Infrared Reflectance Sampling Device

Abstract

A reflectance sampling device for Fourier transform infrared (FTIR) spectroscopy-based analysis and methods of use for said reflectance sampling device. The reflective sampling device includes a base plate defining an array of wells on its surface, wherein each of the one or more wells arrayed on the surface of the base plate is configured as a concave mirror that reflects in a predetermined wavelength range. The reflective sampling device includes a base plate having an array of wells optimized in depth and radius to create an optimal focal point for minimizing reflective interference and maximizing signal amplitude.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 483,117, filed February 3, 2023, filed as a PCT International Patent Application on February 5, 2024, the disclosure of which is incorporated herein by reference in its entirety. [Background technology]

[0002] Cancer remains a major health challenge worldwide and ranks as the second leading cause of death in the United States. More than 1.9 million people are diagnosed with cancer each year in the United States. Of those, approximately 600,000 will ultimately die from their disease. It is widely known that early-stage cancers are treated with much higher success rates than later-stage cancers. After decades of intensive research and recent advances in treatment strategies, almost all patients with stage IV cancer still ultimately succumb to their disease. It is for this reason that countries with advanced healthcare systems have invested in nationwide cancer screening programs, with the goal of catching cancer early, at a more treatable stage. Evidence has shown that, when properly designed, these programs lead to lower mortality rates.

[0003] One of the most effective strategies against cancer is early detection, which can lead to improved treatment options and better patient outcomes. However, existing methods for early cancer detection present challenges: they are often invasive, expensive, and not without risks, thereby complicating the fight against cancer. Generally, cancer screening is primarily recommended for individuals considered at high risk due to factors such as age, smoking, alcohol consumption, and environmental exposures. Nevertheless, cancer rates are rising even among those who do not fall into these risk categories, for example, in the case of breast cancer, human papillomavirus-related head and neck cancer, and colorectal cancer among young adults. Common diagnostic tools such as CT scans, X-rays, and PET scans can paradoxically expose individuals to potentially cancer-causing radiation. Procedures such as endoscopy and biopsy can be invasive and uncomfortable, preventing people from undergoing timely screening. Furthermore, traditional methods such as histopathological analysis have a significant chance of producing false negatives or false positives, leading to misdiagnosis. This is often due to histological similarities between different tumor types and the challenge of identifying poorly differentiated cells, making it difficult to pinpoint the tissue of origin.

[0004] Optical spectroscopy is emerging as a method for early cancer diagnosis, with Fourier transform infrared (FTIR) spectroscopy showing potential despite the low detection sensitivity and specificity of many cancer biomarkers in the infrared (IR) or near-infrared (NIR) bands (900-3080 nm range). FTIR is advantageous due to its simplicity, speed, accuracy, cost-effectiveness, non-destructiveness, and compatibility with automated processes, offering improved diagnostic and management methods over traditional cancer screening. Infrared spectroscopy can serve as a valuable tool for early disease detection, supporting timely decision-making and improving patient outcomes.

[0005] Although NIR spectroscopy is not new in cancer screening, recent research has promoted the use of higher wavelengths and multifaceted approaches for the analysis of various sample types, traditionally between 600-1100 nm. However, longer wavelengths often encounter absorption by water, weakening the detection of other substances. Despite the optical transparency of water, it is highly absorbing in the wavelength region below 20 nm, and in some parts of the near-infrared and even more so in the mid-IR to far-IR regions. The optical IR (OIR) sampling device described in this disclosure provides a novel approach for rapidly producing dried microlayers of samples ready for high-throughput reflectance IR spectroscopy analysis, such as for liquid biopsies, to determine one or more optical properties of the sample.

[0006] Liquid biopsy is another noninvasive technique being explored for cancer detection, such as the method described in U.S. Patent No. 10,288,615, which involves blood analysis. Differentiation of the water content of normal and cancerous cells has also been attempted, as detailed in U.S. Patent No. 7,706,862, which utilizes near-infrared (NIR) spectral optical imaging to identify changes in water content between cancerous and normal tissue at key water absorption wavelengths. Key "fingerprint" wavelengths include 980 nm, 1195 nm, 1456 nm, 1944 nm, 2880 nm, 3360 nm, and 4720 nm, with reference wavelengths such as 4500 nm, 2230 nm, 1700 nm, 1300 nm, 1000 nm, and 800 nm to aid in image comparison. Summary of the Invention [Problem to be solved by the invention]

[0007] Despite NIR's ability to measure water absorption in cells, its reliability and specificity are inconsistent due to water's wide absorption range across many wavelengths. Molecular fingerprinting, which seeks out differences in specific biomarkers that constitute a disease fingerprint, requires a comprehensive method based on spectral signatures from broad-spectrum spectroscopy to identify not only the presence but also the type of cancer. Previous research supports this approach, demonstrating that IR spectroscopy can identify changes in serum components that create distinct spectral signatures for various health conditions. Nevertheless, early research has focused excessively on longer-wavelength IR absorption to analyze serum biomolecules, demonstrating the urgent need for noninvasive, affordable, and safe methods for early, on-site cancer detection. [Means for solving the problem]

[0008] Examples presented herein relate to a reflective sampling device for FTIR-based analysis, which includes a base plate defining an array of wells on its surface, wherein each of the one or more wells disposed on the surface of the base plate is configured as a concave mirror that reflects in a predetermined wavelength range.

