, is moved to different positions. The chip comprises a plurality of sites. An antireflection condition for each of the plurality of sites is calculated based on the plurality of images.

Description

PATENTAttorney Docket No. 096027-1518887-3-24071WOLAMBDA THETA REFLECTOMETRY FOR BIOSENSING APPLICATIONSCROSS REFERENCE TO RELATED APPLICATION

[0001] This application is related to and claims priority benefit from U.S. Provisional Patent Application No. 63 / 682,140, filed on August 12, 2024, entitled LAMBDA THETA REFLECTOMETRY FOR BIOSENSING APPLICATIONS, which is incorporated in its entirety for all purposes.BACKGROUND

[0002] Clinicians and researchers desire custom protein detection of disease-specific biomarkers. Quantitative measurements of protein biomarkers inform disease diagnosis and treatment. Some accurate methods for detecting biomarkers are label-free and preserve native protein binding interactions. Label-free biomolecular interaction analysis includes reflectometry, a family of techniques that measure protein binding via changes in optical thickness when light interacts with phase boundaries of a device.BRIEF SUMMARY

[0003] This disclosure, without limitation, relates to detection of biomarkers. In some configurations, a system for lambda theta reflectometry comprises a light source; a chip with a plurality of sites arranged on the chip, wherein the chip is arranged to be moved about an axis to enable independent measurements of individual sites; a detector; a lens system arranged to focus light from the light source incident on the chip to the detector; and / or one or more memory devices comprising instructions that, when executed, cause one or more processors to perform the following steps: generating a plurality of images using the detector while the chip is moved to different positions about the axis, and calculating an antireflection condition for each of the plurality of sites based on the plurality of images. In some configurations, the instructions, when executed, cause the one or more processors to detect a biomarker in human serum, plasma, saliva, urine, whole blood, cerebral spinal fluid, tumor aspirate, infection site aspirate, or Medium Enriched for Secreted Antibodies at one or more of the plurality of sites; the instructions, when executed, cause the one or more processors to detect a protein or an antibody at one or more of the plurality of sites; an angle of rotation of the chip about the axisis equal to or less than 5 degrees; the plurality of sites are arranged in an array for testing for multiple different proteins or antibodies concurrently; a peak wavelength of light from the light source incident upon the chip is varied during generating the plurality of images; the peak wavelength of light from the light source incident upon the chip is between 300 nm and 1000 nm; the system further comprises a grating optically between the chip and detector; the system further comprises an aperture optically between the chip and the grating; and / or the aperture is one of a plurality of apertures between the chip and the grating.

[0004] In some configurations, a method for lambda theta reflectometry comprises directing light from a light source to a chip with a plurality of sites arranged on the chip, wherein the chip is arranged to move about an axis; focusing light reflected from the chip on a detector using a lens system; generating a plurality of images using the detector while the chip is moved to different positions about the axis; and / or calculating an antireflection condition for each of the plurality of sites based on the plurality of images. In some configurations, the method further comprises detecting a biomarker in human serum at one or more of the plurality of sites, and / or detecting a protein or an antibody at one or more of the plurality of sites. In some configurations, an angle of rotation of the chip about the axis is equal to or less than 5 degrees; the plurality of sites are arranged in an array for testing for multiple different proteins or antibodies concurrently; a peak wavelength of light from the light source incident upon the chip is varied during generating the plurality of images; the peak wavelength of light from the light source incident upon the chip is between 300 nm and 1000 nm; a grating is optically between the chip and detector; and / or an aperture is optically between the chip and the grating.

[0005] In some configurations, a system for lambda theta reflectometry comprises a light source; a chip with a plurality of sites arranged on the chip, wherein the chip is arranged to be moved; a detector; a lens system arranged to focus light from the light source incident on the chip to the detector; and / or one or more memory devices comprising instructions that, when executed, cause one or more processors to perform the following steps: generating a plurality of images using the detector while the chip is moved to different positions, wherein an angle of rotation is equal to or less than five degrees, and the plurality of images are generated while a peak wavelength of light from the light source incident upon the chip is varied, calculating an antireflection condition for each of the plurality of sites based on the plurality of images, and / or detecting a protein or an antibody at one or more of the plurality of sites.BRIEF DESCRIPTION OF THE DRAWINGS

[0006] The present disclosure is described in conjunction with the appended figures.

[0007] FIG. l is a graph of an embodiment of changes in reflectivity as a target binds to a probe.

[0008] FIG. 2 is an image of an embodiment of a substrate with baseline probe deposition at different optical thickness.

[0009] FIG. 3 shows an embodiment of a calibration curve of reflectance vs thickness.

[0010] FIG. 4 depicts an embodiment of a technique used to measure a unique angle and wavelength of light combination forming an antireflective condition and calculate target binding thickness for each probe on a substrate.

[0011] FIGs. 5A and 5B depict an embodiment of an optical design for a single-channel instrument.

