Interferometric sensors for biochemical testing.

Abstract

To provide an interference sensor used for a biochemical test, in which the performance of the interferometer is improved in contrast to the conventional biosensors of thin-film interferometers that provide functionality.SOLUTION: An interference sensor includes an interference layer fixed along the surface of a monolithic substrate. Analyte-binding molecules can be coated along the surface of the interference layer. In the process of a biochemical test, a biolayer is formed as an analyte molecule in a sample bound to the analyte-binding molecules. The refractive index of the monolithic substrate is higher than the refractive index of the interference layer. The refractive index of the interference layer can be designed so as to be substantially the same as the refractive index of the biolayer.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] Various embodiments relate to interferometric sensors to which analyte molecules in a sample can bind over the course of a biochemical test. [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 diagnostic tests can be used to detect whether an analyte molecule is present in a sample, the amount of analyte molecules in a sample, or the rate at which an analyte molecule binds to an analyte-binding molecule. Together, an analyte-binding molecule and its corresponding analyte molecule form an analyte-antianalyte binding pair (or simply a "binding pair"). Examples of binding pairs include complementary strands of nucleic acid, antigen-antibody pairs, and receptor-receptor binding agents. The analyte can be either member of the binding pair, and the anti-analyte can be the other member of the binding pair.

[0003] Historically, diagnostic tests have used solid, flat surfaces with immobilized analyte-binding molecules. Analyte molecules in a sample bind to these analyte-binding molecules with high affinity within a defined detection zone. In this type of assay, known as a "solid-phase assay," the solid surface is exposed to the sample under conditions that promote binding of the analyte molecules to the analyte-binding molecules. Generally, the binding event is detected directly by measuring a change in mass, reflectance, thickness, color, or another property indicative of the binding event. For example, if the analyte molecule is labeled with a chromophore, fluorescent label, or radioactive label, the binding event can be detected based on the amount of label (if present) that can be detected within the detection zone. Alternatively, the analyte molecule can be labeled after binding to the analyte-binding molecule within the detection zone.

[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., capture molecule) coated directly on its distal end to which the substance binds. The distal end is then immersed in a sample containing the analyte. Binding of the analyte to the reagent layer produces an interference pattern that 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 between them, and a second optical element, acting as an interference layer having a thickness greater than 50 nanometers (nm), is then attached to the distal end of the first element. The biolayer is formed on the peripheral surface of the second optical element. An additional reflective surface layer, 5-50 nm thick and with a refractive index greater than 1.8, is coated between the interference layer and the first element. The principle of detecting an analyte in a sample based on changes in spectral interference is described in this reference, which is incorporated herein by reference.

[0006] U.S. Patent No. 7,319,525 discloses a different configuration in which a section of optical fiber is mechanically attached to a distal connector consisting of one or more optical fibers with a gap between the proximal end of the optical fiber section and the distal connector. An interference layer and then a biolayer are built on the distal surface of the optical fiber section.

[0007] Although the prior art provides functionality in the application of biosensors based on thin film interferometers, there is a need for improvement in the performance of these interferometers. [Brief explanation of the drawings]

[0008] [Figure 1A] 1 shows a biosensor interferometer including a light source, a detector, a waveguide, and an optical assembly (also called a "probe"). [Figure 1B] 1 shows an example of a conventional probe. [Figure 2]1 illustrates an example of a probe according to various embodiments. [Figure 3] 1 illustrates another example of a probe according to various embodiments. [Figure 4A] 1 shows the detection principle in a thin film interferometer. [Figure 4B] 1 shows the detection principle in a thin film interferometer. [Figure 5] 1 shows examples of slides according to various embodiments. [Figure 6] 1 illustrates another example of a slide according to various embodiments. [Figure 7] 1 shows a flow diagram of a process for manufacturing a probe. [Figure 8A] 1 includes a side perspective view of a probe, according to various embodiments. [Figure 8B] 1A-1D include bottom perspective views according to various embodiments. [Figure 8C] 1A-1D include top perspective views according to various embodiments. [Figure 9] Figure 1 shows the binding curves (with shifts in nm) of protein A in the APS probe with a conventional probe assigned to channels (CH) 1-4 (i.e., the bottom four curves) and an MgF2 probe assigned to CH5-8 (i.e., the top four curves). [Figure 10] Binding curves of human IgG on Protein A probes with conventional probes assigned to CH1-4 (i.e., the bottom four curves) and MgF2 probes assigned to CH5-8 (i.e., the bottom four curves) are shown.

[0009] Various features of the present technology will become apparent to those skilled in the art from a study of the detailed description in conjunction with the drawings. The drawings illustrate, for illustrative purposes only, various embodiments described throughout the detailed description. While specific embodiments are shown by way of example, the present technology is susceptible to various modifications and alternative forms. The intention is not to limit the technology to the particular embodiments that have been illustrated and / or described. DETAILED DESCRIPTION OF THE INVENTION

[0010] Several entities have developed systems designed to perform biochemical tests. Figures 1A and 1B show one example of such a system. In particular, Figure 1A shows a biosensor interferometer 100 (or simply "interferometer") that includes a light source 102, a detector 104, a waveguide 106, and an optical assembly 108 (also called a "probe"). The probe 108 can be connected to the waveguide 106 via a coupling medium.

[0011] The light source 102 may emit light that is guided by the waveguide 106 toward the probe 108. For example, the light source 102 may be a light-emitting diode (LED) configured to generate light over a range of at least 50 nanometers (nm), 100 nm, or 150 nm within a given spectrum (e.g., below 400 nm to above 700 nm). Alternatively, the interferometer 100 may use multiple light sources with different characteristic wavelengths, such as LEDs designed to emit light of different wavelengths within the visible range. The same function can be achieved by a single light source with appropriate filters to direct light with different wavelengths onto the probe 108.

[0012] Detector 104 is preferably a spectrometer, such as an Ocean Optics USB4000, that can record the spectrum of the interference light received from probe 108. Alternatively, if light source 102 operates to direct different wavelengths onto probe 108, detector 104 can be a simple photodetector that can record the intensity at each wavelength. In another embodiment, detector 104 can include multiple filters that allow for detection of the intensity at each of multiple wavelengths.

[0013] Waveguide 106 may be configured to carry light emitted by light source 102 to probe 108 and then carry light reflected by surfaces within probe 108 to detector 104. In some embodiments, waveguide 106 is a bundle of optical fibers (e.g., single-mode fiber optic cable), while in other embodiments, waveguide 106 is a multi-mode fiber optic cable.

[0014] As shown in FIG. 1B , the probe 108 includes a monolithic substrate 114, a thin film layer (also referred to as an "interference layer"), and a biomolecule layer (also referred to as a "biolayer") including analyte molecules 122 bound to analyte-binding molecules 120. The monolithic substrate 114 includes a transparent material through which light can pass. The interference layer also includes a transparent material. When light is irradiated onto the probe 108, the proximal surface of the interference layer can act as a first reflective surface, and the biolayer can act as a second reflective surface. As described further below, the light reflected by the first and second reflective surfaces can form an interference pattern that can be monitored by the interferometer 100.