[0009] In another example provided herein, the concave mirror has an IR-reflective coating made of a material with high infrared reflectivity. In a further example provided herein, the material is selected from a group including aluminum, copper, nickel, chromium, silver, and gold. In yet another example provided herein, the wells are optimized in depth and radius to create an optimal focal spot for minimizing reflection interference and maximizing signal amplitude. In a further example provided herein, the depth of each well is selected from the range of 0.2 to 2 mm, and the radius of each well is selected from the range of 1.5 to 5 mm. In yet another example provided herein, the base plate is a 96-well plate.

[0010] In yet another example provided herein, the surface of the IR-reflective well is ion-treated to ensure a consistent and smooth thin layer of liquid sample is created. In yet another example provided herein, the reflective surface is created by a process selected from the group consisting of vacuum metallization, the use of a reflective embossed metallized film, and an aluminum foil laminate.

[0011] Another example presented herein relates to a reflective sampling device for FTIR-based analysis, which includes a base plate with an array of wells optimized in depth and radius to create an optimal focal spot for minimizing reflective interference and maximizing signal amplitude.

[0012] In other examples provided herein, the depth of each well is selected from the range of 0.2 to 2 mm, and the radius of each well is selected from the range of 1.5 to 5 mm. In a further example provided herein, the base plate is a 96-well plate.

[0013] In yet another example provided herein, the array of wells is configured as an array of concave mirrors. In a further example provided herein, the concave mirrors have an IR-reflective coating made of a material with high infrared reflectivity. In yet another example provided herein, the IR-reflective coating is applied by a process selected from the group consisting of vacuum metallization, use of a reflective embossed metallized film, and an aluminum foil laminate.

[0014] Another example provided herein relates to a method for making an infrared (IR) reflective array plate for high-throughput optical cancer fingerprinting, the method comprising the steps of obtaining a base plate including an array of concave wells; and applying an IR reflective coating to one or more wells of the array of concave wells, such that each of the one or more wells is a concave mirror.

[0015] In other examples provided herein, the IR-reflective coating is selected from the group including aluminum, copper, nickel, silver, and gold. In further examples provided herein, the step of applying the IR-reflective coating includes vacuum metallizing. In yet other examples provided herein, the method further includes ion treating the concave mirror surface of one or more wells. In yet other examples provided herein, each well of the array of concave wells is optimized in depth and radius to create an optimal focal point for minimizing reflection interference and maximizing signal amplitude.

[0016] In other examples provided herein, the method further includes loading one or more samples into one or more wells; performing an analysis of the one or more samples; and separating the IR-reflective layer from the base plate. In a further example provided herein, the method further includes applying a separate IR-reflective coating to one or more wells of the array of recessed wells.

[0017] Yet another example presented herein relates to a method for improving the signal-to-noise ratio in FTIR-based analysis using diffuse reflectance from the reflectance sampling device described above.

[0018] In the following description, various additional inventive aspects will be described. Inventive aspects can relate to individual features and to combinations of features. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the broad inventive concept on which the embodiments disclosed herein are based. [Brief explanation of the drawings]

[0019] The accompanying drawings, which are incorporated in and constitute a part of the description, illustrate several aspects of the present disclosure. A brief description of the drawings follows: [Figure 1A] FIG. 1A is an example of an infrared (IR) reflecting sample plate assembly according to an embodiment of the present disclosure. [Figure 1B] FIG. 1B shows an example of a vacuum metallized IR-reflective sample plate 156 assembly 105 embodying the subject matter of the present disclosure. [Figure 2A] FIG. 2A is a cross-sectional view of a portion of an IR-reflective sample plate according to an embodiment of the present disclosure. [Figure 2B] FIG. 2B is a cross-sectional view of the layers that make up an individual reflective well, such as would be made by either method of FIG. 2A, to include a base layer and a top coat. [Figure 3] FIG. 3 is a flowchart of an example method for assembly of an IR-reflective sample plate, according to an embodiment of the present disclosure. [Figure 4A] 4A and 4B are diagrams illustrating examples of the variation of target focal spot placement based on angle of incidence. [Figure 4B] 4A and 4B are diagrams illustrating examples of target focus placement variations based on angle of incidence. DETAILED DESCRIPTION OF THE INVENTION

[0020] Detailed Description of the Invention Embodiments of the present disclosure relate to biomedical analytical devices, particularly those used to detect cancer and other diseases through Fourier transform infrared (FTIR) spectroscopy.

[0021] For purposes of this specification, the following terms are specifically defined as follows:

[0022] An "infrared absorption spectrum" refers to a spectrum proportional to the wavelength dependence of a sample's infrared absorption coefficient, absorbance, or similar measure of IR absorption properties. An example of an infrared absorption spectrum is an absorption measurement produced by a Fourier transform infrared (FTIR) spectrometer, i.e., an FTIR absorption spectrum. Generally, infrared light may be absorbed (i.e., part of an infrared absorption spectrum), transmitted (i.e., part of an infrared transmission spectrum), or reflected. The reflected or transmitted spectrum of the collected probe light may have a different intensity at each wavelength compared to the intensity at that wavelength of the probe light source. Note that IR measurements are often plotted showing the amount of transmitted light instead of the amount of absorbed light. For the purposes of this definition, an IR transmission spectrum and an IR absorption spectrum are considered equivalent as two data sets because there is a simple relationship between the two measurements.

