Interferometric sensor for biochemical tests

Abstract

Described herein are interferometric sensors that can be used for biochemical testing. Each interferometric sensor includes an interference layer immobilized along a surface of a monolithic substrate. Analyte binding molecules can be coated along a surface of the interference layer. During biochemical testing, when analyte molecules in a sample bind to the analyte binding molecules, a biological layer will form. The monolithic substrate has a higher refractive index than the interference layer. Furthermore, the interference layer can be designed to have a refractive index that is substantially similar to a refractive index of the biological layer.

Description

Technical Field

[0001] Various embodiments involve interferometric sensors, to which analyte molecules in a sample can bind during biochemical testing. Background Technology

[0002] Diagnostic tests based on binding events between analyte molecules and analyte-binding molecules are widely used in medical, veterinary, agricultural, and research applications. These tests can be used to detect the presence of analyte molecules in a sample, the amount of analyte molecules in the sample, or the binding rate between analyte molecules and analyte-binding molecules. An analyte-binding molecule and its corresponding analyte molecule together form an analyte-antianalyte binding pair (or simply a "binding pair"). Examples of binding pairs include complementary nucleic acid chains, antigen-antibody pairs, and receptor-receptor binding agents. The analyte can be any member of the binding pair, while the antianalyte can be the other member.

[0003] Historically, diagnostic tests have employed solid, planar surfaces on which analyte-binding molecules are immobilized. Analyte molecules in the sample bind with these analyte-binding molecules with high affinity within a defined detection region. In this type of assay, known as a "solid-phase assay," the solid surface is exposed to the sample under conditions that promote the binding of analyte molecules to their analyte-binding molecules. Binding events are typically detected directly by measuring changes in mass, reflectance, thickness, color, or other characteristics that indicate binding events. For example, when analyte molecules are labeled with chromophores, fluorescent markers, or radioactive markers, binding events can be detected based on how much (if any) of the label is detected within the detection region. Alternatively, analyte molecules can be labeled after binding with analyte-binding molecules within the detection region.

[0004] U.S. Patent No. 5,804,453 discloses a method for determining the concentration of a substance in a sample solution using an optical fiber having a reagent (i.e., a trapping molecule) directly coated on the distal end of the fiber to which it binds. The distal end is then immersed in a sample containing the analyte. The binding of the analyte to the reagent layer produces an interference pattern, which is detected by a spectrometer.

[0005] U.S. Patent No. 7,394,547 discloses a biosensor in which a first optically transparent element is mechanically attached to the tip of an optical fiber with an air gap therebetween, and then a second optical element with a thickness greater than 50 nanometers (nm) is attached as an interference layer to the distal end of the first element. A biosensor layer is formed on the outer peripheral surface of the second optical element. An additional reflective surface layer with a thickness between 5 and 50 nm and a refractive index greater than 1.8 is coated between the interference layer and the first element. The principle of detecting analytes in a sample based on changes in spectral interferometry is described in this reference and is incorporated herein by reference.

[0006] U.S. Patent No. 7,319,525 discloses a different configuration in which a portion of an optical fiber is mechanically attached to a tip connector consisting of one or more optical fibers, wherein the one or more optical fibers have an air gap between the proximal end of the optical fiber portion and the tip connector. An interference layer and then a biological layer are constructed on the distal surface of the optical fiber portion.

[0007] Although existing technologies provide the capability to utilize thin-film interferometer-based biosensors, improvements to the performance of these interferometers are still needed. Attached Figure Description

[0008] Figure 1A A biosensor interferometer is described, which includes a light source, a detector, a waveguide, and optical components (also known as a "probe").

[0009] Figure 1B illustrates an example of a conventional probe.

[0010] Figure 2 Examples of probes according to various embodiments are depicted.

[0011] Figure 3 Another example of a probe according to various embodiments is depicted.

[0012] Figures 4A-B illustrate the detection principle of the thin-film interferometer.

[0013] Figure 5 Examples of glass slides according to various embodiments are depicted.

[0014] Figure 6 Another example of a glass slide according to various embodiments is depicted.

[0015] Figure 7 A flowchart depicts the process used to manufacture the probe.

[0016] Figure 8A -C includes a side view, a bottom perspective view, and a top perspective view of the probe according to various embodiments.

[0017] Figure 9 The binding curves of protein A on the APS probe were plotted (where the offset is in nm), with the conventional probe assigned to channels (CH) 1-4 (i.e. the bottom four curves) and the MgF2 probe assigned to CH 5-8 (i.e. the top four curves).

[0018] Figure 10 The binding curves of human IgG on the protein A probe were plotted, with the conventional probe assigned to CH 1-4 (i.e., the bottom four curves) and the MgF2 probe assigned to CH 5-8 (i.e., the bottom four curves).

[0019] By studying the detailed embodiments in conjunction with the accompanying drawings, those skilled in the art will understand the various features of the present technology. The accompanying drawings depict various embodiments described throughout the detailed embodiments section for illustrative purposes only. Although specific embodiments have been shown by way of example, the technology can be modified and substituted in various ways. It is not intended to limit the technology to the specific embodiments that have been described and / or illustrated. Detailed Implementation

[0020] Some entities have developed systems designed for conducting biochemical tests. Figure 1A -B illustrates an instance of such a system. Specifically, Figure 1A A biosensor interferometer 100 (or simply "interferometer") is depicted, comprising a light source 102, a detector 104, a waveguide 106, and an optical assembly 108 (also referred to as a "probe"). The probe 108 can be connected to the waveguide 106 via a coupling medium.

[0021] The light source 102 can emit light that is guided by the waveguide 106 to the probe 108. For example, the light source 102 can be a light-emitting diode (LED).

[0022] (LEDs), configured to produce light in the range of at least 50 nanometers (nm), 100 nm, or 150 nm within a given spectrum (e.g., 400 nm or less to 700 nm or greater). Optionally, the interferometer 100 may employ multiple light sources with different characteristic wavelengths, such as LEDs designed to emit light of different wavelengths in the visible light range. The same functionality can be achieved with a single light source having a suitable filter used to direct light of different wavelengths onto the probe 108.

[0023] Detector 104 is preferably a spectrometer, such as a Marine Optics USB4000, capable of recording the spectrum of interference light received from probe 108. Alternatively, if light source 102 operates to direct different wavelengths onto probe 108, detector 104 may be a simple photodetector capable of recording the intensity of each wavelength. In another embodiment, detector 104 may include multiple filters that allow the detection of the intensity of each of a plurality of wavelengths.

[0024] Waveguide 106 can be configured to transmit light emitted by light source 102 to probe 108, and then transmit light reflected by surfaces within probe 108 to detector 104. In some embodiments, waveguide 106 is a bundle of optical fibers (e.g., a single-mode fiber optic cable), while in other embodiments, waveguide 106 is a multimode fiber optic cable.