[0015] The interference layer typically includes multiple layers combined in such a manner as to improve the detectability of the interference pattern. Here, for example, the interference layer includes a tantalum pentoxide (TaO) layer 116 and a silicon dioxide (SiO) layer 118. The tantalum pentoxide layer 116 may be thin (e.g., about 10-40 nm) because its primary purpose is to improve the reflectivity at the proximal surface of the interference layer. Meanwhile, the silicon dioxide layer 118 may be relatively thick (e.g., about 650-900 nm) because its primary purpose is to increase the distance between the first and second reflecting surfaces.

[0016] To perform a diagnostic test, the probe 108 can be suspended in a microwell 110 (or simply a "well") containing a sample 112. Analyte molecules 122 within the sample 112 bind to analyte-binding molecules 120 along the distal end of the probe 108 during the course of the diagnostic test, and these binding events result in an interference pattern that can be observed by the detector 104. The interferometer 100 can monitor the thickness of the biolayer formed along the distal end of the probe 108 by detecting a shift in the phase characteristics of the interference pattern.

[0017] However, such a design has several disadvantages. One drawback is the low signal intensity observed during biochemical tests involving these probes. Another drawback is the negative shift in the binding curve that can occur when the biolayer is grown for extended periods of time (e.g., several tens of cycles over 20-40 minutes).

[0018] Presented herein is an interferometric sensor (also referred to as an "interferometric biosensor" or "sensing device") that addresses these shortcomings. Specifically, the interferometric sensor can include a monolithic substrate having first and second surfaces disposed substantially parallel to each other at opposite ends of the monolithic substrate, an interference layer coated on the second surface of the monolithic substrate, and a layer of analyte-binding molecules coated on the interference layer. The interference layer can include magnesium fluoride (MgF2). A first interface between the monolithic substrate and the interference layer serves as a first reflective surface when light is irradiated onto the interferometric sensor, while a second interface between a biolayer formed by analyte molecules in the sample that bind to the analyte-binding molecules and a solution containing the sample serves as a second reflective surface when light is irradiated onto the probe. As described above, the thickness of the biolayer can be estimated based on the interference pattern of light reflected by the first and second reflective surfaces.

[0019] Interferometric sensor embodiments may be described in the context of a probe designed to be suspended in a solution containing a sample for illustrative purposes, however, those skilled in the art will recognize that these features are equally applicable to other sensing surfaces, such as planar surfaces (e.g., slides) on which a biolayer is formed by flowing a solution over the planar surface over the course of a biochemical test.

[0020] definition The term "about" means within ±10% of the stated value.

[0021] The term "analyte-binding molecule" refers to any molecule that can participate 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.

[0022] The term "interferometric sensor" refers to any sensing device on which a biolayer forms 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 of an interferometric sensor is a slide with a planar surface on which a biolayer can form over the course of a biochemical test.

[0023] The term "probe" refers to a monolithic substrate having an aspect ratio (length to width) of at least 2 to 1 with a thin film layer coated on the sensing side.

[0024] The term "monolithic substrate" refers to a solid piece of material having a uniform composition, such as glass, quartz, or plastic, with one refractive index.

[0025] The term "waveguide" refers to a device (e.g., a duct, coaxial cable, or optical fiber) designed to confine and direct the propagation of electromagnetic waves (as light). One example of a waveguide is a metal tube for channeling ultra-high frequency waves.

[0026] Probe Overview 2 shows an example of a probe 200 according to various embodiments. The probe 200 includes an interference layer 204 immobilized along the distal end of a monolithic substrate 202. Analyte binding molecules 206 may be deposited along the distal surface of the interference layer 204. During the course of a biochemical test, a biolayer will form as analyte molecules 208 in the sample bind to the analyte binding molecules 206.

[0027] As shown in FIG. 2 , the monolithic substrate 202 has a proximal surface (also referred to as the “coupling side”) that can be coupled to, for example, a waveguide of an interferometer, and a distal surface (also referred to as the “sensing side”) onto which additional layers are deposited. Typically, the monolithic substrate 202 has a length of at least 3 millimeters (mm), 5 mm, 10 mm, or 15 mm. In preferred embodiments, the aspect ratio (length to width) of the monolithic substrate 202 is at least 5:1. In such embodiments, the monolithic substrate 202 may be said to have a columnar morphology. The cross-section of the monolithic substrate 202 may be circular, elliptical, square, rectangular, triangular, pentagonal, etc. The monolithic substrate 202 preferably has a refractive index substantially higher than that of the interference layer 204, such that the proximal surface of the interference layer 204 effectively reflects light directed onto the probe 200. The preferred refractive index of the monolithic substrate may be greater than 1.5, 1.8, or 2.0. Thus, the monolithic substrate 202 may comprise a high refractive index material such as glass (refractive index of 2.0), while some embodiments of the monolithic substrate 202 may comprise a low refractive index material such as quartz (refractive index of 1.46) or plastic (refractive index of 1.32-1.49). Examples of transparent plastics include polypropylene, polyurethane, acrylic, polycarbonate, etc.

[0028] The interference layer 204 includes at least one transparent material coated on the distal surface of the monolithic substrate 202. These transparent materials are deposited on the distal surface of the monolithic substrate 202 in the form of a thin film ranging in thickness from a fraction of a nanometer (e.g., a monolayer) to several micrometers. The interference layer 204 may have a thickness of at least 500 nm, 700 nm, or 900 nm. Exemplary thicknesses are 500 to 5,000 nm (and preferably 800 to 1,200 nm). Herein, for example, the interference layer 204 has a thickness of approximately 900 to 1,000 nm, or 940 nm.

[0029] In contrast to conventional probes, the interference layer 204 has a refractive index substantially similar to that of the biolayer. 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 interference layer 204 and the analyte-binding molecules 206. Typically, the biolayer 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.

[0030] In some embodiments, the interference layer 204 includes magnesium fluoride (MgF), while in other embodiments, the interference layer 204 includes potassium fluoride (KF) having a refractive index of 1.36, lithium fluoride (LiF) having a refractive index of 1.39, sodium fluoride (NaF) having a refractive index of 1.32, lithium calcium aluminum fluoride (LiCaAlF) having a refractive index of 1.39, strontium fluoride (SrF) having a refractive index of 1.37, aluminum fluoride (AlF) having a refractive index of 1.38, sodium aluminum hexafluoride (NaAlF) (also known as "frozen stone") having a refractive index of 1.34, sodium aluminum fluoride (NaAlF) having a refractive index of 1.34, 14 ) (also known as "chiolite"). Additionally or alternatively, the interference layer 204 may include a polymer with a refractive index less than 1.4, such as FICOLL® (a copolymer of sucrose and epichlorohydrin). Magnesium fluoride has a refractive index of 1.38, which is substantially identical to the refractive index of the biolayer formed along the distal end of the probe 200. For comparison, the interference layer of conventional probes typically includes silicon dioxide, with the refractive index of pure silicon dioxide being approximately 1.46. Less pure forms of silicon dioxide have higher refractive indices (e.g., approximately 1.5 in the visible range). Typically, the refractive index of the interference layer 204 is 1.32-1.42, 1.36-1.42, or 1.38-1.40. Because the interference layer 204 and the biolayer have similar refractive indices, light will experience minimal scattering when traveling from the interference layer 204 into the biolayer and then back from the biolayer into the interference layer 204.