[0023] "Infrared source" and "source of infrared radiation" refer to one or more light sources that generate or emit radiation in the infrared wavelength range, generally between 2 and 25 microns. The radiation source can be one of a large number of sources, including thermal or globar sources, supercontinuum laser sources, frequency combs, difference frequency generators, sum frequency generators, harmonic generators, optical parametric oscillators (OPOs), optical parametric generators (OPGs), quantum cascade lasers (QCLs), interband cavity lasers (ICLs), synchrotron infrared radiation sources, nanosecond, picosecond, femtosecond, and attosecond laser systems, CO2 lasers, microscopic heaters, electrically or chemically generated sparks, and / or any other source that produces infrared radiation emissions. In a preferred embodiment, the source emits infrared radiation, but may also emit in other wavelength ranges, for example from ultraviolet to terahertz (THz). -1 Less than or 1cm -1 It may be narrowband, having a spectral width of less than, for example, 10 cm -1 Super, 100cm -1 Over or 500cm -1 The infrared source can be broadband, with a spectral width of more than 100 kHz. Broadband sources can be narrowed by filters, monochromators, and other devices. The infrared source can also be made of one of a number of discrete radiations, for example, tuned to a specific absorption band of the target species.

[0024] "Interacting" in the context of interacting with a sample means that light illuminating the sample is at least one of scattered, refracted, absorbed, aberrated, diverted, diffracted, transmitted, and reflected by, through, and / or from the sample.

[0025] "Optical property" refers to an optical property of a sample, including, but not limited to, refractive index, absorption coefficient, reflectance, absorptance, real and / or imaginary components of the refractive index, real and / or imaginary components of the sample dielectric function, and / or any property mathematically derivable from one or more of these optical properties.

[0026] "Optical response" refers to the result of the interaction of radiation with a sample. The optical response is related to one or more of the optical properties defined above. The optical response can be the absorption of radiation, a temperature increase, thermal expansion, photoinduced forces, light reflection and / or scattering, or any other response of a material upon interaction with illuminating radiation.

[0027] "Probe source," "probe light source," or "probe radiation source" refers to a radiation source that can be used for sensing the optical properties of a sample. A probe light source can be used to detect a sample's response to light incident from an infrared light source. The radiation source can include a gas laser, a laser diode, a superluminescent diode (SLD), a near-infrared laser, a UV and / or visible laser beam, for example, generated by sum or difference frequency generation. It can also include any or other source of near-infrared, UV, and / or visible light that can be focused to a spot on the scale of less than 2.5 micrometers, or less than 1 micrometer, or less than 0.5 micrometers. In some embodiments, the probe light source can be operated at a wavelength outside the tuning or emission range of the infrared light source, but the probe light source can also be a fixed wavelength source at a selected wavelength that actually overlaps with the tuning range of the infrared light source. A "probe light beam" or "sensing light beam" is the beam originally emitted from the probe light source.

[0028] A "probe beam" is a beam of light or radiation directed at a sample to detect photothermal distortions or other optical changes resulting from the interaction of IR radiation with the sample, e.g., to detect absorption of IR radiation by the sample.

[0029] "Indicative signal" refers to a signal mathematically related to a property of interest. The signal can be an analog signal, a digital signal, and / or one or more numbers stored in a computer or other digital electronics. The signal can be a voltage, a current, or any other signal that can be easily converted and recorded. The signal can be mathematically identical to the property being measured, for example, an explicit absolute phase signal or an absorption coefficient. It can also be a signal mathematically related to one or more properties of interest, for example, involving linear or other scaling, offsets, inversions, or more complex mathematical manipulations.

[0030] A "spectrum" refers to the measurement of one or more properties of a sample as a function of wavelength, or equivalently (and more generally) as a function of wavenumber.

[0031] Terms such as "about" or "approximately" are synonymous and are used to indicate that the value modified by the term has an understood range associated with it, which range can be ±20%, ±15%, ±10%, ±5%, or ±1%.

[0032] The term "substantially" is used to indicate that a result (e.g., a measurement) is close to a targeted value, where close can mean, for example, that the result is within 80% of the value, within 90% of the value, within 95% of the value, or within 99% of the value.

[0033] In optical systems, such as fiber optics, and in wireless communication systems, reflections can cause interference. This interference occurs when a signal reflects off a surface or interface within the system, creating an echo or ghost signal. These reflected signals can interfere with the original signal, leading to distortion, loss of clarity, and data loss. In complex systems, multiple reflections exacerbate these problems and can make it difficult to separate and transmit a clear signal. Without a clear signal, it is difficult to evaluate the optical properties of a sample.

[0034] In many communications and signal processing systems, maintaining optimal signal amplitude is crucial for clear transmission and reception. Problems arise when the signal strength is too low, causing loss of information, or too high, causing signal distortion. The automatic gain control used in some cases to address this problem can be inefficient or too slow to adapt to rapidly changing conditions.

[0035] The infrared (IR) reflecting device disclosed herein utilizes digital signal processing techniques to identify and cancel reflected signals and alter the physical design of the system to minimize reflection points. The invention focuses on more precisely and dynamically adjusting signal amplitude by deploying sophisticated algorithms for automatic gain control that can quickly adapt to changing signal conditions and implementing a feedback system that continuously monitors signal quality and adjusts accordingly.

[0036] Disclosed herein is an infrared (IR) reflectance sampling device for FTIR-based molecular fingerprinting to detect cancer and other diseases. In embodiments, the sampling device includes a reflective high-throughput biopsy plate. In some examples, the reflective element includes a concave mirror or an N x N array of concave mirrors, e.g., N = 1 to 400.

[0037] As used throughout this application, the term "reflective" means substantially more reflective than absorptive or transmissive. For example, a reflective material may be 99% or more reflective, or 95% or more reflective, or 90% or more reflective. In addition, reflectivity is often a function of wavelength. For example, water is highly absorbing in the ultraviolet regime while being nearly transparent in the visible regime. The devices described herein are used to test infrared absorption by biological samples, and therefore this disclosure generally describes materials that are reflective in the infrared regime. However, the devices described herein may be used for other types of tests where visible or ultraviolet light is important. In either case, there may be a predetermined wavelength range of interest (e.g., 500-1500 nm, or 780-1000 nm, etc.) for which the devices described herein are best suited. Depending on the range of interest, different materials or different thicknesses of those materials may be used that will provide more reflection than transmission and absorption combined. Although not explicitly described herein, one skilled in the art will understand how to substitute materials that reflect at other wavelengths to achieve the results described herein for other given wavelength ranges.