[0025] As shown in Figure 1B, the probe 108 includes a monolithic substrate 114, a thin film layer (also referred to as the "interference layer"), and a biomolecular layer (also referred to as the "biolayer") containing analyte molecules 122 that have bound to analyte-binding molecules 120. The monolithic substrate 114 comprises a transparent material through which light can propagate. The interference layer also comprises a transparent material. When light shines on the probe 108, the proximal surface of the interference layer can serve as a first reflecting surface, and the biolayer can serve as a second reflecting surface. As further described below, the light reflected by the first and second reflecting surfaces can form an interference pattern that can be monitored by the interferometer 100.

[0026] Interference layers typically comprise multiple layers combined in a manner that enhances the detectability of the interference pattern. Here, for example, the interference layer comprises a tantalum pentoxide (Ta₂O₅) layer 116 and a silicon dioxide (SiO₂) layer 118. The tantalum pentoxide layer 116 can be thin (e.g., approximately 10-40 nm) because its primary purpose is to increase the reflectivity of the proximal surface of the interference layer. Meanwhile, the silicon dioxide layer 118 can be relatively thick (e.g., on the order of 650-900 nm) because its primary purpose is to increase the distance between the first and second reflective surfaces.

[0027] For diagnostic testing, probe 108 can be suspended in a micropore 110 (or simply "pore") containing sample 112. During the diagnostic test, analyte molecules 122 in sample 112 bind to analyte-binding molecules 120 along the distal end of probe 108, and these binding events result in an interference pattern that can be observed by detector 104. Interferometer 100 can monitor the thickness of the biolayer formed along the distal end of probe 108 by detecting the shift in the phase characteristics of the interference pattern.

[0028] However, such a design has several drawbacks. One drawback is the poor signal intensity observed during biochemical tests involving these probes. Another drawback is the potential for a negative shift in the binding curve when the biolayer grows over extended periods (e.g., dozens of cycles over 20–40 minutes).

[0029] This section introduces an interferometric sensor (also known as an "interferometric biosensor" or "sensing device") that addresses these shortcomings. Specifically, the interferometric sensor may include a monolithic substrate having first and second surfaces arranged substantially parallel to each other at opposite ends; an interference layer coated on the second surface of the monolithic substrate; and an analyte-binding molecule layer coated on the interference layer. The interference layer may include magnesium fluoride (MgF2). When light shines on the interferometric sensor, the first interface between the monolithic substrate and the interference layer acts as a first reflective surface, while when light shines on the probe, the second interface between the biolayer formed by the binding of analyte molecules and analyte-binding molecules in the sample and the solution containing the sample acts as a second reflective surface. As described above, the thickness of the biolayer can be estimated based on the interference pattern of the light reflected by the first and second reflective surfaces.

[0030] For illustrative purposes, embodiments of the interferometric sensor can be described in the context of a probe designed to be suspended in a solution containing the sample. However, those skilled in the art will recognize that these characteristics are equally applicable to other sensing surfaces, such as planar surfaces (e.g., glass slides), where a biological layer is formed on the planar surface (e.g., a glass slide) by allowing a solution to flow through it during a biochemical test.

[0031] definition

[0032] The term “about” means within ±10% of the stated value.

[0033] The term "analyte-binding molecule" refers to any molecule capable of participating in a binding reaction with an analyte molecule. Examples of analyte-binding molecules include, but are not limited to, (i) antigen molecules; (ii) antibody molecules; (iii) protein molecules; (iv) ligands; and (v) single-stranded nucleic acid molecules.

[0034] The term "interferometric sensor" refers to any sensing device on which a biological layer is formed to produce an interference pattern. One example of an interferometric sensor is a probe designed to be suspended in a solution containing a sample with analyte molecules. Another example is a glass slide with a flat surface on which a biological layer can be formed during biochemical assays.

[0035] The term "probe" refers to a monolithic substrate with an aspect ratio of at least 2:1, coated with a thin film layer on the sensing side.

[0036] The term "monolithic substrate" refers to a sheet of solid material with a uniform composition, such as glass, quartz, or plastic, and a refractive index.

[0037] The term "waveguide" refers to a device (e.g., a pipe, coaxial cable, or optical fiber) designed to confine and guide the propagation of electromagnetic waves (such as light). An example of a waveguide is a metal tube used to guide ultra-high frequency waves.

[0038] Probe Overview

[0039] Figure 2 Examples of probe 200 according to various embodiments are depicted. Probe 200 includes an interference layer 204 fixed along the distal end of a monolithic substrate 202. Analyte-binding molecules 206 can be deposited along the distal surface of the interference layer 204. During biochemical testing, a biolayer is formed when analyte molecules 208 in the sample bind to the analyte-binding molecules 206.

[0040] like Figure 2 As shown, the monolithic substrate 202 has a proximal surface (also called the "connection side") that can be coupled to a waveguide, such as an interferometer, and a distal surface (also called the "sensing side") on which an additional layer is deposited. Typically, the monolithic substrate 202 has a length of at least 3 mm, 5 mm, 10 mm, or 15 mm. In a preferred embodiment, the aspect ratio (length-to-width ratio) of the monolithic substrate 202 is at least 5:1. In such an embodiment, the monolithic substrate 202 can be described as having a columnar shape. The cross-section of the monolithic substrate 202 can be circular, elliptical, square, rectangular, triangular, pentagonal, etc. The monolithic substrate 202 preferably has a refractive index significantly higher than that of the interferometer layer 204, such that the proximal surface of the interferometer layer 204 effectively reflects the light from the guiding probe 200. The preferred refractive index of the monolithic substrate can be higher than 1.5, 1.8, or 2.0. Therefore, the monolithic substrate 202 may include a high refractive index material, such as glass (refractive index 2.0), although some embodiments of the monolithic substrate 202 may include low refractive index materials, such as quartz (refractive index 1.46) or plastics (refractive index 1.32-1.49). Examples of transparent plastics include polypropylene, polyurethane, acrylic, polycarbonate, etc.

[0041] Interference layer 204 comprises at least one transparent material coated on the distal surface of monolithic substrate 202. These transparent materials are deposited on the distal surface of monolithic substrate 202 in the form of thin films ranging in thickness from a fraction of a nanometer (e.g., a monolayer) to several micrometers. Interference layer 204 may have a thickness of at least 500 nm, 700 nm, or 900 nm. Exemplary thicknesses are between 500 and 5,000 nm (and preferably 800-1,200 nm). Here, for example, interference layer 204 has a thickness of approximately 900-1,000 nm or 940 nm.

[0042] Compared to conventional probes, the interferometer layer 204 has a refractive index substantially similar to that of the biological layer. This ensures that reflections from the distal end of the probe 200 are primarily due to the analyte molecules 208 rather than the interface between the interferometer layer 204 and the analyte-binding molecules 206. Typically, the biological layer has a refractive index of approximately 1.36, although this can vary depending on the type of analyte-binding molecules (and therefore analyte molecules) along the distal end of the probe 200.

[0043] In some embodiments, the interference layer 204 comprises magnesium fluoride (MgF2), while in other embodiments, the interference layer 204 comprises potassium fluoride (KF) with a refractive index of 1.36, lithium fluoride (LiF) with a refractive index of 1.39, sodium fluoride (NaF) with a refractive index of 1.32, lithium calcium aluminum fluoride (LiCaAlF6) with a refractive index of 1.39, strontium fluoride (SrF2) with a refractive index of 1.37, and aluminum fluoride with a refractive index of 1.38.