[0031] The thickness of the biolayer is designed to optimize overall sensitivity based on the interferometer hardware (e.g., optical components). Conventional immobilization chemistries can be used to attach the analyte-binding molecules 206 covalently (e.g., chemically) or non-covalently (e.g., by adsorption) to the distal surface of the interference layer 204.

[0032] The layer of analyte-binding molecules 206 is preferably formed under conditions in which the distal end of probe 200 is densely coated, such that binding of analyte molecules 208 to analyte-binding molecules 206 results in a change in the thickness of the biolayer rather than filling the layer. The layer of analyte-binding molecules 206 can be a monolayer or a multilayer matrix.

[0033] During a biochemical test, the probe 200 may be suspended within a cavity (e.g., a well) containing a sample. An example of a probe-based detection technique 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 the course of a biochemical test, a biolayer forms along the distal end of the probe 200 as analyte molecules 208 bind to the analyte-binding molecules 206.

[0034] When light is shone onto the probe 200, the proximal surface of the interference layer 204 can act as a first reflecting surface, and the distal surface of the biolayer can act as a second reflecting surface. The presence, concentration, or binding rate of analyte molecules 208 to the probe 200 can be estimated based on the interference of the light beams reflected by these two reflecting surfaces. As analyte molecules 208 attach to (or detach from) analyte-binding molecules 206, the distance between the first and second reflecting surfaces changes. Because the dimensions of all other components within the probe 200 remain the same, the interference patterns formed by the light reflected by the first and second reflecting surfaces are out of phase with respect to changes in biolayer thickness due to binding events.

[0035] 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 the refractive index of the interference layer 204. For example, the refractive index of the monolithic substrate 202 may be at least 0.1, 0.2, 0.4, 0.5, or 0.6 higher than the refractive index of the interference layer 204. Because the monolithic substrate 202 is a solid piece of material with a uniform composition, it is easy to select a material with a higher refractive index than the interference layer 204. In contrast, optical fibers are typically circular cross-section dielectric waveguides having a dielectric material (also called a "core material") surrounded by another dielectric material (also called a "cladding") with a lower refractive index, making it difficult to manipulate its refractive index.

[0036] During operation, an incident light signal 210 emitted by a light source is transmitted through the monolithic substrate 202 toward the biolayer. Within the probe 200, the light is reflected off a first reflective surface, resulting in a first reflected light signal 212. The light is also reflected off a second reflective surface, resulting in a second reflected light signal 214. The second reflective surface initially corresponds to the interface between the analyte binding molecules 206 and the sample in which the probe 200 is immersed. When binding occurs during a biochemical test, the second reflective surface becomes the interface between the analyte molecules 208 and the sample.

[0037] The first and second reflected optical signals 212, 214 form a spectral interference pattern, as shown in FIG. 4A. When an analyte molecule 208 binds to an analyte-binding molecule 206 on the distal surface of the interference layer 204, the optical path of the second reflected optical signal 214 lengthens. As a result, the spectral interference pattern shifts from T0 to T1, as shown in FIG. 4B. By continuously measuring the phase shift in real time, a kinetic binding curve can be plotted as the shift versus time. The association rate of the analyte molecule to the analyte-binding molecule immobilized on the distal surface of the interference layer 204 can be used to calculate the analyte concentration in the sample. Thus, measuring the phase shift is the detection principle of thin-film interferometry.

[0038] 4A, the performance of a thin film interferometer can be improved by maximizing the alternating current (AC) component and minimizing the direct current (DC) offset. In other words, since the AC component represents the signal of interest and the DC offset represents noise, increasing the AC-to-DC ratio can improve the performance of a thin film interferometer. To achieve these goals, (1) the efficiency with which the incident optical signal 210 and the reflected optical signals 212, 214 travel through the probe 200 can be increased, (2) the coupling efficiency between the light source and the probe 200 can be increased, and / or (3) the coupling efficiency between the spectrometer and the probe 200 can be increased.

[0039] Substantially matching the refractive index of the interference layer 204 and the biolayer achieves the first of these goals, i.e., by reducing reflections from other surfaces within the probe 200 as much as possible (e.g., the interface between the interference layer 204 and the analyte-binding molecules 206). When the refractive index of the interference layer 204 approaches that of the biolayer (e.g., 1.38 for the interference layer 204 and 1.36 for the biolayer), the shift in the spectral interference pattern increases as the biolayer builds. This is because the delta between T0 and T1 is based on the difference in refractive index between the interference layer 204 and the surrounding material (e.g., the sample). However, as the refractive index of the interference layer 204 decreases, the magnitudes of T0 and T1 also decrease. There is a trade-off between magnitude and shift in the spectral interference pattern. At a high level, the goal is to have a magnitude large enough to distinguish the two peaks while separating them as much as possible. As an example, lowering the refractive index of the interference layer 204 results in more shift, but a smaller AC to DC ratio (i.e., a larger DC component and / or a smaller AC component resulting in a "noisier" signal).

[0040] 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 positioned between the monolithic substrate 202 and the interference layer 204. The reflective layer may comprise a material with a higher refractive index than either the monolithic substrate 202 or the interference layer 204, as its primary purpose is to ensure that the first reflected optical signal 212 reflects at the interface between the monolithic substrate 202 and the interference layer 204. For example, the reflective layer may be made of zinc sulfide (ZnS) having a refractive index of 2.3 to 2.4, titanium dioxide (TiO2) having a refractive index of 2.3 to 2.4, titanium monoxide (TiO) having a refractive index of 2.2 to 2.3, titanium trioxide (Ti2O3) having a refractive index of 1.9 to 2.3, titanium oxide (Ti3O5) having a refractive index of 2.2 to 2.3, tantalum oxide (Ta2O3) having a refractive index of 216, tantalum pentoxide (Ti3O5) having a refractive index of 2.16, silicon monoxide (SiO) having a refractive index of 1.8 to 1.9, aluminum sesquioxide (Al2O3) having a refractive index of 1.67, or titanium monoxide (TiO) having a refractive index of 1.97 to 2.05. The reflective layer may include zirconium dioxide (ZrO2) having a refractive index of 2.01, zinc monoxide (ZnO) having a refractive index of 2.01, lanthanum titanium trioxide (LaTiO3) having a refractive index of 2.1, indium tin oxide (ITO) having a refractive index of 1.8, niobium pentoxide (Nb2O5) having a refractive index of 2.1 to 2.3, zinc selenide (ZnSe) having a refractive index of 2.58, cerium dioxide (CeO2) having a refractive index of 2.35, yttrium oxide (YO3) having a refractive index of 1.87, hafnium oxide (HfO2) having a refractive index of 1.95, or gadolinium oxide (Gd2O3) having a refractive index of 1.8. The reflective layer may be very thin compared to the interference layer 204. For example, the reflective layer may have a thickness of about 3 to 10 nm.