[0012] FIG. 5C depicts another embodiment of a single-channel instrument.

[0013] FIG. 5D depicts an embodiment of an elliptical aperture used in the single-channel instrument of FIG. 5C.

[0014] FIG. 6 depicts an embodiment of a single pixel lineout from a simulated image.

[0015] FIGs. 7A and 7B depict an embodiment of an optical ray-trace modeling of an instrument.

[0016] FIG. 8 depicts a couple of charts of an embodiment showing that a position of reflectance null in lambda / theta space.

[0017] FIG. 9 shows a graph of an embodiment of the antireflective condition as a function of layer thickness.

[0018] FIG. 10 shows a graph of an embodiment of the antireflective condition at a specific thickness as a function of angle and wavelength.

[0019] FIG. 11 shows graphs of the antireflective condition as a function of incident angle and wavelength for an embodiment of Lambda Theta Reflectometry (LTR) images at different film layer thicknesses.

[0020] FIG. 12 is a block diagram of an embodiment of the single channel LTR instrument.

[0021] FIG. 13 depicts an embodiment of using an image set from a multi-channel LTR.

[0022] FIG. 14 depicts a block diagram of an embodiment of a spatially resolved high throughput LTR.

[0023] FIG. 15 depicts an embodiment of a process for multi-channel LTR.

[0024] In the appended figures, similar components and / or features may have the same reference label. Further, various components of the same type may be distinguished by following the reference label by a dash and a second label that distinguishes among the similar components. If only the first reference label is used in the specification, the description isapplicable to any one of the similar components having the same first reference label irrespective of the second reference label.DETAILED DESCRIPTION

[0025] The ensuing description provides preferred exemplary embodiment s) only, and is not intended to limit the scope, applicability, or configuration of the disclosure. Rather, the ensuing description of the preferred exemplary embodiment s) will provide those skilled in the art with an enabling description for implementing a preferred exemplary embodiment. It is understood that various changes may be made in the function and arrangement of elements without departing from the spirit and scope as set forth in the appended claims.

[0026] The global market value of biomarker discovery, validation, and diagnostic technology may reach an approximate value of $100 billion in the next several years. Need is driven by developments in diagnosing and treating cancer, but can be extended to various immunologically complex chronic diseases. Multiplex array-based protein detection technologies are a tool for biomarker discovery. The most commonly used commercially available methods for measuring protein biomarkers rely on a fluorescent label to detect target molecules, which limits measurements to one subclass of proteins within a single assay. Simultaneous quantification of inflammatory proteins, antibodies, and cytokines in human serum can advance research by providing a more informative snapshot of the immune response in specific disease contexts. Label-free optical techniques for biomolecular interaction analysis introduce a pathway towards achieving this goal.

[0027] Determination of the presence and concentration of proteins contained in biological serum can be accomplished by optically monitoring the surface reflectivity of a multi-layer- thin-film substrate containing capture antibodies. This label-free biomolecular interaction analysis includes reflectometry, a family of techniques that measure the binding of protein targets via changes in optical thickness when light interacts with phase boundaries of the device. The signal can be measured as a change in polarization (ellipsometry, Oblique Incidence Reflectivity Difference (OI-RD)), interference spectrum (Reflectometric Interference Spectroscopy (RIfS), Bio Layer Interferometry (BLI), Spectral Reflectance Imaging Biosensor / Interferometric Reflectance Imaging Sensor (SRIB / IRIS)), or destruction of an antireflective condition (Arrayed Imaging Reflectometry (AIR) and Brewster Angle Interferometry (BASI)).

[0028] Arrayed Imaging Reflectometry (AIR) can detect low-abundance proteins because it is highly sensitive near an antireflective condition. AIR is a multiplexed technique in whichan array of different capture antibodies is imaged using a light of a single wavelength and angle of incidence at tightly controlled SiO2 layer thickness. However, the stringent requirements for substrate production for AIR can be difficult and costly to achieve. Lambda Theta Reflectometry (LTR) has the potential to relax manufacturing tolerances of substrates, decrease production costs, and / or increase sensitivity of an assay relative to AIR. LTR can achieve this by introducing flexibility in incident angle and / or wavelength to identify an antireflective condition for each probe on a substrate. The ability for clinicians and researchers to design highly sensitive custom multiplex protein-detection arrays in-house could elevate research and clinical practice and / or increase understanding of the complex proteomes and immunomes involved in disease.