[0044] Sodium aluminum hexafluoride (Na3AlF6) with a refractive index of 1.34 (also known as "cryolite"), and sodium aluminum fluoride (Na5Al3F6) with a refractive index of 1.34. 14 (Also known as "chiolite") etc. Alternatively or alternatively, the interference layer 204 may comprise a polymer with a refractive index less than 1.4, such as... (A copolymer of sucrose and epichlorohydrin). Magnesium fluoride has a refractive index of 1.38, which is essentially the same as the refractive index of the biological layer formed along the distal end of probe 200. In contrast, the interference layer of conventional probes typically contains silica, which has a refractive index of approximately 1.46. Lower-purity forms of silica have higher refractive indices (e.g., approximately 1.5 in the visible light range). Generally, the refractive index of interference layer 204 is between 1.32 and 1.42, 1.36 and 1.42, or 1.38 and 1.40. Because interference layer 204 and the biological layer have similar refractive indices, light will experience minimal scattering as it travels from interference layer 204 to the biological layer and back to interference layer 204.

[0045] The thickness of the biological layer is designed to optimize the overall sensitivity of the interferometer-based hardware (e.g., optical components). Conventional immobilization chemistry can be used to covalently (e.g., chemically) or non-covalently (e.g., by adsorption) attach analyte-binding molecules 206 to the distal surface of the interferometer layer 204.

[0046] The analyte-binding molecular layer 206 is preferably formed under conditions where the distal end of the probe 200 is densely coated, such that the binding of analyte molecules 208 to analyte-binding molecules 206 results in a change in the thickness of the biological layer rather than being filled within the layer. The analyte-binding molecular layer 206 can be a monolayer or a multilayer matrix.

[0047] During biochemical testing, probe 200 can be suspended within a cavity (e.g., a pore) that includes the sample. An example of probe-based detection technology is described in U.S. Patent No. 8,597,578, entitled "Optical Sensor of Bio-Molecules using Thin-Film Interferometer," which is incorporated herein by reference in its entirety. During biochemical testing, as analyte molecule 208 binds to analyte-binding molecule 206, a biolayer is formed along the distal end of probe 200.

[0048] When light shines on probe 200, the proximal surface of interference layer 204 serves as a first reflective surface, while the distal surface of the biolayer serves as a second reflective surface. The presence, concentration, or binding rate of analyte molecule 208 with probe 200 can be estimated based on the interference of the light beams reflected by these two reflective surfaces. As analyte molecule 208 attaches to (or separates from) analyte-bound molecule 206, the distance between the first and second reflective surfaces changes. Because all other components in probe 200 remain the same size, the interference pattern formed by the light reflected from the first and second reflective surfaces undergoes a phase shift according to the change in biolayer thickness due to the binding event.

[0049] Using a monolithic substrate 202 instead of an optical fiber offers several advantages. As mentioned above, the refractive index of the monolithic substrate 202 is preferably higher than that of the interferometer layer 204. For example, the refractive index of the monolithic substrate 202 can be at least 0.1, 0.2, 0.4, 0.5, or 0.6 higher than that of the interferometer layer 204. Since the monolithic substrate 202 is a solid material with a uniform composition, it is easier to select a material with a higher refractive index than that of the interferometer layer 204. In contrast, optical fibers are typically dielectric waveguides with a circular cross-section, whose dielectric material (also called the "core") is surrounded by another dielectric material with a lower refractive index (also called the "cladding"), making it difficult to manipulate their refractive index.

[0050] During operation, the incident light signal 210 emitted by the light source is transmitted to the biological layer through the monolithic substrate 202. Within the probe 200, the light is reflected at a first reflective surface, generating a first reflected light signal 212. The light is also reflected at a second reflective surface, generating a second reflected light signal 214. The second reflective surface initially corresponds to the interface between the analyte-binding molecule 206 and the sample into which the probe 200 is immersed. Due to binding occurring during biochemical testing, the second reflective surface becomes the interface between the analyte molecule 208 and the sample.

[0051] The first and second reflected light signals 212 and 214 form a spectral interference pattern, as shown in Figure 4A. When the analyte molecule 208 binds to the analyte-binding molecule 206 on the distal surface of the interference layer 204, the optical path of the second reflected light signal 214 becomes longer. As a result, the spectral interference pattern shifts from T0 to T1, as shown in Figure 4B. By continuously measuring the phase shift in real time, a kinetic binding curve can be plotted as a graph of the shift versus time. The binding rate of the analyte molecule to the analyte-binding molecule fixed on the distal surface of the interference layer 204 can be used to calculate the analyte concentration in the sample. Therefore, this phase shift measurement is the detection principle of the thin-film interferometer.

[0052] Referring to Figure 4A, the performance of the thin-film interferometer can be improved by maximizing the alternating current (AC) component and minimizing the direct current (DC) offset. In other words, the performance of the thin-film interferometer can be improved by increasing the AC / DC ratio, since the AC component represents the signal of interest and the DC offset represents noise. To achieve these goals, the efficiency of the incident light signal 210 and the reflected light signals 212, 214 passing through the probe 200 can be improved; the coupling efficiency between the light source and the probe 200 can be improved; and / or the coupling efficiency between the spectrometer and the probe 200 can be improved.

[0053] Essentially, matching the refractive indices of the interferometer layer 204 and the biological layer achieves the first of these goals: reducing reflections as much as possible from other surfaces within the probe 200 (e.g., the interface between the interferometer layer 204 and the analyte-binding molecule 206). Because the refractive index of the interferometer layer 204 is close to that of the biological layer (e.g., the refractive index of the interferometer layer 204 is 1.38, and that of the biological layer is 1.36), the shift in the spectral interference pattern will increase as the biological layer is established. This is because the increment between T0 and T1 is based on the difference between the refractive indices of the interferometer layer 204 and the surrounding material (e.g., the sample). However, note that as the refractive index of the interferometer layer 204 decreases, the amplitudes of T0 and T1 will also decrease. There is a trade-off between the amplitude and the shift in the spectral interference pattern. At a high level, the goal is to have a sufficiently large amplitude to identify both peaks while keeping them as far apart as possible. As an example, reducing the refractive index of the interferometer layer 204 will result in a more shifted but smaller AC / DC ratio (i.e., a larger DC component and / or a smaller AC component, which results in a “noisier” signal).

