Multiplexed photonic biosensor devices, systems and methods

Abstract

The photonic biosensor device comprises a sample addition zone in fluid communication with the wicking zone and the sample detection zone, at least one optical input port disposed within the sample detection zone, the optical input port configured to optically couple to a light source, at least one optical output port disposed within the sample detection zone, the optical output port configured to optically couple to a photodetector via a fiber bundle, and at least one photonic integrated circuit (PIC) disposed directly on the substrate, the at least one photonic integrated circuit including at least one first grating coupler aligned with the optical input port, at least two second grating couplers aligned with the optical output port, at least one waveguide between the first grating coupler and the second grating coupler, and at least one detection element disposed within the at least one waveguide.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 341,586, filed May 13, 2022, which is incorporated by reference herein in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT Research or Development This invention was made with Government support under Grant No. FA8650-15-2-5220 awarded by the Department of Defense. The United States Government has certain rights in this invention. [Background technology]

[0003] The use of photonic biosensors to measure changes in refractive index in a sample is known. Detection of the analyte is provided by detecting a change in refracted light of the sample. In some known optical structures of biosensors, a change in refractive index as a result of binding of the analyte to optical surfaces and / or reagents produces a detectable change in the optical resonance frequency. These known biosensors provide sensitive, label-free detection of the desired analyte.

[0004] Typically, biosensor sensing elements are fabricated on silicon substrates using conventional silicon-based nanoscale fabrication processes, such as complementary metal oxide semiconductor ("CMOS") fabrication processes. The use of silicon-based fabrication processes creates biosensor sensing elements for integrated photonics with exceptional optical and biochemical properties. For example, silicon-based fabrication processes allow the precise and / or complex optical structures of the sensing elements to be fabricated in silicon or silicon nitride. Optical structures include, for example, ring resonators, spiral waveguides, grating couplers, and Mach-Zehnder interferometers ("MZIs"). These structures typically require a nearly defect-free optical path to ensure that results are not affected by material impurities or structural defects.

[0005] Although silicon-based processes provide precise biosensor sensing elements on a substrate, known biosensors typically have expensive fluid and optical interconnections. For example, fiber optic coupling of input and output optics is typically required to interface with the optical path of the sensing element. Additionally, many biosensors have complex active fluid delivery mechanisms to bring the sample into contact with the sensing element. Often, the fluid sample is pulled or pushed to the sensing element using an external pump that controls the volume and flow rate of the sample through the biosensor. The complexity of this optical and fluid interconnect increases the cost of the instrumentation and the biosensor itself. While the increased cost may be acceptable for some medical applications, point-of-care ("PoC") and mainframe laboratory diagnostic applications are generally cost sensitive, especially for disposable products such as disposable biosensor examples slides, cassettes, membranes, fibrous substrates, or test cards.

[0006] Thus, there is a need in the art for improved multiplexed photonic biosensors and related methods. Summary of the Invention

[0007] Several embodiments of the invention disclosed herein are described below, and any combination of these embodiments (or portions thereof) may be made to define further embodiments.

[0008] In one aspect, the photonic biosensor device comprises a sample addition zone in fluid communication with the wicking zone and the sample detection zone, the sample detection zone being between the sample addition zone and the wicking zone, and at least one photonic integrated circuit (PIC) disposed directly on the substrate and optically coupled to a light source and a photodetector via a fiber bundle, the at least one photonic integrated circuit including at least one first grating coupler, at least two second grating couplers, at least one waveguide between the first grating coupler and the second grating coupler, and at least one detection element disposed within the at least one waveguide.

[0009] In one embodiment, the PIC is optically coupled through the substrate.

[0010] In one embodiment, the device further includes at least one optical input port disposed within the sample detection zone, the optical input port configured to optically couple to a light source, and at least one optical output port disposed within the sample detection zone, the optical output port configured to optically couple to a photodetector via a fiber bundle, wherein at least one first grating coupler is aligned with the optical input port and at least two second grating couplers are aligned with the optical output port.

[0011] In one embodiment, the fiber bundle includes a plurality of individual fibers, and each of the at least one second grating coupler is mapped to an individual fiber of the fiber bundle.

[0012] In one embodiment, at least one of the individual fibers of the fiber bundle comprises a multimode fiber.

[0013] In one embodiment, at least one of the individual fibers of the fiber bundle comprises a single mode fiber.

[0014] In one embodiment, the individual fibers of the fiber bundle include at least one of a single mode fiber and a multimode fiber.

[0015] In one embodiment, the individual fibers of the fiber bundle are positioned in a hexagonal close-packed configuration.

[0016] In one embodiment, the individual fibers of the fiber bundle are positioned in a square close-packed configuration.

[0017] In one embodiment, the PIC is configured for surface-side coupling, where the PIC is coupled to a top surface of the substrate opposite the fiber bundle.

[0018] In one embodiment, the PIC is configured for backside coupling, where the PIC is coupled to the bottom surface of the substrate between the substrate and the fiber bundle.

[0019] In one embodiment, the device is configured to detect three or more analytes simultaneously.

[0020] In one embodiment, the substrate comprises at least one of a cassette, a slide, a membrane, a fibrous substrate, or a test card.

[0021] In one embodiment, the light source and the light detector are contained within a read head of at least one of a laboratory analyzer or a point-of-care ("PoC") analyzer.

[0022] In one embodiment, the device further comprises at least one of a fluid path, a paper path, or a membrane path fluidly connecting the sample application zone, the detection zone, and the wicking zone.