[0029] Existing single-antigen point-of-care tests exist for Influenza, Covid-19, and Group A streptococcal pharyngitis, and are highly effective at rapidly diagnosing patients and accelerating a path to effective treatment. There are many diseases for which a multipleantigen or antibody test would be useful to the same end. Autoimmune and chronic inflammatory diseases constitute a large group of conditions for which diagnosis and treatment could benefit from a panel of protein and antibody measurements. An array -based diagnostic tool could be useful for informing clinical diagnosis of these diseases and could aid in understanding risk factors and disease progression. Clinicians have demonstrated interest in the use and development of array-based protein detection to detect protein biomarkers for specific diseases. Clinicians have requested custom panels for Dermatomyositis (DM), Bullous Pemphigoid, Seborrheic Dermatitis, Atopic Dermatitis, Staphylococcus aureus infections, and cancer antigens. These targets are present in human samples at a wide range of concentrations, and quantitative information can be used for diagnostic or prognostic ends. For example, the ability to measure specific antibodies implicated in DM currently exists only as a qualitative assay. For DM antibody profiling, serum samples are shipped to an external facility that has 3 -week or more turnaround to perform an assay which produces yes / no results. There is a need for a more rapid and quantitative assay to speed treatment administration and to advance research into the relationships between DM-specific antibody titers and disease severity. Some configurations can bridge this gap by simplifying rapid development of custom arrays. In some embodiments, target binding to each probe on the array is measured with exceptional sensitivity, which improves the ability to measure low-abundance targets in human samples

[0030] LTR can be used to measure thin film layer thickness by identifying a surface reflectivity null as a function of incident light wavelength and angle of incidence (e.g., onto asilicon dioxide / silicon substrate). The foundational AIR technology relies on sensor substrates manufactured to a fixed thickness, and uses incident light at a fixed angle and wavelength in order to measure changes in reflected light intensity as protein targets bind. In practice, it is difficult to achieve the manufacturing tolerances used to achieve the highest binding sensitivity with AIR. LTR technology can quantitatively measure captured target protein without constraining initial substrate or probe thickness. LTR can measure the antireflective condition for each probe spot by illuminating the sensor chip with multiple wavelengths (e.g., spanning 600-700 nm) over a range of angles (e.g., 1, 2, 3, or 5 degrees). The reflected light can be spectrally analyzed to identify a unique angle and wavelength parameters that produce the antireflection condition for each probe / target binding location. These parameters contain information used to calculate thickness as a label-free measurement of a target-probe protein binding event. Thus, an optical biosensing device can detect biological interactions in a repeatable and expected manner. Benefits can include relaxing thin-film sensor substrate manufacturing tolerances and / or ensuring a high sensitivity measurement for each probe on the array. This can simplify the production of custom arrays and / or increase the ability to detect low concentration proteins.

[0031] In some configurations, a single channel version of LTR demonstrates functionality and tests the feasibility of assembling optics and acquiring a reliable measurement. The single channel version can be used to validate techniques by measuring silicon / silicon dioxide substrates with varying silicon dioxide thicknesses. The single channel version can be used to test the functionality of LTR in the presence of linking chemistry and probe / target protein. For example, IgG molecules can be deposited onto the substrate and incubated with anti-IgG antibody to construct a serial dilution curve. This can provide information about the integrity of LTR for detecting proteins at varying concentrations. A Langmuir binding curve is expected, which is typical of antigen-antibody interactions.

[0032] In some configurations, a multichannel LTR system can be used. A strength of this technique, in some embodiments, is the ability to create custom multiplex protein arrays to detect biomarkers for specific diseases. In some configurations, a 5-plex Dermatomyositis (DM) array recombinant proteins can be tested against five autoantibodies known to be biomarkers of DM: MDA-5, Mi-2, SAE, NXP2, and TIF-ly. The multichannel LTR system can be valuable for multiplexed detection of complex combinations of biomarkers in human serum and other biological samples. The ability to design custom arrays in-house can elevate research and clinical practice (e.g., by improving understanding of complex immune states underpinning specific diseases).

[0033] In FIG. 1, AIR measures large changes in reflectivity with small changes in thickness as target binds to a probe near the antireflective condition, in one embodiment. Reflectivity vs angle of incidence under illumination with 632.8 nm is shown. Of various reflectometric techniques, AIR, in which an array of different capture antibodies is simultaneously imaged using a light of a single wavelength and angle of incidence, has the capability for detecting low-abundance proteins. AIR is highly sensitive near the antireflective condition such that small changes in optical thickness (target binding) are measured as large increases in the intensity of reflected light, as shown in FIG. 1. AIR measures protein binding by measuring the reflectivity of a sensor surface illuminated with single, polarized, monochromatic source at fixed angle of incidence designed to produce an antireflection condition prior to protein capture. Target molecule binding adds to layer thickness and disrupts the antireflection condition resulting in an increased reflectivity that is detected quantitatively through spatial imaging of the sensor surface.