[0054] In some embodiments, a reflective layer (not shown) is deposited along the distal end of the monolithic substrate 202, such that the reflective layer is located between the monolithic substrate 202 and the interference layer 204. Since its primary purpose is to ensure that the first reflected light signal 212 is reflected at the interface between the monolithic substrate 202 and the interference layer 204, the reflective layer may comprise a material having a higher refractive index than either the monolithic substrate 202 or the interference layer 204. For example, the reflective layer may comprise zinc sulfide (ZnS) with a refractive index of 2.3-2.4, titanium dioxide (TiO2) with a refractive index of 2.3-2.4, titanium monoxide (TiO) with a refractive index of 2.2-2.3, titanium trioxide (Ti2O3) with a refractive index of 1.9-2.3, titanium oxide (Ti3O5) with a refractive index of 2.2-2.3, tantalum oxide (Ta2O3) with a refractive index of 216, tantalum pentoxide (Ti3O5) with a refractive index of 2.16, silicon monoxide (SiO) with a refractive index of 1.8-1.9, or aluminum oxide (TiO2) with a refractive index of 1.67. The reflective layer can be very thin compared to the interference layer 204. For example, the reflective layer can have a thickness of approximately 3-10 nm. The reflective layer can be Al₂O₃, zirconium dioxide (ZrO₂) with a refractive index of 1.97-2.05, zinc monoxide (ZnO) with a refractive index of 2.01, lanthanum titanium trioxide (LaTiO₃) with a refractive index of 2.1, indium tin oxide (ITO) with a refractive index of 1.8, niobium pentoxide (Nb₂O₅) with a refractive index of 2.1-2.3, zinc selenide (ZnSe) with a refractive index of 2.58, cerium dioxide (CeO₂) with a refractive index of 2.35, yttrium oxide (Y₂O₃) with a refractive index of 1.87, hafnium oxide (HfO₂) with a refractive index of 1.95, and gadolinium oxide (Gd₂O₃) with a refractive index of 1.8.

[0055] Figure 3 Another example of a probe 300 according to various embodiments is depicted. Figure 3 The probe 300 can be basically similar to Figure 2 The probe 200. However, here, the probe 300 includes an adhesion layer 310 deposited along the distal surface of the interference layer 304, which is attached to a monolithic substrate 302. The adhesion layer 310 may include a material that promotes adhesion of the analyte-binding molecules 306. An example of such a material is silicon dioxide. Compared to the interference layer 304, the adhesion layer 310 is typically very thin, so its influence on light traveling to or returning from the biological layer will be minimal. For example, the adhesion layer 310 may have a thickness of about 3-10 nm, while the interference layer 304 may have a thickness of about 800-1,000 nm. The biological layer formed by the analyte-binding molecules 306 and analyte molecules 308 is typically a few nanometers thick. Figure 2 The probe 200 is very similar. Figure 3The probe 300 may also have a reflective layer (not shown) deposited along the distal end of the monolithic substrate 302, such that the reflective layer is located between the monolithic substrate 302 and the interference layer 304. The thickness of the reflective layer may be approximately the same as the thickness of the adhesion layer 310.

[0056] As mentioned above, these characteristics also apply to sensing surfaces of other forms. One example of such a sensing surface is a glass slide (also known as a "chip") with a flat surface on which a biological layer is formed by flowing a solution through the flat surface during a biochemical test. See below for reference. Figure 5-6 Several examples of flat surfaces are discussed.

[0057] Figure 5 Examples of glass slides 500 according to various embodiments are depicted. The glass slide 500 includes a substrate 502 on which an interference layer 504 is deposited. In some embodiments, the interference layer 504 is deposited along the entire upper surface of the substrate 502, while in other embodiments, the interference layer 504 is deposited along a portion of the upper surface of the substrate 502. For example, the interference layer 504 may be deposited within channels or holes formed within the upper surface of the substrate 502. As described above, Figure 2-3 The height of the monolithic substrates 202 and 302 is typically much larger than their width. However, in this case, the reverse may be true. In fact, the width of substrate 502 can be 5, 7.5, 10, or 20 times larger than its length. As an example, the substrate could be approximately 75 × 26 mm with a height / thickness of approximately 1 mm.

[0058] During diagnostic testing, analyte molecule 508 can bind to analyte-binding molecule 506, which has been immobilized along the upper surface of interference layer 504 to form a biolayer. To determine the thickness of the biolayer, light can be shone onto the upper surface of slide 500, such as... Figure 5 As shown. More specifically, the incident light signal 510 emitted by the light source can be displayed at the biological layer formed along the upper surface of the slide 500. This may require the incident light signal 510 to pass through an ambient medium 516, which can be a vacuum, air, or a solution. The incident light signal 510 will be reflected at a first reflective surface, producing a first reflected light signal 512. The first reflective surface can represent the interface between the biological layer and the ambient medium 516. The incident light signal 510 will also be reflected at a second reflective surface, producing a second reflected light signal 514. The second reflective surface can represent the interface between the interference layer 504 and the substrate 502. As described above, the first and second reflected light signals 512, 514 form a spectral interference pattern, which can be analyzed to determine the thickness of the biological layer. Note that because the incident light signal 510 does not propagate through the substrate 502, the substrate 502 can be transparent or opaque (e.g., opaque).

[0059] Figure 6 Another example of a glass slide 600 according to various embodiments is depicted. Figure 6 The 600 slide can be used with Figure 5 The slide 500 is very similar. Therefore, the slide 600 may include a substrate 602, an interference layer 604, and an analyte-binding molecule 606 deposited on the substrate. During diagnostic testing, the analyte molecule 608 can bind to the analyte-binding molecule 606 to form a biolayer.

[0060] However, here, the incident light signal 610 is displayed on the lower surface of the slide 600. In operation, the incident light signal 610 is transmitted to the biological layer through the substrate 602. Within the slide 600, the light is reflected at a first reflective surface, generating a first reflected light signal 612. The first reflective surface may represent the interface between the interference layer 604 and the substrate 602. The light is also reflected at a second reflective surface, generating a second reflected light signal 614. The second reflective surface may represent the interface between the biological layer and the environmental medium 616. As described above, the first and second reflected light signals 612 and 614 form a spectral interference pattern, which can be analyzed to determine the thickness of the biological layer.

[0061] Although Figure 5-6 Although not shown, slides 500 and 600 may include a reflective layer disposed between substrates 502 and 602 and interference layers 504 and 604 to increase reflectivity along the interface and / or an adhesion layer disposed along the upper surface of interference layers 504 and 604 to fix analyte-binding molecules 506 and 606.

[0062] Figure 7 A flowchart of process 700 for manufacturing an interferometric sensor is depicted. First, the manufacturer obtains a monolithic substrate (step 701). For example, the manufacturer may select a monolithic substrate from a plurality of monolithic substrates designed for different biochemical tests, analyte binding molecules, etc. The preferred refractive index of the monolithic substrate may be higher than 1.5, 1.8, or 2.0. Therefore, the monolithic substrate obtained by the manufacturer may include high refractive index materials, such as glass (refractive index 2.0), or low refractive index materials, such as quartz (refractive index 1.46) or plastic (refractive index 1.32-1.49). As described above, in some embodiments, the monolithic substrate has a columnar form (e.g., Figure 2-3 In other embodiments, the monolithic substrate 202, 302), while in other embodiments, the monolithic substrate has a planar form (e.g., Figure 5-6 (502, 602 single-layer substrates).

[0063] The manufacturer can then deposit a transparent material on the surface of the monolithic substrate to form an interference layer (step 702). For example, the transparent material can be deposited on the distal surface of the monolithic substrate in the form of a thin film with a thickness ranging from a fraction of a nanometer (e.g., a monolayer) to a few micrometers. Typically, the interference layer has a thickness of at least 500 nm, 700 nm, or 900 nm. Exemplary thicknesses are between 500 and 5,000 nm (and preferably 800 to 1,200 nm).