[0038] 1A, an example of an assembly 100 of an infrared (IR) reflective sample plate 116 embodying the subject matter of the present disclosure is shown. The assembly 100 of the IR reflective sample plate 116 may include creating or obtaining a suitable base plate 102, applying an IR reflective layer 104, and ionizing an IR reflective well 106.

[0039] At 102, the assembly 100 begins with a suitable base plate 108. The suitable base plate 108 may be designed or selected according to considerations such as throughput, sample size, and use settings.

[0040] The base plate 108 can be made of any suitable material, allowing for the use of the plate in FTIR analysis. Some non-limiting examples of suitable materials for the base plate 108 include acrylonitrile butadiene styrene (ABS), polystyrene, polypropylene, polycarbonate, polyetherimide, glass, quartz, and other thermally stable materials. In embodiments, the base plate 108 can be made of disposable, reusable, or other recyclable materials. The base plate 108 can be formed by several different processes, such as injection molding or vacuum forming, e.g., vacuum metallization.

[0041] In examples, the base plate 108 is designed according to experimental and sample parameters. Some non-limiting examples of suitable base plate designs include single sample well plates and multi-well plates, such as 1-, 12-, 24-, 48-, 96-, or 384-well formats. In some examples, a standard design is easily accommodated by a standard plate reader, such as a plate reader for an existing design of an FTIR spectrometer.

[0042] Each well 110 of one or more wells in the base plate 108 may be configured to create a focal point 112 during placement to eliminate reflective interference and achieve a desired signal amplitude. In an example, the desired signal amplitude may be an optimal signal amplitude. In an embodiment, wells with a shallow depth and a curved cross-section may be preferred. For example, each well may have a depth (d), which may also be understood as the well height, and a parabolic cross-section with a radius (r) configured to optimize the location of the focal point 112. In an example referring to a 96-well format, the well depth (d) may range from 0.2 to 2 mm, and the radius (r) may range from 1.5 to 5 mm, the values ​​being optimized to create an optimal focal point to eliminate reflective interference and achieve optimal signal amplitude.

[0043] At 104, an IR reflective layer 114 is applied to the base plate 108. In examples, the IR reflective layer 114 may be a coating, film, or foil such as aluminum, copper, nickel, silver, gold, a dielectric mirror, a multilayer optical film, or the like.

[0044] The IR-reflective layer 114 can be applied to cover individual wells, such as well 110, or to cover the entire plate, such as base plate 108. In some embodiments, the IR-reflective layer 114 can be applied over the entire surface of the base plate, such that all plate surfaces are covered, or it can be applied only to one or more wells, such that non-well surfaces and one or more wells may be uncovered. To ensure a smooth and consistent IR-reflective coating or layer, an underlying basecoat can be applied to the base plate before the IR-reflective coating. The undercoat, or basecoat, can be a material compatible with the IR-reflective uppercoat or can act as a primer to enhance adhesion of the IR-reflective uppercoat. The basecoat can be an ultraviolet (UV)-curable basecoat, an epoxy-based, a polyurethane basecoat, and others. In one embodiment, PARYLENE® brand primer can be used, a material that provides a barrier layer to prevent moisture, corrosion, and solvent evaporation while also providing smoothing for adjacent layers.

[0045] While the reflective properties of materials such as aluminum, copper, nickel, chromium, silver, and gold in the visible light spectrum are well known, their behavior in the infrared (IR) spectrum is particularly important for a variety of applications. These metals have electrons in their conduction bands that can vibrate in response to electromagnetic radiation such as IR light.

[0046] In the IR spectrum, materials such as gold and silver efficiently reflect IR radiation due to their high reflectivity at longer wavelengths. Gold reflects up to 98% of IR radiation, making it very efficient for applications requiring thermal insulation. Despite tarnishing, silver also offers excellent IR reflectivity when cleaned. Aluminum is less reflective than gold and silver, but still reflects a significant amount of IR radiation and is more cost-effective. Copper and nickel have more moderate reflectivity in the IR range, but are still useful due to their other physical properties.

[0047] The uses of these materials in the IR spectrum are multifaceted. Gold's excellent reflectivity has led to its use in satellite and space telescope components to protect against the sun's heat. Silver's high IR reflectivity has led to its frequent use in insulation and coatings to improve energy efficiency. Aluminum, due to its cost-effectiveness and good IR reflectivity, is widely used in rescue heating blankets and architectural design to reflect IR and reduce heating. Copper has found use in heat exchangers due to its ability to reflect IR and its excellent thermal conductivity. Nickel, with its moderate IR reflectivity, is often used as a coating on other materials to add protection from IR radiation while providing corrosion resistance.

[0048] Several methods for applying the IR-reflective layer 114 to the base plate 108 are contemplated. The IR-reflective layer 114 can be, for example, aluminum and can be vacuum-metallized to the base plate 108. This method may be preferred in some instances, such as when cost-effectiveness is an important consideration, due to its simplicity and low cost. In vacuum metallization, also known as physical vapor deposition, metal is evaporated in a vacuum environment and then allowed to condense on the substrate surface to form a thin film. This method is widely used to create reflective surfaces on items such as mirrors, automotive parts, and decorative items. It provides a uniform coating and can be used with a variety of metals, such as aluminum, which is often chosen for its reflective properties. Vacuum metallization of wells and / or plates with aluminum or other materials to make the plate reflective is a simpler, lower-cost process that is more cost-effective than adding reflective metallized film or aluminum foil.