[0034] In FIG. 2, an image of an embodiment of an AIR substrate with baseline probe deposition at different optical thicknesses is shown. It is difficult to formulate each probe to deposition at the antireflective condition. AIR has demonstrated the ability to detect proteins at low concentrations under tightly regulated manufacturing conditions. However, stringent tolerances for substrate production are difficult and costly to achieve. Furthermore, covalently bound probe molecules are deposited at different optical thicknesses and are not sometimes optimally tuned to achieve the antireflective condition across the multiplexed chip, despite attempts to do so. This can result in loss of sensitivity and / or introduce further complexity into the production process. These variations in initial thickness, prior to protein capture, can be challenging and time-consuming to quantify, reduce the yield of useable substrates, complicate the array production process, and / or form a barrier to easily producing and validating custom arrays.

[0035] FIG. 3 shows an embodiment of an AIR calibration curve of reflectance vs thickness demonstrating the challenge of achieving optimal probe deposition when incident light angle and wavelength are fixed. Three probe and target binding scenarios with different starting conditions are envisioned. In scenario 1 (red), the probe was properly constructed an increased reflectivity is correlated to build thickness. In scenario 2 (black), initial build was slightly under and protein capture results in no change in reflectivity. In scenario 3 (green), initial build is significantly under and protein binding results in decreased reflectivity. In some configurations, LTR does not rely on optimization of initial probe deposition thickness and will reduce or eliminate these concerns. An ability to relax the manufacturing tolerances ofsubstrates (e.g., silicon / silicon dioxide) can decrease production costs and / or increase sensitivity of the assay.

[0036] FIG. 4 depicts an embodiment of LTR used to measure a unique angle and wavelength of light combination forming the antireflective condition and calculate target binding thickness for each probe individually on the sensor substrate. LTR illuminates the substrate with wavelengths spanning 600-700 nm over a 2-5° range of angles simultaneously, therefore enabling a precise measurement of the optical thickness for each probe / binding site. This approach has the potential to relax thin-film manufacturing tolerances, improve limits of detection by an order of magnitude or more by working within the range of highest sensitivity, and / or enhance the ability to measure different proteins at high and low concentrations within the sample simultaneously.

[0037] The theory behind multi-layer thin film reflectance is well understood and documented. An optical thin film reflectance calculation algorithm was developed based on derivations by A. Thelen in Design of Optical Interference Coatings, McGraw-Hill 1989. The reflectance calculation algorithm calculates the reflectivity of a thin film stack with user- defined number, thickness, and material properties of boundaries as a function of incidence wavelength, polarization, and input angle. Using the reflectance calculation algorithm, a variety of instrumental approaches were explored based on identifying the reflectance null as a function of angle and wavelength of s-polarized incident light.

[0038] FIGs. 5A and 5B depict an embodiment of an optical design of a single-channel LTR 500. In FIG. 5A, a side view shows an angular dispersive axis. An assembly 504 is tilted about an axis 506, centered on the biosensor while the image sensor 508 remains fixed, so that a rotation of the assembly 504 changes a focus of light on the images sensor 508 for a given site in the theta dimension (e.g., changes location of focus for the site in the y dimension; after passing through one or more lenses 510 of lenes and / or an aperture (e.g., pinhole 512; not necessarily the aperture stop)). In FIG. 5B, a top view shows the spectral dispersion axis (e.g., lambda dispersion along the x and / or z axes, after passing through, or reflected by, a grating 514).

[0039] FIG. 5C depicts another embodiment of a single-channel LTR 550. The singlechannel LTR comprises three sections: illumination 552, localization 554, and imaging 556. In the embodiment shown, a length of the illumination 552 and a length of the localization 554 sections are equal (e.g., both are 2, 4, 6, 8, 10, 12, 14, 16, or 20 inches long). The Singlechannel LTR 550 was optimized using OSLO (Optics Software for Layout and Optimization) and checked with FRED (Fred Optical Engineering Software). Light from an illuminationsource 560 is imaged on a target 564. For example, the illumination source is an output surface of an optical fiber. The optical fiber is a multimode optical fiber (e.g., having a core diameter equal to or greater than 10, 100, or 200 and / or equal to or less than 500, 750, or 1000 microns, including as 200, 250, 300, or 350 pm). Spatial locations emit multiple wavelengths at multiple angles. Light incident on the surface of the target 564 is reflected as multiple wavelengths at multiple angles. Light reflected from the target 564 is imaged through an aperture 568 to localize a region of interest. FIG. 5D depicts an embodiment of the aperture 568. The aperture 568 is elliptical (e.g., and not circular). The aperture 568 comprises a wall 570 with an opening 572 defined in the wall 570. FIGs 5C and 5D are not to scale.

[0040] Given typical initial substrate thickness variations, (~1 nm) and anticipated total thickness ranges to be measured (10 nm), a prototype design can be specified. The LTR optical setup comprised a fiber coupled white light lamp, optics for polarization and collimation of the beam, the aperture to eliminate scattered light, and the grating 514 to spectrally disperse the reflected light. By tilt scanning the protein array and corresponding collection optics, a two- dimensional image is produced that maps probe site reflectivity as a function of wavelength and angle of incidence.