[0041] FIG. 3 illustrates another example of a probe 300 according to various embodiments. The probe 300 of FIG. 3 may be substantially similar to the probe 200 of FIG. 2. However, as used herein, the probe 300 includes an adhesion layer 310 deposited along the distal surface of an interference layer 304 immobilized on a monolithic substrate 302. The adhesion layer 310 may include a material that promotes adhesion of the analyte-binding molecules 306. One example of such a material is silicon dioxide. The adhesion layer 310 is generally very thin compared to the interference layer 304, and therefore has minimal effect on light traveling toward or returning from the biolayer. For example, the adhesion layer 310 may have a thickness of approximately 3-10 nm, while the interference layer 304 may have a thickness of approximately 800-1,000 nm. The biolayer formed by the analyte-binding molecules 306 and the analyte molecules 308 typically has a thickness of a few nanometers. 2, the probe 300 of FIG. 3 may also have a reflective layer (not shown) deposited along the distal end of the monolithic substrate 302 such that the reflective layer is positioned 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 adhesive layer 310.

[0042] As noted above, these features are equally applicable to sensing surfaces having other forms. One example of such a sensing surface is a slide (also called a "chip") having a planar surface on which a biolayer is formed by flowing a solution over the planar surface during a biochemical test. Some examples of planar surfaces are discussed below with reference to Figures 5 and 6.

[0043] FIG. 5 shows an example of a slide 500 according to various embodiments. The 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 top surface of the substrate 502, while in other embodiments, the interference layer 504 is deposited along a portion of the top surface of the substrate 502. For example, the interference layer 504 may be deposited within a channel or well formed in the top surface of the substrate 502. As discussed above, the monolithic substrates 202, 302 of FIGS. 2 and 3 are generally much taller than they are wide. However, the reverse may also be true. In fact, the width of the substrate 502 may be 5, 7.5, 10, or 20 times greater than its length. As an example, the substrate may be approximately 75 x 26 mm with a height / thickness of approximately 1 mm.

[0044] During a diagnostic test, analyte molecules 508 can bind to analyte-binding molecules 506 immobilized along the top surface of the interference layer 504, forming a biolayer. To establish the thickness of the biolayer, light can be shone onto the top surface of the slide 500, as shown in FIG. 5. More specifically, an incident light signal 510 emitted by a light source can be seen in the biolayer formed along the top surface of the slide 500. This can require the incident light signal 510 to travel through a surrounding medium 516, which can be a vacuum, air, or a solution. The incident light signal 510 is reflected off a first reflective surface, resulting in a first reflected light signal 512. The first reflective surface can represent the interface between the biolayer and the surrounding medium 516. The incident light signal 510 is also reflected off a second reflective surface, resulting in 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 optical signals 512, 514 form a spectral interference pattern that can be analyzed to establish the thickness of the biolayer. Note that because the incident optical signal 510 is not transmitted through the substrate 502, the substrate 502 may or may not be transparent (e.g., it may be opaque).

[0045] Figure 6 shows another example of a slide 600 according to various embodiments. Slide 600 of Figure 6 may be substantially similar to slide 500 of Figure 5. Accordingly, slide 600 may include a substrate 602 on which an interference layer 604 and analyte binding molecules 606 are deposited. During the course of a diagnostic test, analyte molecules 608 may bind to analyte binding molecules 606 to form a biolayer.

[0046] Here, however, an incident light signal 610 is shown on the underside of the slide 600. In operation, the incident light signal 610 is transmitted through the substrate 602 toward the biolayer. Within the slide 600, the light is reflected off a first reflective surface, resulting in 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 off a second reflective surface, resulting in a second reflected light signal 614. The second reflective surface may represent the interface between the biolayer and the surrounding medium 616. As described above, the first and second reflected light signals 612, 614 form a spectral interference pattern that can be analyzed to establish the thickness of the biolayer.

[0047] Although not shown in Figures 5 and 6, the slides 500, 600 may include a reflective layer disposed between the substrate 502, 602 and the interference layer 504, 604 to improve reflectivity along that interface, and / or an adhesive layer disposed along the top surface of the interference layer 504, 604 to immobilize the analyte binding molecules 506, 606.

[0048] FIG. 7 shows a flow diagram of a process 700 for manufacturing an interferometric sensor. First, a manufacturer acquires a monolithic substrate (step 701). For example, the manufacturer may select a monolithic substrate from among multiple monolithic substrates designed for different biochemical tests, analyte-binding molecules, etc. The preferred refractive index of the monolithic substrate may be greater than 1.5, 1.8, or 2.0. Thus, the monolithic substrate acquired by the manufacturer may include a high-refractive index material, such as glass (refractive index of 2.0), or a low-refractive index material, such as quartz (refractive index of 1.46) or plastic (refractive index of 1.32-1.49). As mentioned above, in some embodiments, the monolithic substrate has a columnar morphology (e.g., monolithic substrates 202, 302 in FIGS. 2 and 3), while in other embodiments, the monolithic substrate has a planar morphology (e.g., monolithic substrates 502, 602 in FIGS. 5 and 6).

[0049] The manufacturer can then deposit a transparent material onto the surface of the monolithic substrate to form an interference layer (step 702). For example, the transparent material may be deposited onto the distal surface of the monolithic substrate in the form of a thin film ranging in thickness from a fraction of a nanometer (e.g., a monolayer) to several micrometers. Typically, the interference layer has a thickness of at least 500 nm, 700 nm, or 900 nm. Exemplary thicknesses are 500 to 5,000 nm (and preferably 800 to 1,200 nm).

[0050] In some embodiments, the manufacturer deposits another transparent material onto the surface of the interference layer to form an adhesion layer (step 703). The adhesion layer may include a material that promotes adhesion of analyte-binding molecules. One example of such a material is silicon dioxide. The adhesion layer is generally very thin compared to the interference layer, so its effect on light traveling along the interferometric sensor is minimal. For example, the adhesion layer may have a thickness of approximately 3-10 nm.

[0051] The manufacturer can then immobilize analyte-binding molecules on the surface of the adhesive layer (step 704). As described above, the layer of analyte-binding molecules can be formed under conditions in which 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 as analyte molecules bind to the analyte-binding molecules during the course of a biochemical test, these binding events result in a change in the thickness of the biolayer rather than filling up the layer of analyte-binding molecules. The layer of analyte-binding molecules can be a monolayer or a multilayer matrix.