[0064] In some embodiments, the manufacturer deposits another transparent material on the surface of the interference layer to form an adhesion layer (step 703). The adhesion layer may include a material that promotes the adhesion of analyte-binding molecules. An example of such a material is silicon dioxide. Compared to the interference layer, the adhesion layer is typically very thin, so its influence on light propagating along the interference sensor will be minimal. For example, the adhesion layer may have a thickness of approximately 3-10 nm.

[0065] Subsequently, the manufacturer can immobilize the analyte-binding molecules onto the surface of the adhesion layer (step 704). As described above, the analyte-binding molecule layer can be formed under conditions where the surface of the interferometric sensor (e.g., the distal end of a probe, or the distal surface of a planar chip) is densely coated. This ensures that when analyte molecules bind to the analyte-binding molecules during biochemical testing, these binding events result in changes in the thickness of the biolayer, rather than filling the analyte-binding molecule layer. The analyte-binding molecule layer can be a monolayer or a multilayer matrix.

[0066] Unless contrary to physical possibilities, it is conceivable that the above steps can be performed in various orders and combinations. For example, the manufacturer may choose not to create an adhesion layer along the distal surface of the interference layer. In such an embodiment, the analyte-binding molecules can be directly attached to the distal surface of the interference layer.

[0067] Additional steps may also be performed. For example, the manufacturer may form a reflective layer on the surface of the monolithic substrate. As described above, the reflective layer may comprise a transparent material having a higher refractive index than the monolithic substrate and the interference layer. Due to its location, this transparent material may be deposited onto the surface of the monolithic substrate before the interference layer is formed (i.e., before performing step 702). As another example, the manufacturer may cure the interference layer (e.g., using heat, air, radiation, etc.) before forming the adhesion layer. Similarly, the manufacturer may (i) cure the reflective layer before attaching the adhesion layer thereto and / or (ii) cure the adhesion layer before attaching the analyte-bound molecules thereto. As another example, the manufacturer may polish the first and second surfaces of the monolithic substrate, which are arranged substantially parallel to each other at opposite ends. Polishing may be performed to improve the adhesion of the interference layer to the monolithic substrate.

[0068] Figure 8AIncludes a side view of a probe 800 according to various embodiments. Figure 8B Including the bottom perspective view of probe 800, and Figure 8C The image includes a top perspective view of probe 800. Probe 800 includes a rod portion 802 (also referred to as a "rod member") and a flexible support member 804 (also referred to as a "flexible skirt"). The flexible support member 804 can be centrally positioned along the length of rod portion 802 such that a first portion of rod portion 802 extends from the top side of flexible support member 804 and a second portion of rod portion 802 extends from the bottom side of flexible support member 804. Therefore, flexible support member 804 can be located at the central portion of rod portion 802.

[0069] The rod portion 802 can be a monolithic substrate, for example... Figure 2 The monolithic substrate 202. The rod portion 802 may have a length of at least 3 mm, 5 mm, 10 mm, or 15 mm. Note that the first and second portions of the rod portion 802 may have different dimensions. For example, the first portion of the rod portion 802 extending from the top side of the flexible support member 804 may be 2-5 mm, while the second portion of the rod portion 802 extending from the bottom side of the flexible support member 804 may be 5-10 mm.

[0070] The flexible support member 804 may include a flange portion 806 and a sleeve portion 808. In some embodiments, the flange portion 806 and the sleeve portion 808 are joined together after the production of each member. In other embodiments, the flange portion 806 and the sleeve portion 808 are part of a single member formed by a molding process, extrusion process, or the like. The flexible support member 804 may comprise, partially or entirely, silicone rubber, nitrile rubber, or some other elastomer. For example, in some embodiments, the entire flexible support member 804 comprises a flexible material, while in other embodiments, only the flange portion 806 comprises a flexible material.

[0071] like Figure 8B As shown, the bottom side of the flexible support member 804 may include a recess 810 defined by a concave surface 816. An internal extension feature 818 located in the recess 810 may be secured around the rod portion 802. In embodiments including the internal extension feature 818, the recess 810 may take the form of an annular recess extending radially around the rod portion 802.

[0072] As described above, the distal end 812 (also referred to as the "bottom end") of the rod portion 802 may have an interference layer fixed thereon, and analyte-binding molecules may be coated on the interference layer. During biochemical testing, a biolayer is formed when analyte molecules in the sample bind to analyte-binding molecules. When light shines on the proximal end 814 of the probe 800, the proximal surface of the interference layer can serve as a first reflecting surface, while the biolayer can serve as a second reflecting surface.

[0073] When the probe 800 is inserted into the hole, the top surface of the hole applies pressure to the bottom side of the flange portion 806 of the flexible support member 804. This pressure causes the distal end 812 of the rod portion 802 to suspend within the hole. The flange portion 806 may be designed to prevent the distal end 812 of the rod portion 802 from contacting the inner surface of the hole when inserted. The hole may be included in a box comprising a plurality of holes arranged in a linear pattern or in a microplate comprising a plurality of holes arranged in a grid pattern.

[0074] Remark

[0075] For purposes of illustration and description, the foregoing description of various embodiments of the technology has been provided. It is not intended to be exhaustive or to limit the claimed subject matter to the precise forms disclosed.

[0076] Many modifications and variations will be apparent to those skilled in the art. The embodiments were chosen and described in order to best illustrate the principles of the art and its practical application, thereby enabling others skilled in the art to understand the claimed subject matter, various embodiments, and various modifications suitable for the intended particular purpose.

[0077] Example

[0078] The invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention to the specific procedures described herein.

[0079] Example 1. Fabrication of a conventional probe (SiO2 probe)

[0080] Figure 1B shows a conventional probe. 20 mm long, 1 mm diameter quartz rods (refractive index 1.46) are polished to a mirror finish at both ends using an optical polisher. After being washed and cleaned in pure water, the rods are arranged in a fixture and then loaded into an ion beam-assisted physical vapor deposition (PVD) machine. In the PVD machine, an electron beam is used to bombard and vaporize the target material to be coated onto the surface; then an ion beam is applied to deposit the vapor onto the surface to form a thin film layer. These quartz rods are first coated with a 20-nm Ta₂O₅ layer, followed by a 730-nm SiO₂ layer. After the Ta₂O₅ / SiO₂ layers are coated on the surface, the rods are placed in a chemical vapor deposition (CVD) machine (e.g., a Lab Kote manufactured by Yield Engineering) to coat a thin layer of aminopropyltriethoxysilane (APS). The thickness of the APS layer is typically 1-2 nm. APS is deposited to immobilize proteins. The APS adsorbs proteins onto the probe surface through a combination of hydrophobic and ionic interactions. Cross-linking agents can also be used to covalently couple proteins to the amino groups of APS. APS is only a monolayer, so its thickness can be about 7 nm.