[0041] Since the probe protein is arrayed onto the sensor substrate using a piezoelectric arrayer that deposits droplets of liquid, the protein thickness build occurs in a circle within a square detection area. To reduce or eliminate reflected light contributions from background areas on the sensor, the surface illumination is limited to regions that contain probe chemistry. In some embodiments, a digital micromirror device can be used to control the illumination and / or to selectively relay light from the sensed areas on the biosensor. A microscope viewing along a separate line of sight can capture a spatially resolved image of the sensor substrate to evaluate spot integrity, identify defects from scratches and dust, and / or inform micromirror alignment.

[0042] In FIG. 6, an embodiment of a single pixel lineout from a simulated image is shown on the red curve. This lineout is taken at the angle of incidence where minimum reflectivity occurs and includes anticipated detector noise and instrument scattered light levels. An analytical fit to the data is used to ascertain a wavelength corresponding to the reflectivity null with sub-pixel position accuracy. Thickness builds inferred from a fit routine reconstruct an initial model thickness to within + / - 1 picometer over 12 simulated images.

[0043] Theoretical thickness build sensitivity was evaluated using estimated real-world instrument resolution and detector performance parameters. With this model, synthetic LTR detector plane images were generated with given known protein build thickness. Experimentalfits (FIG. 6) to the simulated data were made to recover the inferred thickness. Expected measurement accuracy was quantified by differencing the model input thickness from the measured thickness recovered from the synthetic data over a set of 12 independent simulations. Typical measurement error was less than 1 picometer. This represents a potential order of magnitude improvement in build thickness sensitivity compared to the original AIR technology. Because binding to the surface follows a Langmuir curve, this potentially represents much greater than an order of magnitude improvement in the achievable limit of detection.

[0044] FIGs. 7A and 7B depict an embodiment of an optical ray-trace modeling of an instrument 700 viewed along the spectrally dispersed axis. In FIG. 7A, a single channel measurement is relayed using three lenses to produce a spectrally dispersed line of the biosensor surface reflection. The single channel measurement shown in FIG. 7A can be similar to the layout shown in FIG. 5B. In FIG. 7B, Measurement multiplexing is accomplished using lens array optics. A single 9 element row out of a 9 x 9 array is shown.

[0045] The optical layout was modeled using ray trace software to verify that the first order layout produces good imaging properties using readily available and manufacturable optical components. The area sampled on the sensor is based on the illumination beam geometry. A 1 mm x 1 mm sensor illumination area was specified. Extending a single probe site measurement to an array can be accomplished using monolithic lens array optics. This multichannel design specifies a 1 mm array pitch enabling simultaneous measurement of 81 probe sites spanning a 9 mm x 9 mm substrate.

[0046] Lambda theta reflectometry can be used for identifying a reflectivity null as a function of incident light wavelength and angle of incidence (e.g., onto a silicon di oxide / silicon substrate). In some configurations, surface reflectivity is measured over a range of wavelengths (lambda) and angle of incidence (theta) to find a position of a reflectance null along those two dimensions. Some configurations enable a position measurement of the reflectance null in lambda and theta space, from which thickness can be calculated using mathematics of thin film optical coatings.

[0047] FIG. 8 depicts a couple of charts of an embodiment showing that a position of reflectance null in lambda / theta space contains a very sensitive constraint to film thickness with an error of 7.5 mA + 2.5 mA over 12 repeat modeled measurements.

[0048] FIGs. 5A, 5B, and 5C depict embodiments of a single channel instrument applying the concept of Lambda Theta Reflectometry (LTR): A localized, spatially integrated, reflectivity measurement with continuous angular and spectral resolution. FIGs. 7A and 7Bdepict an embodiment of a multi-channel that generates an array of reflectivity images with continuous angular and spectral resolution corresponding to a plurality of locations on the chip. FIGs. 13 and 14 depict an embodiment of a multichannel - High Throughput LTR: A spatially resolved reflectivity measurement with set of images each with a discrete spectral and angular illumination. Both instruments are intended to be used to for bio sensing applications, but the concept of lambda theta reflectometry is general and will have uses beyond biological sensing.

[0049] There are a handful of reflectometry techniques used to measure the binding of biological molecules (e.g., Fechner P, Gauglitz G, Proll, G. Through the looking-glass — Recent developments in reflectometry open new possibilities for biosensor applications. TrAC 2022;156: 116708), but LTR offers a unique and highly sensitive way to measure static biological binding events. Some reflectometric techniques measure a change in polarization of light (ellipsometry), or a position measurement change in the wavelength at which an interference pattern can be measured (e.g., Reflectometric Interference Spectroscopy, Biolayer Interferometry, Total Reflectometric Interference Spectroscopy), or a change in the reflected light intensity at a single or narrow band of wavelength at fixed angle (Spectral Reflectance Imaging Biosensor, Ik Reflectometry, Arrayed Imaging Reflectometry).