[0052] It is contemplated that the above steps may be performed in various orders and combinations, provided that this is not contrary to physical feasibility. For example, a manufacturer may choose not to create an adhesive layer along the distal surface of the interference layer. In such an embodiment, the analyte-binding molecule may be immobilized directly on the distal surface of the interference layer.

[0053] Additional steps may also be performed. For example, a manufacturer may form a reflective layer on the surface of the monolithic substrate. As described above, the reflective layer may include 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 on the surface of the monolithic substrate before the interference layer is formed (i.e., before step 702 is performed). As another example, a manufacturer may cure the interference layer (e.g., using heat, air, radiation, etc.) before forming the adhesive layer. Similarly, a manufacturer may (i) cure the reflective layer before fixing the adhesive layer to the reflective layer, and / or (ii) cure the adhesive layer before fixing the analyte binding molecule to the adhesive layer. As another example, a manufacturer may polish first and second surfaces of the monolithic substrate, which are disposed substantially parallel to each other at opposite ends of the monolithic substrate. Polishing may be performed to improve adhesion of the interference layer to the monolithic substrate.

[0054] FIG. 8A includes a side view of a probe 800 according to various embodiments. FIG. 8B includes a bottom perspective view of the probe 800, while FIG. 8C includes a top perspective view of the probe 800. The probe 800 includes a rod section 802 (also referred to as a "rod component") and a flexible support component 804 (also referred to as a "flexible skirt"). The flexible support component 804 may be centrally located along the length of the rod section 802 such that a first portion of the rod section 802 extends from an upper side of the flexible support component 804 and a second portion of the rod section 802 extends from a lower side of the flexible support component 804. Thus, the flexible support component 804 may be located in a central portion of the rod section 802.

[0055] Rod section 802 may be a monolithic substrate, such as monolithic substrate 202 of FIG. 2. Rod section 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 rod section 802 may be different sizes. For example, the first portion of rod section 802 extending from the upper side of flexible support component 804 may be 2-5 mm, while the second portion of rod section 802 extending from the lower side of flexible support component 804 may be 5-10 mm.

[0056] Flexible support component 804 can include a flange section 806 and a sleeve section 808. In some embodiments, flange section 806 and sleeve section 808 are coupled to one another after each component is manufactured. In other embodiments, flange section 806 and sleeve section 808 are part of a single component formed via a molding process, an extrusion process, or the like. Flexible support component 804 can partially or entirely comprise silicone rubber, nitrile rubber, or some other elastomer. For example, in some embodiments, the entire flexible support component 804 comprises a flexible material, while in other embodiments, only flange section 806 comprises a flexible material.

[0057] 8B, the underside of the flexible support component 804 may include a recess 810 defined by an inner concave surface 816. An inner extension feature 818 located within the recess 810 may be secured around the rod section 802. In embodiments including the inner extension feature 818, the recess 810 may take the form of an annular recess extending radially around the rod section 802.

[0058] As described above, the distal end 812 (also referred to as the "bottom end") of the rod section 802 may have an interference layer immobilized thereon, and analyte-binding molecules may be coated on the interference layer. During the course of a biochemical test, a biolayer will form as analyte molecules in the sample bind to the analyte-binding molecules. When light is irradiated onto the proximal end 814 of the probe 800, the proximal surface of the interference layer may act as a first reflective surface, and the biolayer may act as a second reflective surface.

[0059] When probe 800 is loaded into a well, pressure is exerted by the top surface of the well against the bottom surface of flange section 806 of flexible support component 804. Such pressure suspends distal end 812 of rod section 802 within the well. Flange section 806 may be designed to prevent distal end 812 of rod section 802 from contacting the interior surface of the well when loaded into the well. The well may be contained in a cartridge containing multiple wells arranged in a linear format or in a microplate containing multiple wells arranged in a grid format.

[0060] remarks The foregoing description of various embodiments of the present technology has been provided for purposes of illustration and description and is not intended to be exhaustive or to limit the claimed subject matter to the precise form disclosed.

[0061] Many modifications and variations will be apparent to those skilled in the art. The embodiments have been chosen and described in order to best explain the principles of the technology and its practical applications, so that those skilled in the art can understand the claimed subject matter, various embodiments, and various modifications that are suited to the particular applications contemplated. [Example]

[0062] The present invention is further illustrated by the following examples which should not be construed as limiting the invention in scope to the specific procedures described therein.

[0063] Example 1. Preparation of a conventional probe (SiO2 probe) A conventional probe is shown in Figure 1B. Both ends of 20 mm long, 1 mm diameter quartz (refractive index 1.46) rods were polished to a mirror finish using an optical polisher. After cleaning and rinsing the rods in purified water, they were aligned in a fixture and then loaded into an ion-beam-assisted physical vapor deposition (PVD) machine. In a PVD machine, an electron beam is used to bombard the target material to be coated on the surface, vaporizing the target material. An ion beam is then applied to deposit the vapor on the surface, forming a thin film layer. These quartz rods were first coated with a 20 nm Ta2O5 layer, followed by a 730 nm SiO2 layer. After coating the surface with the Ta2O5 / SiO2 layer, the rods were placed in a chemical vapor deposition (CVD) machine (e.g., Lab Kote, manufactured by Yield Engineering) and coated with a thin layer of aminopropyltriethoxysilane (APS). The APS layer is typically 1–2 nm thick. APS is deposited to enable protein immobilization. APS adsorbs proteins to the surface of the probe through a combination of hydrophobic and ionic interactions. Proteins can also be covalently linked to the amino groups of APS using cross-linking reagents. APS can be monolayer-only, therefore, approximately 7 nm thick.

[0064] Example 2A. Preparation of a probe according to one embodiment of the present invention (FIG. 3) An example of a probe of the present invention is shown in Figure 3. The probe may be referred to as an "MgF2 probe" or "MgF2-APS probe." Both ends of a 20 mm long, 1 mm diameter glass (refractive index 2.0) rod were polished to a mirror finish using an optical polisher. After cleaning and rinsing the rods in purified water, they were aligned in a fixture and then loaded into an ion-beam-assisted physical vapor deposition (PVD) machine. In a PVD machine, an electron beam is used to bombard a target material to be coated on a surface, vaporizing the target material. An ion beam is then applied to deposit the vapor on the surface, forming a thin film layer. The glass rod was 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 was placed in a chemical vapor deposition (CVD) machine and coated with a thin layer of APS, typically 1-2 nm thick.

[0065] Example 2B. Preparation of a probe according to another embodiment of the present invention (FIG. 2) Another example of a probe of the present invention is shown in Figure 2. The probe may be referred to as an "MgF2 probe" or "MgF2-APS probe." Both ends of a 20 mm long, 1 mm diameter glass (refractive index 2.0) rod were polished to a mirror finish using an optical polisher. After cleaning and rinsing the rods in purified water, they were aligned in a fixture and then loaded into an ion-beam-assisted physical vapor deposition (PVD) machine. In a PVD machine, an electron beam is used to bombard a target material to be coated on a surface, vaporizing the target material. An ion beam is then applied to deposit the vapor on the surface, forming a thin film layer. The glass rod was coated with a single layer of 940 nm of MgF2 without a thin layer of SiO2. After coating the surface with the MgF2 layer, the rod was placed in a chemical vapor deposition (CVD) machine and coated with a thin layer of APS, typically 1-2 nm thick.