[0049] FIG. 1B illustrates an example assembly 105 of a vacuum metallized IR-reflective sample plate 156 embodying the subject matter of this disclosure. Similar to the example assembly 100, one or more wells 110 are configured to create a focal point 112 during alignment to eliminate reflective interference and achieve a desired signal amplitude. An IR-reflective coating material 154 is applied to one or more wells 110 or to the base plate 108, typically by vacuum metallization. The IR-reflective coating material 154 creates a thin, reflective layer that conforms to the concave shape and curves of the wells 110, resulting in the metallized wells 110 becoming concave mirrors. In this manner, the reflective sample plate 156 is prepared as an array of concave mirrors.

[0050] Similar to vacuum metallization, sputtering involves coating a substrate with a thin, reflective metal layer. However, instead of evaporating metal, sputtering uses high-energy particles to eject atoms from a target material, which then deposit on the substrate. This method is used for applications requiring highly controlled film structure.

[0051] In chemical vapor deposition (CVD), a substrate is exposed to one or more volatile precursors that react and / or decompose on the substrate surface to produce the desired deposit. For reflective surfaces, materials such as silicon or titanium can be deposited to form a reflective layer.

[0052] Electroplating involves the deposition of a metal coating onto an object by passing an electric current through an electrolyte solution in which the object is submerged. This can be used to create reflective surfaces, particularly for metals such as chromium.

[0053] Spray coating involves spraying a solution containing metal particles onto a substrate. After the solvent evaporates, a reflective metal layer remains. This method is less precise than vacuum metallization, but is more cost-effective for large items.

[0054] Metallized films are typically made by coating a thin layer of metal onto a plastic film. In one embodiment, the metal layer can be aluminum due to its reflective properties and cost-effectiveness. These films can be applied to a variety of surfaces to provide a reflective finish and are commonly used in packaging, insulation, and decorative applications.

[0055] The reflective embossed metallized film can be used as a substrate for forming one or more wells as a foil laminate, such as an aluminum foil laminate, and can be applied to a polymer film or paper that can also be embossed with small wells corresponding to the final shape of the device to prevent wrinkling, warping, or cracking that might otherwise occur upon application to the base, as shown in Figure 1A or 2A. Concave lens films can be used, such as by being metallized and then cut to size. In an example, the IR-reflective layer 114 can be configured to be separated from the base plate for disposal after sample analysis.

[0056] FIG. 2A is a cross-sectional view of a portion of an IR-reflective sample plate, showing one of the active sampling wells (i.e., one of the 96 concave mirrors, 96X, A), according to an embodiment of the present disclosure. Other features surrounding and underlying the active sampling well were designed to form a rigid base plate using a minimal amount of material. When manufactured from a polymer, such as by injection molding, the resulting base plate is very sturdy and inflexible, ready to receive an IR-reflective coating with or without an undercoat. The plate is designed for ease of handling, can be covered with a standard microtiter or customized lid, and is compatible with industry-standard plate readers.

[0057] FIG. 2A shows an IR-reflective coating 114 disposed across the top surface of the substrate of the wells 112. As shown in FIG. 2A, the base plate 108 below the IR-reflective coating provides mechanical support to the wells 112 and includes any necessary posts or other support structures to maintain the shape and position of the wells 112. However, other areas of the wells may be substantially hollow. The material of the base plate 108 and the placement of the support structures are selected according to several criteria. First, it may be desirable for the base plate 108 to be the same overall size as other 96-well plates (or plates with other well counts) so as to be interoperable with existing testing equipment. Second, the base plate 108 can be made using environmentally friendly materials, such as recyclable or recycled materials. Additionally, the design and materials can be selected to prioritize the location and shape of the wells 112. This means that appropriate support should be provided beneath or adjacent to the wells 112 to ensure they do not shift or bend during normal use conditions (e.g., sample insertion or movement into, out of, or within a testing device).

[0058] Figure 2B is a cross-sectional view of a single well 112, such as the well 112 depicted in Figure 2A. It should be understood that Figure 2B is a detailed cross-sectional view and that not all features shown therein are to scale. Rather, features in Figure 2B may be exaggerated for a better conceptual understanding of the disclosure.

[0059] FIG. 2B shows three layers: a smoothing layer 113 , the IR-reflective layer 114 mentioned above, and a base 108 that provides support for an upper coat 115 .

[0060] The smoothing layer 113 can be a layer to provide good bonding, adhesion, and smoothness between the base 108 and the IR-reflective layer 114, as described elsewhere herein. In some embodiments, such as when applying a foil layer to act as the IR-reflective layer 114, wrinkling, stretching, or other deformation of the IR-reflective layer 114 may occur. Even when using other, generally more precise methodologies (such as sputtering or CVD) to provide the IR-reflective layer 114, if there is insufficient wetting and adhesion to the base 108, the IR-reflective material 154 can bead, pool, or form other discontinuities, which can change the focus of the wells 112 or cause the wells 112 to lose focus entirely.

[0061] Thus, smoothing layer 113 can be provided in some embodiments as a primer and adhesion promoter. Smoothing layer 113 can be selected from materials that exhibit good bonding properties with both the material used as base 108 and the material selected for IR-reflective layer 114 (i.e., IR-reflective material 154). Thus, smoothing layer 113 can be a different material depending on whether base 108 is made of a polar or non-polar polymer, a metal, or some other material. Similarly, a different material can be used for smoothing layer 113 for aluminum than gold or copper, which have very different melting temperatures and may respond differently to adhesives, solders, or other materials that may be used as smoothing layers.