[0050] LTR is a measurement of an angle and a wavelength of the antireflective condition of a thin film optical coating, from which the thickness change due to a biological binding event can be measured. The antireflective condition can have a very steep drop in reflected light intensity, and as a biosensor, measurements are most sensitive at this condition. Small changes in thickness cause large changes in reflected light very close to the antireflective condition. FIG. 9 shows a graph of an embodiment of the antireflective condition as a function of layer thickness at a fixed incident light angle and a fixed wavelength. The antireflective condition at fixed incident light angle and wavelength is a steep drop in reflectivity. Instruments applying the concept of LTR can capitalize on this specificity. Instruments using LTR can measure a location of the antireflective condition 904 so that sensitivity won’t change with thickness, which can enable maximum sensitivity measurements for each probe spot on a multiplex array. FIG. 10 shows a graph of an embodiment of the antireflective condition at a specific substrate thickness as a function of incident light angle and wavelength. The antireflective condition is a unique combination of substrate film thickness and incident light angle and wavelength. FIGs. 9 and 10 are simulations assuming infinite S / P polarization contrast resulting in 10 orders of magnitude of signal level change, without taking the camera noise floor, scattered light, or substrate surface heterogeneity into consideration. FIG. 11 shows graphs of the antireflective condition as a function of incident angle and wavelength foran embodiment of LTR single channel images at different film layer thicknesses, which demonstrate movement of antireflective condition in angle and wavelength space. FIG. 11 shows a theoretical reflectivity null with 30,000: 1 s:p polarized light. This does not take into account the read noise of the camera or diffraction limitations for spectral and angular resolution. FIG. 8 depicts a more accurate model of the measurable depth of the reflectivity null.

[0051] LTR, in some embodiments, offers two advances in the field of reflectometric optical biosensing. First, by making a position measurement of the antireflective condition for each probe spot, the instrument ensures a measurement at the region of highest sensitivity each time. This may enable label-free sensing of small proteins of low abundance in human serum, such as cytokines and chemokines alongside larger and more abundant antibodies, in a single multiplexed assay. Second, the ability to measure the antireflective condition over a range of angles and wavelengths loosens the manufacturing tolerance for the sensor chips, which improves the commercial potential for this technique. This has a potential to compete with established bead-based immunoassay techniques by offering label-free measurements of biomolecules at the highest regime of sensitivity over a large concentration range of biomolecular targets in a single sample.

[0052] A device is designed such that light of varying angles and wavelengths is simultaneously incident on the sensor substrate. Light is reflected from the sensor substrate into a CCD camera, and an image of reflected light intensity is captured that maps to incident angle and incident wavelength. Therefore, each pixel of the image can be mapped to a unique combination of wavelength and angle of incident light. By modeling a thin film (e.g., including a protein layer) the location of the intensity minimum (antireflective condition in angle and wavelength) can then be used to calculate the thickness change of the probe layer due to a protein binding event. This technique has the potential to be expanded to other types of molecules by changing the parameters of the thin film model to match the material being sensed.

[0053] An embodiment for a single channel version (e.g., FIGs. 5A, 5B, and 5C) can be used to measure molecular binding events. For example, a probe protein is coated over the entire substrate (e.g., sometimes referred to as a chip) surface or spotted on the sensor substrate in a discrete spot of about 200 micrometers in diameter. The substrate is then incubated in a target solution with specific affinity for the probe. This incubation protocol occurs outside of the instrument, and chips are washed and dried before being placed on the instrument stage for imaging. Multiplex chips are possible with this single channel instrument as different probescan be spotted onto the chip in individual locations, or probe spots. During imaging, incident light is centered upon a single probe spot for each individual measurement, and the chip is moved between measurements. This process can limit the single-channel prototype to small, multiplexed arrays (e.g., a handful of probe spots at most).

[0054] FIG. 12 is a block diagram of an embodiment of the single channel LTR instrument.

[0055] FIG. 13 depicts an embodiment of using an image set from a multi-channel LTR. A multichannel, high throughput LTR instrument can be used to make rapid measurements of large, multiplexed arrays (-100 to 250 probe spots) to collect a “data cube” of images covering the range of incident light angles and wavelengths over the chip (e.g., over a portion or the entire chip). An automatic data processing engine can incorporate image analysis to identify an angle and a wavelength corresponding to the antireflective condition for each individual probe spot. A set of images contains data at each pixel location of the surface reflectivity as a function of both AOI and illumination wavelength (center). Reflectivity is measured at discrete points spanning a range of wavelengths and angle of incidence (AIO). A best fit surface map is generated using thin film modeling to match the data (left). Sampling frequency can be adjusted to gather additional data around the reflectivity null as needed. In FIG. 13, a null centroid moves along a diagonal [A, 9] space, so that spatial imaging many chips incrementing [X, 9] in subsequent images acquires a 3D [x, y, ( , 9)] data cube. A reflectivity chart (right) shows reflectance along a diagonal 1394 of the best fit surface map. 3D [x, y, ( , 9)] data cube is a spatial resolved surface thickness map for 1 99’s of sites.