[0081] Example 2A. Preparation of a probe according to an embodiment of the present invention ( Figure 3 )

[0082] Examples of the probe of the present invention Figure 3 As shown. This probe can be called a "MgF2 probe" or a "MgF2-APS probe". A 20mm long, 1mm diameter glass rod (refractive index 2.0) is polished to a mirror finish at both ends using an optical polisher. After cleaning and rinsing in pure water, the rods are arranged in a fixture and then loaded into an ion beam-assisted physical vapor deposition (PVD) machine. In the PVD machine, an electron beam is used to bombard and vaporize the target material to be coated onto the surface; then an ion beam is applied to deposit the vapor onto the surface to form a thin film layer. The glass rod is first coated with a 940-nm MgF2 layer, followed by a 5-nm SiO2 layer. After coating the surface with the MgF2 / SiO2 layer, the rod is placed in a chemical vapor deposition (CVD) machine to coat an APS thin layer, typically 1-2nm thick.

[0083] Example 2B. Preparation of a probe according to another embodiment of the present invention ( Figure 2 )

[0084] Another example of the probe of the present invention Figure 2 As shown. This probe can be called a "MgF2 probe" or a "MgF2-APS probe". A 20mm long, 1mm diameter glass rod (refractive index 2.0) was polished to a mirror finish at both ends using an optical polisher. After cleaning and rinsing in pure water, the rods were arranged in a fixture and then loaded into an ion beam-assisted physical vapor deposition (PVD) machine. In the PVD machine, an electron beam is used to bombard and vaporize the target material to be coated onto the surface; then an ion beam is applied to deposit the vapor onto the surface to form a thin film layer. The glass rod was coated with a single 940nm layer of MgF2, without a SiO2 thin layer. After coating the surface with the MgF2 layer, the rod was placed in a chemical vapor deposition (CVD) machine to coat an APS thin layer, typically 1-2nm thick.

[0085] Example 3. Comparison of protein A binding between MgF2 probe and conventional probe

[0086] For side-by-side comparative studies, a conventional APS probe (Example 1) and a MgF2-APS probe (Example 2A) were immobilized with protein A for binding assays.

[0087] The two probes undergo three steps in a 96-well plate, as shown in Table 1.

[0088] Table 1. Combined with test parameters.

[0089]

[0090] This experiment was conducted using a Gator interferometer instrument and software version 1.3 from Probe Life, Inc. The results are as follows: Figure 9 The results are shown and summarized in Table 2. Figure 9 The binding curves of protein A on the APS probe were plotted (where the offset is in nm), with the conventional probe assigned to channels (CH) 1-4 (i.e. the bottom four curves) and the MgF2 probe assigned to CH 5-8 (i.e. the top four curves).

[0091] Table 2. Results based on signal analysis

[0092] The binding signal of protein A (nm offset) conventional probe 2.5nm <![CDATA[MgF2 probe]]> 5.6nm

[0093] The results show that the MgF2-APS probe generates a binding signal (nm offset) that is more than 2.24 times greater than that of a conventional probe, as shown in the upper limit of the wavelength offset (in nm).

[0094] Example 4. Comparison of IgG / protein A binding between the MgF2 probe and a conventional probe

[0095] Because protein A has five Ig-binding domains, and in the case of the human VH3 family, it binds heavy chains within the Fc and Fab regions, we can repeatedly immobilize human IgG (Equitech-Bio SLH56) and protein A on the probe surface to test the upper limit of nm offset.

[0096] Two APS probes (a conventional probe, Example 1; and a MgF2 probe, Example 2A) were used to cycle steps 1 through 4 50 times in a 96-well plate.

[0097] 1. K buffer (PBS, 0.02% BSA, 0.002% Tween-20, 200 μL) was incubated at 1000 rpm for 10 seconds.

[0098] 2.2 μg / mL whole human IgG in K buffer (200 μL) at 1000 rpm for 60 seconds

[0099] 3. K buffer (200 μL) was added at 1000 rpm for 10 seconds.

[0100] 4.10 μg / mL protein A in K buffer (200 μL) at 1000 rpm for 60 seconds

[0101] The experiment was conducted using a Gator instrument and software version 1.3 from Probe Life, Inc. The results are as follows: Figure 10 shown. Specifically, Figure 10The binding curves of human IgG on the protein A probe were plotted, with the conventional probe assigned to CH1-4 (i.e., the bottom four curves) and the MgF2 probe assigned to CH5-8 (i.e., the top four curves).

[0102] Figure 10 The results showed that the MgF2 probe achieved a wavelength shift of 120 nm without turning negative, while the conventional probe only achieved a wavelength shift of 7 nm before showing a negative nm shift. The signal (nm shift) of the MgF2 probe was significantly higher than that of the conventional probe. Example 5. Comparison of protein binding and regeneration between the MgF2 probe and the conventional probe.

[0103] Preparation of anti-mouse Fc coated probe

[0104] Probes coated with streptavidin were prepared by immersing two APS probes (Examples 1 and 2A) in 50 μg / mL streptavidin (Invitrogen, 21122) in PBS buffer of a 96-well plate at 1000 rpm for 10 minutes.

[0105] In this experiment, affinity-purified goat anti-mouse IgG Fc-γ fragment specificity (Jackson-Immuno, 115-005-071) was used. This anti-mouse Fc exhibits minimal cross-reactivity with human, bovine, and equine serum proteins. Anti-mouse IgG was biotinylated using a standard protocol with EZ-link NHS-PEG4-Biotin (Thermo Scientific, A39259). The biotinylated antibody was diluted in K buffer (Probe Life, 120011). The streptavidin-coated probe was immersed in 0.5 mg / ml biotin-anti-mouse Fc for 10 min, followed by washing in K buffer for 30 s to remove any non-specific binding interactions on the probe surface.

[0106] Measurement

[0107] The anti-mouse Fc-coated dry probe was immersed in Q buffer (PBS + 0.2% BSA + 0.02% Tween-20) and hydrated for 5 minutes before any assay.

[0108] Mouse IgG was generated at concentrations ranging from 0.5 to 200 μg / ml using mouse IgG dissolved in Q buffer. This concentration series was used to test the conventional probe and the MgF2 probe side-by-side to compare their performance in binding capacity, signal intensity, and regeneration. For regeneration of both probes, 10 mM glycine (pH 1.75) and 150 mM NaCl were used as regeneration solutions.

[0109] The experiment was performed using a Gator instrument (GA007) and software version 1.3 from Probe Life, Inc. Samples and regeneration solutions were prepared in microplates from Greiner Bio (Ref#655209).

[0110] The reaction and regeneration protocols are shown in Table 3. Regeneration was repeated 10 times.

[0111] Table 3. Regeneration Test Results

[0112]

[0113] result

[0114] A side-by-side comparison of the binding capabilities of conventional probes and MgF2 probes was conducted to understand the binding strength, binding rate, and regeneration. The results are summarized in Tables 4 and 5.

[0115] Table 4 shows that the MgF2 probe has a much higher signal (nm wavelength offset) and a faster binding rate than the conventional probe.