[0062] In some embodiments, the smoothing layer 113 can be applied to the base 108, and then the IR-reflective layer 114 can be applied directly on top of the smoothing layer 113. In embodiments, a further processing step, such as heating the entire structure of these layers, can be used to activate the smoothing layer 113 and provide the bonding and smoothing properties described above. For example, this can be useful when a hot melt adhesive is used as the smoothing layer 113.

[0063] The IR-reflective layer 114 has been described in detail with reference to other figures, and the discussion therein applies equally to Figure 2B. The IR-reflective layer 114 can be applied as a sheet, via deposition, or by any other method for adding a layer on top of the base 108.

[0064] The upper coat 115 is an optional layer provided on top of the IR-reflective layer. The upper coat 115 can be provided, for example, if the IR-reflective layer 114 is susceptible to oxidation or other reaction with the ambient environment or with the sample expected to be tested. The upper coat 115 can be, for example, a polymeric material. Generally speaking, the upper coat 115 will be thin enough to prevent distortion or reflection that would otherwise occur at the interface between two layers with different refractive indices. Additionally, the upper coat 115 can be reflective at wavelengths of interest for testing the sample (e.g., in the infrared regime) or transparent at these wavelengths. The upper coat 115 will generally not exhibit high levels of absorption at the wavelengths of interest to avoid excessively reducing the focused optical signal after interaction with the sample.

[0065] Although not shown in FIG. 2B , various surfaces (108, 113, 114, 115) between or on top of the layers can be treated. For example, ionization treatment can be used in various embodiments to increase adhesion between any of the layers. Additionally, the upper surface of the upper coat 115 can be treated to provide better interaction with the expected sample (e.g., to prevent meniscus or droplet formation). One particularly important bond is between the infrared-reflective layer 114 and the upper coat 115 in those embodiments in which the upper coat 115 is used. As mentioned above, the upper coat 115 should ideally not affect the reflected light by distorting or absorbing portions of it. The better the adhesion between the infrared-reflective layer 114 and the upper coat 115, the less likely the reflected signal will lose intensity or focus.

[0066] Referring now to FIG. 3, a flow chart of an example method 200 of assembly of an IR-reflective sample plate according to an embodiment of the present disclosure is shown.

[0067] At 202, a suitable baseplate is prepared or obtained. The suitable baseplate may be designed or selected according to considerations such as throughput, sample size, and use setting. In an example, the baseplate may be baseplate 108 of FIG. 1A or FIG. 1B.

[0068] Of particular interest may be consideration of focal spot placement when samples in the wells of a plate are interrogated. Wells within a plate may be configured to reflect light in a particular direction or toward a particular point, for example, to improve analysis by reducing noise. Each well may be configured such that once the well is made reflective, it reflects light to create a focal spot within the placement to eliminate reflective interference and achieve a desired signal amplitude.

[0069] In an example, the desired signal amplitude may be the optimal signal amplitude. In an embodiment, wells with a shallow depth and a curved cross-section may be preferred. For example, each well may have a parabolic cross-section with a depth (d) and radius (r) (see FIG. 4B) configured to optimize the location of the focal point. As disclosed herein, the optics and focus are configured to optimize the interaction between the sample, which may be a thin-layer sample, and an interrogation source, such as IR radiation, which is directly correlated to the angle of incidence of the IR beam. The selected curvature of the well surface depends on the angle of incidence of the source IR beam or other interrogation beam and the optimal sample drying time. Therefore, the respective ranges of well depth and well radius depend on the range of IR beam incidence angle and sample drying time. This range also depends on the well array configuration, i.e., whether there are 96 wells, 386 wells, or some other number of wells per plate. In an example referring to a 96-well format, the depth (d) may range from 0.2 to 2 mm, and the radius (r) may range from 1.5 to 5 mm.

[0070] At 204, an IR reflective layer is applied to the base plate. The IR reflective layer can be applied with or without a base or primer coat, for example, by vacuum metallizing the base plate and laminating an embossed metallized film to a substrate, or by using a metallized concave lens film.

[0071] Several methods of applying the IR-reflective layer 114 to the base plate 108 are contemplated. The IR-reflective layer 114 can be, for example, aluminum and vacuum-metallized to the base plate 108. This method may be preferred in some cases, such as when cost-effectiveness is an important consideration, due to its simplicity and low cost. A reflective embossed metallized film may be used as a substrate to form one or more wells, such as a foil laminate, such as an aluminum foil laminate applied to a polymer film or paper that can also be embossed with small wells. A concave lens film may be used, such as by being metallized and then cut to size.

[0072] In embodiments, vacuum metallization may be a preferred method of applying the IR-reflective layer. For example, vacuum metallization may support process scalability by supporting mass production with relatively low-cost materials. Vacuum metallization can be consistently achieved with very thin layers of aluminum. Thin layers may be preferred because they avoid altering the precise curvature of the substrate wells. In some instances, application of the reflective layer may create variations in the precise curvature of the wells, creating plates with wells with different variations in curve. Applying the reflective layer in very thin layers, such as by vacuum metallization, may help avoid such variations. In addition, vacuum metallization is thermally compatible, meaning that the reflective layer remains stable, e.g., less prone to air pockets or other distortions, when the plate is subsequently heated during the work process, e.g., to dry samples.