[0066] Example 3. Comparison of Protein A binding between MgF2 probe and conventional probe For a side-by-side comparative study, the conventional APS probe (Example 1) and the MgF2-APS probe (Example 2A) were immobilized with protein A for binding studies.

[0067] As shown in Table 1, two types of probes were subjected to three steps in a 96-well plate. [Table 1]

[0068] Experiments were performed using a ProbeLife Gator interferometer instrument and software version 1.3. The results are shown in Figure 9 and summarized in Table 2. Figure 9 shows the binding curves (with shifts in nm) of protein A on the APS probe with a conventional probe assigned to channels (CH) 1-4 (i.e., the bottom four curves) and an MgF2 probe assigned to CHs 5-8 (i.e., the top four curves). [Table 2]

[0069] The results show that the MgF2-APS probe obtained 2.24 times more binding signal (nm shift) compared to the conventional probe, as indicated by the upper limit of wavelength shift in nm.

[0070] Example 4. Comparison of IgG / Protein A binding of MgF2 probe and conventional probe Because Protein A has five Ig-binding domains and binds to heavy chains within the Fc region and, in the case of the human VH3 family, also within the Fab region, we were able to repeatedly immobilize human IgG (Equitech-Bio SLH56) and Protein A on the probe surface to test the upper limit of the nm shift.

[0071] Two APS probes (a conventional probe, Example 1, and an MgF2 probe, Example 2A) were cycled through steps 1-4 50 times in a 96-well plate. 1. K buffer (PBS, 0.02% BSA, 0.002% Tween-20, 200 μL) at 1000 rpm for 10 seconds 2. 2 μg / mL human whole IgG in K buffer (200 μL) was mixed at 1000 rpm for 60 seconds. 3. Add 200 μL of K buffer solution and mix at 1000 rpm for 10 seconds. 4. 10 μg / mL of protein A in K buffer (200 μL) was added to the centrifuge tube at 1000 rpm for 60 seconds.

[0072] The experiments were performed using a ProbeLife Gator instrument and software version 1.3. The results are shown in Figure 10. In particular, Figure 10 shows the binding curves of human IgG to a Protein A probe with a conventional probe assigned to CH1-4 (i.e., the bottom four curves) and an MgF2 probe assigned to CH5-8 (i.e., the top four curves).

[0073] The results in Figure 10 show that the MgF2 probe reached a wavelength shift of 120 nm without turning negative, whereas the conventional probe only reached a wavelength shift of 7 nm before showing a negative nm shift. The signal (nm shift) was much higher for the MgF2 probe than for the conventional probe.

[0074] Example 5. Comparison of protein binding and regeneration of MgF2 probes and conventional probes Preparation of anti-mouse Fc-coated probes Streptavidin-coated probes were made by immersing two APS probes (Examples 1 and 2A) in 50 μg / mL streptavidin (Invitrogen, 21122) in PBS buffer in a 96-well plate for 10 minutes at 1000 rpm.

[0075] Affinity-purified goat anti-mouse IgG Fc-γ fragment specific (Jackson-Immuno, 115-005-071) was used in the experiments. This anti-mouse Fc has minimal cross-reactivity with human, bovine, and equine serum proteins. The anti-mouse IgG was biotinylated with EZ-link NHS-PEG4-biotin (Thermo Scientific, A39259) using standard protocols. The biotinylated antibody was diluted in K buffer (Probe Life, 120011). Streptavidin-coated probes were immersed in 0.5 mg / mL biotin-anti-mouse-Fc for 10 minutes and washed in K buffer for 30 seconds to remove any nonspecific binding interactions on the probe surface.

[0076] Assay Dried probes coated with anti-mouse Fc were immersed in Q buffer (PBS+0.2% BSA+0.02% Tween-20) and allowed to hydrate for 5 minutes before any assay.

[0077] Mouse IgG dissolved in Q buffer was used to generate mouse IgG concentrations ranging from 0.5 to 200 μg / ml. This concentration series was used to test the conventional probe and the MgF2 probe in parallel and compare the performance of both probes in terms of binding capacity, signal intensity, and regeneration. To regenerate both probes, 10 mM glycine pH 1.75 with 150 mM NaCl was used as the regeneration solution.

[0078] The experiments were carried out using a Probe Life Gator instrument (GA007) and its software version 1.3. Samples and regeneration solutions were prepared in microplates from Greiner Bio (reference number 655209).

[0079] The reaction and regeneration protocol are shown in Table 3. The regeneration is repeated 10 times. [Table 3]

[0080] result A side-by-side comparison of the binding capacity of conventional probes and MgF2 probes was performed to understand binding strength, binding kinetics, and regeneration. The results are summarized in Tables 4 and 5.

[0081] Table 4 shows that the MgF2 probe has a much higher signal (nm wavelength shift) and faster binding kinetics than the conventional probe. [Table 4]

[0082] 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, whereas the conventional probe retained only 29% (30 μg / mL mIgG) and 30% (3 μg / mL mIgG) of the original signal intensity. [Table 5]

[0083] Example 6. Comparison of small molecule binding of MgF2 probes and conventional probes In this example, the binding of the enzyme carbonic anhydrase II (CAII) to one of its inhibitors, furosemide, was detected using the MgF2 probe from Example 2B and compared to a conventional biolayer interferometry (BLI) sensor with a SiO2 optical layer. An antibody, anti-estradiol, was also tested for binding to its antigen, estradiol. Furosemide and estradiol have molecular weights of 330 and 272 daltons, respectively, making them excellent models for small molecule label-free detection.

[0084] Preparation of materials Biotin labeling of bovine carbonic anhydrase II (CAII) and human anti-estradiol antibody. The biotinylation reaction used CAII (Sigma-Aldrich), anti-estradiol (US Biological), and NHS-LC-LC-biotin (ThermoFisher). No further purification of the materials was performed before the labeling reaction. CAII and anti-fluorescein antibodies were labeled at a molar coupling ratio (MCR) of 1. NHS-LC-LC-biotin was dissolved using anhydrous DMF and immediately added to the respective proteins, vortexed, and allowed to proceed at room temperature for 1 hour. After the labeling reaction, the biotinylated proteins were purified using a PD-10 column (GE Healthcare).