[0056] The Multi-channel, High Throughput LTR enables measurements of order 100’s to 1000’ s individual probe sites spatially arrayed on a single sample chip by measuring the surface reflectivity over a range of illumination wavelength and AIO. A set of spatially resolved images are taken at discrete AOI’s and illumination wavelengths. Each image measures the reflectivity of the entire sample surface at a particular wavelength and AIO. By varying the AOI and / or wavelength and recording a new image, a 5 dimensional data set is created that contain the reflectivity values, R, as a function at (x, y, ( ,9)) where X and Y are the spatial dimensions on the biochip surface, X is the illumination wavelength, and 9 is the illumination angle. Using this data set, a reflectivity vs A, 9 surface map for each location on the sample is generated. Through a fitting process, the location of the reflectivity null can be ascertained in ,9 space enabling the calculation of layer thickness with great precision. In some embodiments, a data set with an of order 1 9 images is sufficient to measure thickness changes with sub angstrom precision over a range of up to -199 A change in thickness. Thickness measurement range and resolution can be adjusted by changing the increment of wavelengths and angles that are scanned. Totaltime to collect a data set of ~100’s images is in the range of 1 about 10 minutes, in some configurations. Large format imaging can be used to expand throughput by simultaneously recording multiple bio chips arrayed at the sample holder image plane. In some embodiments, up to ~30 chips can be imaged simultaneously leading to 10,000 to 100,000 probe sites to be measured in about 1-10 minutes. FIG. 14 depicts a block diagram of an embodiment of a spatially resolved high throughput LTR.

[0057] FIG. 15 depicts an embodiment of a process 1500 for LTR. Process 1500 begins in step 1510 with directing light from a light source to a chip with a plurality of sites arranged on the chip. For example, light from a lamp in FIG. 12 or a laser in FIG. 14 is directed to a holder (e.g., assembly 504 in FIG. 5A). The chip is arranged to move (e.g., rotate, translate, and / or tip) about an axis (e.g., the chip 504 is arranged to rotate about axis 506 in FIG. 5A).

[0058] In step 1520, light reflected from the chip is focused on a detector (e.g., sensor 508 in FIG. 5 A) using a lens system (e.g., one or more lenses 510 in FIG. 5 A).

[0059] In step 1530, a plurality of images is generated using the detector, while the chip is moved to different positions about the axis (e.g., as shown in FIG. 13). In step 1540, an antireflection condition for each of the plurality of sites is calculated based on the plurality of images (e.g., as shown in FIG. 11).

[0060] In some embodiments, the method further comprises detecting a biomarker in human serum at one or more of the plurality of sites and / or detecting a protein or an antibody at one or more of the plurality of sites. In some embodiments, movement of the chip about the axis is equal to or less than 2, 3, 4, or 5 degrees; the plurality of sites are arranged in an array for testing for multiple different proteins or antibodies concurrently (e.g., see FIG. 2); a peak wavelength of light from the light source incident upon the chip is varied during generating the plurality of images; the peak wavelength of light from the light source incident upon the chip is between 300 nm and 1000 nm (or between 600 nm and 700); a grating (e.g., grating 514 in FIG. 5 A) is optically between the chip and detector; and / or an aperture (e.g., pinhole 512 in FIG. 5A used as a field stop) is optically between the chip and the grating.

[0061] Embodiments were chosen and described in order to explain the principles of the invention and practical applications to thereby enable others skilled in the art to best utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. Various configurations may omit, substitute, or add various procedures or components as appropriate. For instance, in alternative configurations, the methods may be performed in an order different from that described, and / or various stages may be added, omitted, and / or combined. Also, features described with respect to certain configurations maybe combined in various other configurations. Different aspects and elements of the configurations may be combined in a similar manner.

[0062] In some configurations, techniques described herein may be used to measure film thickness deposited during and / or after a thin film manufacturing processes. For example, LTR can be used as an optical monitor technique for the calibration and / or observation of the deposition rate and / or thickness of coating materials. While LTR can share some similarities to existing spectroscopic optical monitoring approaches, LTR, in some configurations, offers increased film thickness measurement sensitivity due to its additional resolution in the angle of incidence dimension. LTR can share similarities and / or overlap with ellipsometry from an application point of view, but LTR can be different in the way the instrument works and the way measurements are made.

[0063] Specific details are given in the description to provide a thorough understanding of exemplary configurations including implementations. However, configurations may be practiced without these specific details. For example, well-known circuits, processes, algorithms, structures, and techniques have been shown without unnecessary detail in order to avoid obscuring the configurations. This description provides example configurations only and does not limit the scope, applicability, or configurations of the claims. Rather, the preceding description of the configurations will provide an enabling description for implementing described techniques. Various changes may be made in the function and arrangement of elements without departing from the spirit or scope of the disclosure.