[0116] Table 4. Results of the integration ability

[0117]

[0118] Table 5 shows that after 10 rounds of regeneration, the MgF2 probe retained 52% (30 μg / mL mIgG) and 41% (3 μg / mL mIgG) of the original signal intensity, while the traditional probe retained only 29% (30 μg / mL mIgG) and 30% (3 μg / mL mIgG) of the original signal intensity.

[0119] Table 5. Regeneration Test Results

[0120]

[0121] Example 6. Comparison of small molecule binding between MgF2 probe and conventional probe

[0122] In this example, the binding of the enzyme carbonic anhydrase II (CAII) to its inhibitor furosemide was detected using the MgF2 probe from Example 2B, and compared with a conventional biolayer interferometer (BLI) sensor with a SiO2 optical layer. Furthermore, the binding of the antibody anti-estradiol to its antigen estradiol was also tested. Furosemide and estradiol are excellent models for label-free detection of small molecules because of their molecular weights of 330 and 272 Daltons, respectively.

[0123] Material preparation

[0124] Biotin labeling of bovine carbonic anhydrase II (CAII) and human anti-estradiol antibody

[0125] CAII (Sigma-Aldrich), anti-estradiol (US Biological), and NHS-LC-LC-Biotin (ThermoFisher) were used for the biotinylation reaction. No further purification of the material was performed prior to the labeling reaction. CAII and anti-fluorescein antibody were labeled at a 1 molar coupling ratio (MCR). NHS-LC-LC-Biotin was dissolved in anhydrous DMF, immediately added to the corresponding protein, vortexed, and incubated at room temperature for 1 hour. Following the labeling reaction, the biotinylated protein was purified using a PD-10 column (GE Healthcare).

[0126] Crosslinking Preparation

[0127] Preparation of crosslinks The method is described in U.S. Patent No. 8,309,369. Amination is performed with 88 amines containing 20 mg / ml of PBS. 400kD (Skold Technology) 2ml Add 10 μL of SPDP (Invitrogen, 6-[3-[2-pyridyldithio]propionamido]hexanoic acid succinimide ester) at a concentration of 50 mg / ml to DMF in 400 (Sigma / Aldrich). SPDP and The molecular coupling ratio (MCR) was 15. The mixture was allowed to react at room temperature for 1 hour, followed by dialysis. The thiol incorporation was estimated using standard methods to be approximately [value missing]. 400kD 5.5.

[0128] In order to make SPDP marking Thiol deprotection was performed on a 400 column by adding 30 μL of DTT in 1 mL of 1 mL of PBS (38 mg / mL) and allowing the reaction to proceed at room temperature for two hours. The reaction was then carried out on a PD10 column. Purification is performed.

[0129] The following describes the interaction between SMCC and amination. 400 (88 amines / ) In two preparations: 1.) 10 mg of amination solution was added to 1 ml of PBS. 400 was mixed with 25 μL of SMCC at 10 mg / mL DMF, SMCC / The MCR was 30. The mixture was allowed to react at room temperature for two hours, followed by purification on a PD10 column (GE Healthcare). 2.) 10 mg of the amination was added to 1 mL of PBS. 400 was mixed with 12.5 μl of SMCC at 10 mg / mL DMF, SMCC / The MCR was 15. The mixture was reacted at room temperature for 2 hours, and then purified on a PD10 column.

[0130] In order to 400 and 400 crosslinking was performed in two preparations: 1.) 10 mg of [a specific ingredient] was added to 1 mL of PBS. 400 mg in 1 mL PBS 400 Mix (30 MCR). 2.) Add 10 mg of [the drug] to 1 mL of PBS. 400 mg in 1 mL PBS Mix 400g (15MCR). Allow the mixture to react overnight at 30°C.

[0131] In order to provide 400 and 400, two preparations were performed: 1.) 10 mg of [unspecified ingredient] was added to 1 mL of PBS. 400 mg in 1 mL PBS Mix 400, MCR is 30. 2.) Add 10 mg of [the drug] to 1 mL of PBS. 400 mg in 1 mL PBS Mix 400g (15MCR). Allow the mixture to react overnight at 30°C.

[0132] Streptavidin crosslinking Synthesis of conjugates

[0133] 1 mg of SPDP-labeled crosslinked [product name] was dissolved in 38 mg / mL DTT (ThermoFisher, 20290) in PBS. Deprotection was performed at room temperature for 1 hour, with an MCR of 592. 8 mg of streptavidin (SA) (Prozyme, SA10) was labeled with 4-(N-maleimide methyl)cyclohexane-1-carboxylate (SMCC) dissolved in anhydrous DMF for 1 hour at room temperature, with an MCR of 1. Following SMCC labeling or DTT deprotection, streptavidin (SA) was purified using a PD-10 column (GE Healthcare, 17085101) or cross-linked. Purified crosslinking The mixture was combined with SA in a 50 mL tube, and the coupling reaction was carried out overnight at room temperature. The next day, 12 μL of 16 mg / mL N-ethylmaleimide dissolved in PBS was added to the reaction mixture, and the mixture was reacted at room temperature for 30 minutes to cap unreacted cysteine. After the capping reaction, the reaction mixture was purified on a 4B-CL column.

[0134] Cross-linked with streptavidin MgF2 coated probe

[0135] All shaking speeds were 1000 rpm. The MgF2 probe (Example 2B) was first washed with ethanol for 120 seconds. Then, the probe was washed with PBS for 60 seconds, followed by cross-linking with 100 μg / mL streptavidin. Coat for 600 seconds. Then wash twice with PBS for 30 seconds each time, and then coat the probe with 15% sucrose in PBS for 60 seconds as a preservative for long-term storage. Then dry the probe in a 40°C oven for 20 minutes.

[0136] Biotinylated CAII and biotinylated antiestradiol were loaded onto the MgF2 probe.

[0137] Unless otherwise specified, all shaking speeds are 1000 rpm. Equilibrate the probe first in Q buffer for 120 seconds. Next, load biotinylated CAII or biotinylated antiestradiol at 10 μg / mL on a track shaker at 400 rpm for 1800 seconds. Load 1 mM biotin onto the reference probe (the probe without CAII) for subsequent dual-reference experiments. Perform a final wash for 60 seconds.

[0138] Biotinylated CAII and anti-estradiol antibody were loaded onto a streptoantibiotin-SiO2 probe.

[0139] Unless otherwise specified, all shaking speeds are 1000 rpm. First, equilibrate the Octet SA probe (ForteBio, 18-5019) in Q buffer for 120 seconds. Next, load either biotinylated CAII or anti-estradiol antibody at 10 μg / mL at 400 rpm for 1800 seconds. Load 1 mM biotin onto the reference probe for reference experiments. Perform a final wash for 60 seconds.

[0140] Measurement scheme and data processing

[0141] MgF2 probe measurement

[0142] All shaking speeds were 1000 rpm. Assays and data acquisition were performed on a Gator™ instrument (GatorBio) at 30°C. Furosemide (Acros 448970010) was at a concentration of 10 μM, and estradiol (Sigma-Aldrich, 1250008) was at a concentration of 6.4 nM. Before the binding step, the probe loaded with CAII or anti-estradiol antibody was pre-wetted in assay buffer (PBS + 0.05% DMSO) for 600 seconds. Next, a 60-second baseline was established in assay buffer, followed by a 180-second binding step with furosemide or estradiol in PBS containing 0.05% DMSO. In the reference experiment, the biocytin-loaded probe on the second column was exposed to furosemide.