[0085] Preparation of Cross-Linked FICOLL® A method for preparing crosslinked FICOLL® is described in U.S. Patent No. 8,309,369. To 2 ml of FICOLL® 400 (Sigma / Aldrich) aminated to contain 88 amines per FICOLL® 400 kD (Skold Technology) in 20 mg / ml PBS was added 10 μL of SPDP (Invitrogen, succinimidyl 6-[3-[2-pyridyldithio]-propionamido]hexanoate) at 50 mg / ml in DMF. The SPDP-to-FICOLL® molecular binding ratio (MCR) was 15. The mixture was allowed to react at room temperature for 1 hour before dialysis. Thiol incorporation was estimated by standard methods to be 5.5 per FICOLL® 400 kD.

[0086] To deprotect the thiol on SPDP labeled with FICOLL® 400, 30 μL of DTT at 38 mg / mL in PBS was added to 20 mg in 1 mL of PBS and allowed to react for 2 hours at room temperature. SH-FICOLL® was purified on a PD10 column.

[0087] SMCC was linked to aminated FICOLL® 400 (88 amine / FICOLL®) in two preparations: 1.) 10 mg of aminated FICOLL® 400 in 1 ml of PBS was mixed with 25 μL of SMCC at 10 mg / mL DMF for 30 SMCC / FICOLL® MCR. The mixture was reacted at room temperature for 2 hours and then purified on a PD10 column (GE Healthcare). 2.) 10 mg of aminated FICOLL® 400 in 1 mL of PBS was mixed with 12.5 μL of SMCC at 10 mg / mL DMF for 15 SMCC / FICOLL® MCR. The mixture was reacted at room temperature for 2 hours and then purified on a PD10 column (GE Healthcare).

[0088] To crosslink SH-FICOLL® 400 and SMCC-FICOLL® 400, the following two preparations were made: 1.) 10 mg of SH-FICOLL® 400 in 1 mL of PBS was mixed with 10 mg of SMCC-FICOLL® 400 in 1 mL of PBS (30 mcg). 2.) 10 mg of SH-FICOLL® 400 in 1 mL of PBS was mixed with 10 mg of SMCC-FICOLL® 400 in 1 mL of PBS (15 mcg). The mixtures were allowed to react overnight at 30°C.

[0089] To provide SH-FICOLL® 400 and SMCC-FICOLL® 400, the following two preparations were made: 1.) 10 mg in 1 mL of PBS SH-FICOLL® 400 was mixed with 10 mg in 1 mL of PBS SMCC-FICOLL® 400 (30 mcg). 2.) 10 mg in 1 mL of PBS SH-FICOLL® 400 was mixed with 10 mg in 1 mL of PBS SMCC-FICOLL® 400 (15 mcg). The mixture was allowed to react overnight at 30°C.

[0090] Synthesis of streptavidin-crosslinked FICOLL® conjugates One mg of SPDP-labeled cross-linked FICOLL® was deprotected with 38 mg / mL DTT (ThermoFisher, 20290) dissolved in PBS at an MCR of 592 for 1 hour at room temperature. Eight mg of streptavidin (SA) (Prozyme, SA10) was labeled with succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) dissolved in anhydrous DMF at an MCR of 1 for 1 hour at room temperature. After the SMCC labeling or DTT deprotection reaction, streptavidin (SA) or cross-linked FICOLL® was purified using a PD-10 column (GE Healthcare, 17085101). The purified cross-linked FICOLL® and SA were mixed in a 50 mL tube, and the coupling reaction was allowed to proceed 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 reacted at room temperature for 30 minutes to cap the unreacted cysteines. After the capping reaction, the reaction mixture was purified on a 4B-CL column.

[0091] Coating of MgF2 probes with streptavidin-crosslinked FICOLL® All rocking speeds were 1000 rpm. First, the MgF2 probe (Example 2B) was washed with ethanol for 120 seconds. Next, the probe was washed with PBS for 60 seconds, followed by coating with 100 μg / mL streptavidin-crosslinked FICOLL® for 600 seconds. Two additional PBS washes of 30 seconds each were performed before coating the probe with 15% sucrose in PBS for 60 seconds as a preservative for long-term storage. The probe was then dried in a 40°C oven for 20 minutes.

[0092] Loading of biotinylated CAII and biotinylated anti-estradiol onto MgF2 probes. All rocking speeds were 1000 rpm unless otherwise noted. First, the probes were equilibrated in Q buffer for 120 s. Next, biotinylated CAII or biotinylated anti-estradiol was loaded at 10 μg / mL for 1800 s on an orbital shaker at 400 rpm. 1 mM biocytin was loaded onto the reference probe (probe without CAII) for subsequent double-reference experiments. A final wash of 60 s was performed.

[0093] Loading of biotinylated CAII and anti-estradiol antibody onto streptavidin SiO2 probe Unless otherwise specified, all rocking speeds were 1000 rpm. First, the Octet SA probe (ForteBio, 18-5019) was equilibrated in Q buffer for 120 seconds. Next, biotinylated CAII or anti-estradiol antibody was loaded at 10 μg / mL together for 1800 seconds at 400 rpm. 1 mM biocytin was loaded onto the reference probe for reference experiments. A final wash of 60 seconds was performed.

[0094] Assay protocol and data processing MgF2 probe assay All rocking speeds were 1000 rpm. Assays and data collection were performed on a Gator™ instrument (GatorBio) at 30°C. Furosemide (Acros 448970010) was used at 10 μM, and estradiol (Sigma-Aldrich, 1250008) was used at 6.4 nM. Probes loaded with CAII or anti-estradiol antibodies were prewetted in assay buffer (PBS + 0.05% DMSO) for 600 s before the start of the binding step. A 60-s baseline was then established in assay buffer, followed by a 180-s association step with furosemide or estradiol in PBS with 0.05% DMSO. In a reference experiment, the biocytin-loaded probe on the second column was then exposed to furosemide.

[0095] MgF2 probe data processing Estradiol and furosemide binding data were processed in Gator Data Analysis version 1.7.2 using the reference well subtraction option. The Y-axis was aligned to each baseline and averaged over the last 50 seconds. Savitzky-Golay filtering was applied to remove high-frequency noise from the data. Binding curve data were then calculated and expressed as wavelength shifts in picometers (pm).

[0096] Conventional probe (SiO2) assay All rocking speeds were 1000 rpm. Assays and data collection were performed on an OctetRED instrument (ForteBio) at 30° C. The same assay protocol as described above for the MgF2 probe was used.

[0097] Conventional probe data processing Furosemide data were processed in Octet Data Analysis 10.0 using the reference subtraction option, in which the furosemide binding signal was obtained by subtracting the reference probe from the active furosemide probe.

[0098] Estradiol binding data were processed using the reference probe subtraction option, in which the binding signal was obtained by subtracting the reference probe from the active estradiol probe.

[0099] In both cases, the y-axis was aligned to each baseline and averaged from 1 to 59 seconds. Savitzky-Golay filtering was applied to remove high-frequency noise from the data. Binding curve data were then calculated and presented as wavelength shifts in picometers (pm).