[0064] Having described several example configurations, various modifications, alternative constructions, and equivalents may be used without departing from the spirit of the disclosure. For example, the above elements may be components of a larger system, wherein other rules may take precedence over or otherwise modify the application of the technology. Also, a number of steps may be undertaken before, during, or after the above elements are considered. Accordingly, the above description does not bind the scope of the claims.

[0065] Also, it is noted that the embodiments may be described as a process which is depicted as a flowchart, a flow diagram, a data flow diagram, a structure diagram, or a block diagram. Although a flowchart may describe the operations as a sequential process, many of the operations can be performed in parallel or concurrently. In addition, the order of the operations may be re-arranged. A process is terminated when its operations are completed, butcould have additional steps not included in the figure. A process may correspond to a method, a function, a procedure, a subroutine, a subprogram, etc.

[0066] A recitation of “a”, “an”, or “the” is intended to mean “one or more” unless specifically indicated to the contrary.

[0067] All patents, patent applications, publications, and descriptions mentioned here are incorporated by reference in their entirety for all purposes. None is admitted to be prior art.

Claims

WHAT IS CLAIMED IS:

1. A system for lambda theta reflectometry comprising: a light source; a chip with a plurality of sites arranged on the chip, wherein the chip is arranged to be moved about an axis to enable independent measurements of individual sites; a detector; a lens system arranged to focus light from the light source incident on the chip to the detector; and one or more memory devices comprising instructions that, when executed, cause one or more processors to perform the following steps: generating a plurality of images using the detector while the chip is moved to different positions about the axis; and calculating an antireflection condition for each of the plurality of sites based on the plurality of images.

2. The system of claim 1, wherein the instructions, when executed, cause the one or more processors to detect a biomarker in human serum, plasma, saliva, urine, whole blood, cerebral spinal fluid, tumor aspirate, infection site aspirate, or Medium Enriched for Secreted Antibodies at one or more of the plurality of sites.

3. The system of claim 1, wherein the instructions, when executed, cause the one or more processors to detect a protein or an antibody at one or more of the plurality of sites.

4. The system of claim 1, wherein an angle of rotation of the chip about the axis is equal to or less than 5 degrees.

5. The system of claim 1, wherein the plurality of sites are arranged in an array for testing for multiple different proteins or antibodies concurrently.

6. The system of claim 1, wherein a peak wavelength of light from the light source incident upon the chip is varied during generating the plurality of images.

7. The system of claim 6, wherein the peak wavelength of light from the light source incident upon the chip is between 300 nm and 1000 nm.

8. The system of claim 1, further comprising a grating optically between the chip and detector.

9. The system of claim 8, further comprising an aperture optically between the chip and the grating.

10. The system of claim 9, wherein the aperture is one of a plurality of apertures between the chip and the grating.

11. A method for lambda theta reflectometry comprising: directing light from a light source to a chip with a plurality of sites arranged on the chip, wherein the chip is arranged to move about an axis; focusing light reflected from the chip on a detector using a lens system; generating a plurality of images using the detector while the chip is moved to different positions about the axis; and calculating an antireflection condition for each of the plurality of sites based on the plurality of images.

12. The method of claim 11, further comprising detecting a biomarker in human serum at one or more of the plurality of sites.

13. The method of claim 11, further comprising detecting a protein or an antibody at one or more of the plurality of sites.

14. The method of claim 11, wherein an angle of rotation of the chip about the axis is equal to or less than 5 degrees.

15. The method of claim 11, wherein the plurality of sites are arranged in an array for testing for multiple different proteins or antibodies concurrently.

16. The method of claim 11, wherein a peak wavelength of light from the light source incident upon the chip is varied during generating the plurality of images.

17. The system of claim 16, wherein the peak wavelength of light from the light source incident upon the chip is between 300 nm and 1000 nm.

18. The method of claim 11, wherein a grating is optically between the chip and detector.

19. The system of claim 18, wherein an aperture is optically between the chip and the grating.

20. A system for lambda theta reflectometry comprising: a light source; a chip with a plurality of sites arranged on the chip, wherein the chip is arranged to be moved; a detector; a lens system arranged to focus light from the light source incident on the chip to the detector; and one or more memory devices comprising instructions that, when executed, cause one or more processors to perform the following steps: generating a plurality of images using the detector while the chip is moved to different positions, wherein: an angle of rotation is equal to or less than five degrees; and the plurality of images are generated while a peak wavelength of light from the light source incident upon the chip is varied; calculating an antireflection condition for each of the plurality of sites based on the plurality of images; and detecting a protein or an antibody at one or more of the plurality of sites.

Images