[0143] MgF2 probe data processing

[0144] The estradiol and furosemide binding data were processed using the reference aperture subtraction option in Gator Data Analysis version 1.7.2. The Y-axis was aligned with each baseline and averaged over the last 50 seconds. Savitzky-Golay filtering was applied to remove high-frequency noise from the data. The binding curve data were then calculated and displayed as wavelength offsets in picometers (pm).

[0145] Conventional probe (SiO2) measurement

[0146] All shaking speeds were 1000 rpm. Measurements and data acquisition were performed on an OctetRED instrument (ForteBio) at 30°C. The same measurement protocol as described above for the MgF2 probe was used.

[0147] Conventional probe data processing

[0148] Use the reference subtraction option to process furosemide data in Octet Data Analysis 10.0. In the reference option, furosemide binding signals are obtained by subtracting a reference probe from the active furosemide probe.

[0149] Use the reference probe subtraction option to process estradiol binding data. In this option, the binding signal is obtained by subtracting the reference probe from the active estradiol probe.

[0150] In both cases, the y-axis is aligned with each baseline, with averaging times ranging from 1 to 59 seconds. Savitzky-Golay filtering is applied to remove high-frequency noise from the data. The combined curve data is then calculated and displayed as wavelength offset in picometers (pm).

[0151] Comparison results between MgF2 probe and traditional probe

[0152] Table 6 shows the comparison results of carbonic anhydrase / furosemide binding to MgF2 and conventional SiO2 probes. The binding signal of 10 μM furosemide to CAII on the MgF2 probe was 210.7 pm (picometers), which was 18 times higher than the 11.7 pm on the conventional SiO2 probe.

[0153] Table 6. Results based on combined signals

[0154] Binding signal (pm shift) of 10 μM furosemide conventional probe 11.7 <![CDATA[MgF2 probe]]> 210.7

[0155] Table 7 shows a comparison of the binding results of anti-estradiol / estradiol with MgF2 and conventional SiO2 probes. The conventional SiO2 probe produced a negligible binding signal (2 pm), while the MgF2 probe produced a significant binding signal of 90.9 pm.

[0156] Table 7. Results based on combined signals

[0157] Binding signal (pm shift) of 6.4 nM estradiol conventional probe 2 <![CDATA[MgF2 probe]]> 90.9

Claims

1. An interferometric sensor for detecting an analyte in a sample, the interferometric sensor comprising: A monolithic substrate comprising glass, the monolithic substrate having a first surface and a second surface arranged substantially parallel to each other at opposite ends of the monolithic substrate; An interference layer is coated on the second surface of the monolithic substrate, wherein the interference layer is composed of magnesium fluoride (MgF2) and has a refractive index of 1.38; and An analyte-binding molecular layer coated on the interference layer; The first interface between the monolithic substrate and the interference layer acts as a first reflective surface when light is irradiated onto the interferometric sensor. When light shines on the interferometric sensor, the second interface between the biological layer formed by the combination of analyte molecules and analyte-binding molecules in the sample and the solution containing the sample acts as a second reflective surface. The refractive index of the interference layer is essentially the same as that of the biological layer.

2. The interferometric sensor of claim 1, wherein the monolithic substrate has a length of at least 5 millimeters (mm) as defined from the first surface to the second surface, and wherein the aspect ratio of the monolithic substrate is at least 5:

1.

3. The interferometric sensor according to claim 1, wherein the interferometric layer has a thickness of at least 500 nanometers (nm).

4. The interferometric sensor according to claim 1, further comprising: An adhesion layer comprising silicon dioxide (SiO2) is located between the interference layer and the analyte binding molecular layer.

5. The interferometric sensor according to claim 4, wherein the adhesion layer has a thickness of less than 10 nm.

6. An interferometric sensor, comprising: A monolithic substrate having a first surface and a second surface arranged substantially parallel to each other at opposite ends of the monolithic substrate and having a refractive index greater than 1.5; An interference layer having a refractive index at least 0.1 lower than that of the monolithic substrate, wherein the interference layer is composed of magnesium fluoride (MgF2) with a refractive index of 1.38; and An analyte-binding molecular layer is formed during biochemical testing by analyte molecules in the sample binding to this layer to form a biological layer. The refractive index of the interference layer is essentially the same as that of the biological layer.

7. The interferometric sensor according to claim 6, wherein the thickness of the interferometric layer is between 500 and 5,000 nm.

8. The interferometric sensor according to claim 7, wherein the thickness of the interferometric layer is between 800 and 1,200 nm.

9. The interferometric sensor according to claim 6, wherein the monolithic substrate comprises glass.

10. The interferometric sensor according to claim 6, wherein the refractive index of the monolithic substrate is at least 1.

8.

11. The interferometric sensor according to claim 6, further comprising: The analyte-binding molecular layer is attached to the adhesion layer of the interference layer.

12. The interferometric sensor of claim 11, wherein the adhesive layer comprises silicon dioxide, and wherein the adhesive layer has a thickness of less than 10 nm.

13. The interferometric sensor according to claim 6, wherein the monolithic substrate has a columnar shape.

14. The interferometric sensor according to claim 13, further comprising: The flexible support component is located in the central part of the monolithic substrate. The first portion of the monolithic substrate extends from the top side of the flexible support member, and The second portion of the monolithic substrate extends from the bottom side of the flexible support member.

15. The interferometric sensor according to claim 14, wherein the flexible support component includes a flange and a sleeve located below the flange.

16. The interferometric sensor according to claim 14, wherein the flexible support component comprises silicone rubber.

17. The interferometric sensor of claim 14, wherein the flexible support member is configured to support the interferometric sensor when loaded into a hole including a sample.

18. The interferometric sensor according to claim 6, further comprising: A reflective layer interconnected between the monolithic substrate and the interference layer. The refractive index of the reflective layer is higher than that of the monolithic substrate and also higher than that of the interference layer.

19. A method for manufacturing an interferometric sensor according to claim 1, the method comprising: Obtain a single substrate; Polish the first and second surfaces of the monolithic substrate, the first and second surfaces being arranged substantially parallel to each other at opposite ends of the monolithic substrate; A first transparent material is deposited on the second surface of the monolithic substrate to form an interference layer, wherein the first transparent material is composed of magnesium fluoride (MgF2) with a refractive index of 1.38; and The analyte-binding molecules are coated onto the interference layer. The refractive index of the interference layer is basically the same as that of the biological layer.

20. The method of claim 19, wherein the monolithic substrate comprises glass.

21. The method of claim 19, wherein the interference layer has a thickness of at least 900 nm.

22. The method of claim 19, further comprising: A second transparent material is deposited on the interference layer to form an adhesion layer. The analyte binds to the molecular layer and the adhesive layer.

23. The method of claim 22, wherein the second transparent material is silicon dioxide.

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