[0100] Comparison of MgF2 probe and conventional probe Table 6 shows the results of carbonic anhydrase / furosemide binding comparison between MgF2 and conventional SiO2 probes. The binding signal of 10 μM furosemide to CAII on the MgF2 probe was 210.7 pm (picometers), 18-fold higher than that of 11.7 pm on the conventional SiO2 probe. [Table 6]

[0101] Table 7 shows the results of a comparison of anti-estradiol / estradiol binding with the MgF2 probe and the conventional SiO2 probe. The conventional SiO2 probe produced a negligible binding signal (2 pm), while the MgF2 probe produced a significant binding signal of 90.9 pm. [Table 7] The following is a summary of the claims as originally filed: [1] An interferometric sensor for detecting an analyte in a sample, the interferometric sensor comprising: a monolithic substrate comprising glass having first and second surfaces disposed substantially parallel to one another at opposite ends of the monolithic substrate; Magnesium fluoride (MgF) coated on the second surface of the monolithic substrate 2 an interference layer including a layer of analyte binding molecules coated on the interference layer; a first interface between the monolithic substrate and the interference layer functions as a first reflecting surface when light is irradiated onto the interferometric sensor; An interferometric sensor, wherein a second interface between a biolayer formed by analyte molecules in a sample that bind to the analyte-binding molecules and a solution containing the sample functions as a second reflective surface when the light is irradiated onto the interferometric sensor. [2] The interferometric sensor according to [1], wherein the monolithic substrate has a length of at least 5 millimeters (mm) and an aspect ratio of the monolithic substrate is at least 5 to 1. [3] The interferometric sensor of [1], wherein the interference layer has a thickness of at least 500 nanometers (nm). [4] Silicon dioxide (SiO ) positioned between the interference layer and the layer of analyte-binding molecules. 2 10. The interferometric sensor according to claim 1, further comprising an adhesive layer comprising: [5] The interferometric sensor according to [4], wherein the adhesion layer has a thickness of less than 10 nm. [6] An interferometric sensor, a monolithic substrate having first and second surfaces disposed substantially parallel to one another at opposite ends of the monolithic substrate; an interference layer having a refractive index at least 0.1 less than the refractive index of the monolithic substrate; a layer of analyte-binding molecules to which analyte molecules in the sample bind during a biochemical test to form a biolayer; An interferometric sensor wherein the refractive index of the interference layer is within 0.05 of the refractive index of the biolayer. [7] The interference sensor according to [6], wherein the thickness of the interference layer is 500 to 5,000 nm. [8] The interference sensor according to [7], wherein the thickness of the interference layer is 800 to 1,200 nm. [9] The interferometric sensor of [6], wherein the monolithic substrate comprises glass.

[10] The interferometric sensor according to [6], wherein the interference layer contains magnesium fluoride.

[11] The interferometric sensor according to [6], wherein the refractive index of the monolithic substrate is at least 1.8.

[12] The interferometric sensor of [6], further comprising an adhesive layer connecting the layer of analyte binding molecules to the interference layer.

[13] The interferometric sensor of

[12] , wherein the adhesion layer comprises silicon dioxide and has a thickness of less than 10 nm.

[14] The interferometric sensor according to [6], wherein the monolithic substrate has a columnar morphology.

[15] a flexible support component located in a central portion of the monolithic substrate, a first portion of the monolithic substrate extending from an upper side of the flexible support component;

[14] The interferometric sensor of

[14] , wherein the second portion of the monolithic substrate further includes a flexible support component extending from a bottom side of the flexible support component.

[16] The interferometric sensor of

[15] , wherein the flexible support component includes a flange and a sleeve positioned below the flange.

[17] The interferometric sensor of

[15] , wherein the flexible support component comprises silicone rubber.

[18] The interferometric sensor of

[15] , wherein the flexible support component is configured to support the interferometric sensor when loaded into a well.

[19] A reflective layer interconnected between the monolithic substrate and the interference layer, comprising: [6] The interferometric sensor according to [6], further comprising a reflective layer having a refractive index higher than the refractive index of the monolithic substrate and the refractive index of the interference layer.

[20] A method for manufacturing an interferometric sensor according to [1], the method comprising: Obtaining a monolithic substrate; polishing first and second surfaces of the monolithic substrate disposed substantially parallel to one another at opposite ends of the monolithic substrate; Magnesium fluoride (MgF 2 depositing a first transparent material comprising: and binding an analyte binding molecule to said interference layer.

[21] The method of

[20] , wherein the monolithic substrate comprises glass.

[22] The method of

[20] , wherein the interference layer has a thickness of at least 900 nm.

[23] depositing a second transparent material on the interference layer to form an adhesion layer; 20. The method of claim 20, further comprising forming a layer of the analyte-binding molecule bound to the adhesive layer.

[24] The method of

[23] , wherein the second transparent material is silicon dioxide.

Claims

1. 1. An interferometric sensor comprising: a monolithic substrate having first and second surfaces disposed substantially parallel to one another at opposite ends of the monolithic substrate, wherein the monolithic substrate has a refractive index of at least 1.5; an interference layer having a refractive index at least 0.1 less than that of the monolithic substrate, the refractive index being 1.32 or more and 1.4 or less; a layer of analyte-binding molecules to which analyte molecules in the sample bind during a biochemical test to form a biolayer; An interferometric sensor wherein the refractive index of the interference layer is within 0.05 of the refractive index of the biolayer.

2. 10. The interferometric sensor of claim 1, wherein the thickness of the interference layer is between 500 and 5,000 nm.

3. 3. The interferometric sensor of claim 2, wherein the thickness of the interference layer is between 800 and 1,200 nm.

4. The interferometric sensor of claim 1 , wherein the monolithic substrate comprises glass.

5. The interferometric sensor of claim 1 , wherein the interferometric layer comprises magnesium fluoride.

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

8.

7. The interferometric sensor of claim 1 , further comprising an adhesive layer connecting the layer of analyte binding molecules to the interferometric layer.

8. The interferometric sensor of claim 7 , wherein the adhesion layer comprises silicon dioxide, and the adhesion layer has a thickness of less than 10 nm.

9. The interferometric sensor of claim 1 , wherein the monolithic substrate has a columnar morphology.

10. a flexible support component located in a central portion of the monolithic substrate, a first portion of the monolithic substrate extending from an upper side of the flexible support component; The interferometric sensor of claim 9 , wherein the monolithic substrate second portion further includes a flexible support component extending from an underside of the flexible support component.

11. The interferometric sensor of claim 10 , wherein the flexible support component includes a flange and a sleeve underlying the flange.

12. The interferometric sensor of claim 10 , wherein the flexible support component comprises silicone rubber.

13. The interferometric sensor of claim 10 , wherein the flexible support component is configured to support the interferometric sensor when loaded into a well.

14. a reflective layer interconnected between the monolithic substrate and the interference layer, The interferometric sensor of claim 1 , further comprising a reflective layer, the reflective layer having a refractive index higher than the refractive index of the monolithic substrate and the refractive index of the interference layer.

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