[0073] Vacuum metallization is a process used to apply thin metal layers to various surfaces. The material to be coated (the substrate) is cleaned and prepared to ensure good adhesion of the metal layer to the surface. The substrate is placed inside a vacuum chamber. The chamber is then sealed, and the air is removed to create a vacuum. The vacuum helps remove air and other gases that can interfere with the metallization process. The metal (such as aluminum, silver, or copper) is heated to its vaporization temperature. The metal is typically in the form of a solid wire or pellets. The heat causes the metal to vaporize, forming a metal vapor cloud inside the vacuum chamber. The vaporized metal condenses on the substrate, forming a thin, uniform metal layer. The substrate can be rotated or moved during this process to ensure an even coating. The thickness of the deposited metal layer can be controlled by adjusting parameters such as the temperature of the metal source, the deposition time, and the motion of the substrate. After the desired thickness is achieved, the coated substrate is cooled, and the vacuum chamber is opened. The cooling process helps solidify the thin metal layer on the substrate, creating a durable, adherent coating.

[0074] In embodiments, an upper coating may be applied to the surface of the metal layer to provide further smoothing of the final surface. A number of coatings may be suitable for the upper coating, provided that the coating is transparent and does not interfere with the reflectivity of the concave mirror.

[0075] At 206, the IR reflector plate from 204 is ionized by ion treatment. Ion treatment is applied to increase sample adhesion on the well surface. This process involves bombarding the surface with ions to create a more reactive surface that can more effectively bond with the sample. Several different types of ion treatments can be used, including plasma treatment, which uses a low-pressure plasma to create a reactive surface on the material being treated. Plasma can be generated using various gases, including oxygen, nitrogen, and argon. In another example, corona treatment uses a high-voltage discharge to create a reactive surface on the material being treated. The discharge creates a corona of ions around the material that can then more effectively bond with the sample. In yet another example, flame treatment exposes the surface to a flame, which creates a reactive surface that can more effectively bond with the sample. In yet another example, a self-assembled monolayer can be applied to the reflective layer to promote sample wetting and adhesion.

[0076] At 208, the sample is loaded into the plate. The sample is applied as a thin layer across the curved, well-ion-treated well surface and can be dried for FTIR analysis of disease fingerprints. The combination of ion treatment and well surface geometry can provide improved adhesion of low volume samples to ensure that a consistent and smooth thin layer of liquid sample is created when the sample is added to the well. In embodiments, the wells are ion-treated for sterilization purposes.

[0077] At 210, the sample is analyzed. Sample plates embodying the presently disclosed subject matter can be used in a wide array of known and experimental sample analysis methods. In embodiments, sample plates embodying the present disclosure can be specifically optimized for FTIR analysis of lipids, proteins, glycans, DNA, RNA, or other analytes for fingerprinting for disease detection.

[0078] In embodiments using an IR beam, the analyte must be infrared-active, such as by having a dipole moment that changes during vibration or rotation. The analyte may also have a unique vibrational frequency that can be detected by a detector, such as a detector in an FTIR spectrometer. A "detector" refers to a device that produces a signal indicative of the power, intensity, and / or energy of light / radiation incident on the detector surface. The signal will generally be an electrical signal, such as a voltage, current, and / or charge. The detector may be a photodiode, a phototransistor, or a charge-coupled device (CCD). In some cases, the detector may be a semiconductor detector, such as a silicon PIN photodiode. The detector may be an avalanche photodiode, a photomultiplier tube, or any other device that produces a change in current, voltage, charge, conductivity, etc., upon the incidence of light. The detector may include a single element, multiple detector elements, for example a bi-cell or quad-cell, a linear or two-dimensional array of detector elements, including camera-based detectors.

[0079] The plates disclosed herein are suitable for use in any assay applied to a liquid or thin layer sample that can be fixed or adhered to a well surface for reflectance FTIR spectroscopy. This includes assays for identifying or analyzing chemical or drug fingerprints, molecular fingerprints, or any related vibrational fingerprints by FTIR. The design allows for maximum IR reflectance and interaction with the sample by optimizing IR exposure to target analytes, especially the more complex analytes found in biological samples.

[0080] At 212, the IR reflective layer may be removed from the base plate. Removal of the IR reflective layer may extend the life of the base plate, as additional IR reflective layers may be applied for reuse.

[0081] FIG. 4A illustrates an example of target focal point placement variations based on incidence angle. As described above and otherwise disclosed herein, the optical system and focal point are configured to optimize the interaction between the sample, which may be a thin layer sample, and an interrogation source, such as IR radiation, that directly correlates with the incidence angle of the IR beam. In example 300 of FIG. 4, three example incidence angles 302, 304, and 306 resulting from an IR source 308 are shown. Each of incidence angles 302, 304, and 306 is associated with a particular target focal point 310, 312, and 314. For example, first incidence angle 302 is associated with first focal point 310, while second incidence angle 304 is associated with second focal point 312, and third incidence angle 306 is associated with third focal point 314.

[0082] The curvature of the well design is optimized based on the selected angle of incidence of the IR beam; thus, the curvature or depth of the concave surface can be varied depending on the angle of incidence. For a fixed angle of incidence, the depth (d) and radius (r) of the mirror or concave surface (see Figure 4B) can be scaled within a given range while maintaining the same curvature.

[0083] FIG. 4B shows an example of an incident IR beam (402) illuminating a well 112 having a depth (d) and a radius (r). As discussed herein, illuminating refers to directing radiation toward an object, such as the surface of a sample, the probe tip, and / or the area of ​​probe-sample interaction. Illumination can include radiation in the infrared wavelength range, the visible, and other wavelengths from the ultraviolet to millimeters or longer. Illumination can include any configuration of radiation source, reflecting elements, focusing elements, and any other beam steering or adjusting elements. The incident IR beam 402 can be, for example, a collimated, collinear laser beam that focuses to a single point upon reflection by a parabolic mirror. Indeed, as shown in FIG. 4B, the light interacts with a parabolic mirror that is a well 112 having a radius r and a depth d such that it is focused into an objective lens 404.

[0084] In addition to optical considerations, well 112 is designed to hold a sample, which is often a liquid. Therefore, the parabolic shape of well 112 has a depth d sufficient to act as a bowl to hold this sample. This can be achieved by submerging well 112 into base 108 by a depth d. Alternatively or additionally, and as shown in FIG. 4B, well 112 can be built up by a height h. In this way, the expected sample surface (shown by the dashed line in FIG. 4B) can be at the same level as the surface of the entire well plate without overflow. Height h is therefore that of a kind of shoulder used to prevent sample from spilling out of well 112.

[0085] 4B, incident light 402 passes through the sample (see dashed line) and is reflected to the focal point of objective lens 404. The depth d and radius r can be selected based on the expected angle of incidence θ of light 402 for a particular instrument. In some cases, such as when physical space above the sample plate is limited, a very low angle of incidence θ can be used that would interact with a shoulder, and in such instances the height h can be reduced.

[0086] It should also be understood that while an objective lens 404 is used in FIG. 4B , other types of detectors, sensors, or cameras that collect focused light can be used. Generally, the system collects the radiation of a probe or interrogation light beam that interacts with a sample. In some instances, a detector, such as a photodiode, can have enough surface area that the light does not need to be perfectly focused. Instead, a relatively large spot size for the reflected light can be collected by such a detector or sensor to provide information about the reflected signal that interacts with the sample.

[0087] While preferred embodiments and implementations of the present disclosure have been described, modifications and equivalents of the disclosed concepts may readily occur to those skilled in the art, and such modifications and equivalents are intended to be included within the scope of the claims appended hereto. [Explanation of symbols]

[0088] 100 Assembly 102 base plate 104 IR reflective layer 106 IR Reflective Wells 108 base plate 110 wells 112 Focus 113 Smoothing layer 114 IR reflective layer 115 Upper Court 116 IR Reflectance Sample Plate 154 IR Reflective Materials 156 IR Reflectance Sample Plate 200 ways 300 examples 302, 304, 306 angle of incidence 308 IR source 310, 312, 314 target focus 402 incident IR beam 404 Object θ angle of incidence d depth h height

Claims

1. 1. A reflective sampling device for FTIR-based analysis comprising: a base plate defining an array of wells on its surface; A reflective sampling device, wherein each of one or more wells disposed on a surface of a base plate is configured as a concave mirror that reflects in a predetermined wavelength range.

2. The device of claim 1 , wherein the concave mirror has an IR-reflective coating made of a material with high infrared reflectivity.

3. The device of claim 2 , wherein the material is selected from the group consisting of aluminum, copper, nickel, chromium, silver, and gold.

4. The device of claim 1 , wherein the well is optimized in depth and radius to create an optimal focal point for minimizing reflective interference and maximizing signal amplitude.

5. 5. The device of claim 4, wherein the depth of each well is selected from the range of 0.2 to 2 mm and the radius of each well is selected from the range of 1.5 to 5 mm.

6. The device of claim 5 , wherein the base plate is a 96-well plate.

7. 10. The device of claim 1, wherein the surface of the IR-reflective well is ion-treated to ensure that a consistent and smooth thin layer of liquid sample is created.

8. 10. The device of claim 1, wherein the reflective surface is created by a process selected from the group consisting of vacuum metallization, the use of a reflective embossed metallized film, and an aluminum foil laminate.

9. A reflective sampling device for FTIR-based analysis, comprising a base plate having an array of wells optimized in depth and radius to create an optimal focal spot for minimizing reflective interference and maximizing signal amplitude.

10. 10. The device of claim 9, wherein the depth of each well is selected from the range of 0.2 to 2 mm and the radius of each well is selected from the range of 1.5 to 5 mm.

11. The device of claim 10 , wherein the base plate is a 96-well plate.

12. The device of claim 9 , wherein the array of wells is configured as an array of concave mirrors.

13. 13. The device of claim 12, wherein the concave mirror has an IR reflective coating made of a material with high infrared reflectivity.

14. 14. The device of claim 13, wherein the IR reflective coating is applied by a process selected from the group consisting of vacuum metallization, use of a reflective embossed metallized film, and an aluminum foil laminate.

15. 1. A method for making an infrared (IR) reflectance array plate for high-throughput optical cancer fingerprinting analysis, said method comprising: obtaining a base plate including an array of recessed wells; and applying an IR reflective coating to one or more wells of said array of concave wells, such that each of said one or more wells is a concave mirror.

16. 16. The method of claim 15, wherein the IR reflective coating is selected from the group consisting of aluminum, copper, nickel, silver and gold.

17. The method of claim 16 , wherein the step of applying the IR-reflective coating comprises vacuum metallizing.

18. 16. The method of claim 15, further comprising ion treating the concave mirror surface of the one or more wells.

19. 16. The method of claim 15, wherein each well of the array of concave wells is optimized in depth and radius to create an optimal focal point for minimizing reflective interference and maximizing signal amplitude.

20. 16. The method of claim 15, loading one or more samples into said one or more wells; conducting an analysis of the one or more samples; and The method further includes the step of: separating the IR reflective layer from the base plate.

21. 21. The method of claim 20, further comprising applying a separate IR-reflective coating to one or more wells of the array of recessed wells.

22. 10. A method for improving signal-to-noise ratio in FTIR-based analysis using diffuse reflectance from the device of claim 1.

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