[0023] In one embodiment, the flow path comprises micropillars or protrusions that are substantially perpendicular to the surface of the substrate, the micropillars or protrusions having a height between about 1 μm and 1000 μm, a diameter between about 10 μm and 100 μm, and a reciprocal lattice spacing between the micropillars between about 5 μm and 100 μm, such that lateral capillary flow of the fluid sample is achieved.

[0024] In one embodiment, the detection zone is configured to provide at least one of fluorescence, refractive index shift, Raman signal, absorbance signal, plasmon shift, or colorimetric detection of one or more analytes in the fluid sample.

[0025] In one embodiment, the photonic integrated circuit is attached to the substrate using at least one of a UV curable adhesive, a physical lamination, a lamina, or a tape / glue application.

[0026] In another aspect, a photonic integrated circuit (PIC) includes at least one first grating coupler, at least two second grating couplers, at least one waveguide between the first grating coupler and the second grating coupler, and at least one detector element disposed within the at least one waveguide.

[0027] In one embodiment, at least one detection element comprises at least one capture molecule.

[0028] In one embodiment, at least one first grating coupler is aligned with an optical input port and at least two second grating couplers are aligned with optical output ports.

[0029] In one embodiment, the at least one detection element includes at least one of a ring resonator, a double ring resonator, a cylindrical resonator, a spherical resonator, a spiral waveguide, a Vernier filter, a photonic crystal, a Mach-Zehnder interferometer ("MZI"), or a combination thereof.

[0030] In one embodiment, at least one waveguide comprises a silicon nitride waveguide.

[0031] In one embodiment, the photonic integrated circuit has a right angle prism or rectangular parallelepiped shape with a length between 2 and 20 mm, a width between 0.25 and 10 mm, and a height between 0.1 and 5 mm.

[0032] In one embodiment, at least one waveguide is split into multiple branches from a first grating coupler to at least two second grating couplers.

[0033] In one embodiment, at least one sensing element is positioned on one of the multiple branches.

[0034] In one embodiment, at least one detector element has an extinction ratio under the water soluble cladding of greater than 5 dB.

[0035] In one embodiment, each detector element has a unique extinction ratio.

[0036] In another aspect, a substrate comprises a sample addition zone in fluid communication with the wicking zone and the sample detection zone, the sample detection zone being between the sample addition zone and the wicking zone, and at least one photonic integrated circuit (PIC) disposed directly on the top or bottom surface of the substrate and optically coupled to a light source and a photodetector via a fiber bundle, the at least one photonic integrated circuit including at least one first grating coupler, at least one second grating coupler, at least one waveguide between the first grating coupler and the second grating coupler, and at least one detection element disposed within the at least one waveguide.

[0037] In one embodiment, at least one sensing element is positioned to contact the fluid sample within the sample detection zone.

[0038] In one embodiment, the PIC is optically coupled through the substrate.

[0039] In one embodiment, the substrate further includes at least one optical input port disposed within the sample detection zone, the optical input port configured to optically couple to a light source, and at least one optical output port disposed within the sample detection zone, the optical output port configured to optically couple to a photodetector via a fiber bundle, wherein at least one first grating coupler is aligned with the optical input port and at least two second grating couplers are aligned with the optical output port.

[0040] The above and other objects and features will become apparent from the following description and by reference to the accompanying drawings, which are included to provide an understanding of the invention and are incorporated in and constitute a part of this specification, and in which like numerals represent like elements. [Brief description of the drawings]

[0041] [Figure 1A] FIG. 1 is a diagram of a photonic biosensor including a substrate (cassette, slide, membrane, fibrous substrate, or test card) and a PIC, according to some embodiments. [Figure 1B] FIG. 2 illustrates an exemplary micropillar layout of a fluid flow path of a biosensor, according to some embodiments. [Figure 2A] 1B shows a cutaway view of the exemplary substrate of FIG. 1A at a section of a sample detection zone including an optical input port and an optical output port, according to some embodiments. [Figure 2B] 1B shows a cutaway view of the exemplary substrate of FIG. 1A at a section of a sample detection zone including an optical input port and an optical output port, according to some embodiments. [Diagram 3] 1 illustrates an exemplary PIC, according to some embodiments. [Figure 4] 1 illustrates an exemplary multiplex hub optical system, according to some embodiments. [Diagram 5] 1 illustrates an exemplary computing environment, according to some embodiments. [Figure 6A]1 shows an exemplary experimental setup of a multiplexed PIC mounted on a micropillar card according to some embodiments. [Figure 6B] 1 shows an exemplary experimental setup of a multiplexed PIC mounted on a micropillar card according to some embodiments. [Figure 7] 1 illustrates an exemplary experimental setup for a multiplex hub optical system, according to some embodiments. [Figure 8] 1A-C show exemplary functionalization of a multiplexed disposable photonics PIC according to some embodiments. [Figure 9] 1 illustrates details of an exemplary multiplexed optical hub fiber bundle, according to some embodiments. [Figure 10] 1 shows an experimental IR micrograph of an output grating of an exemplary PIC, according to some embodiments. [Figure 11] FIG. 1 shows a schematic diagram of a multiplexed optical hub photonic biosensing device according to some embodiments. [Figure 12] 1A-C are experimental images showing alignment of a fiber bundle to the output grating of a multiplexed PIC according to some embodiments. [Figure 13] 1 is a plot showing an experimental four-channel spectrum from a properly aligned multiplexed PIC, according to some embodiments. [Figure 14] 1 is a plot showing an example experimental spectrum according to some embodiments. [Figure 15A] 1 is a plot showing the effect of sample diluent on detection of SARS-CoV-2 and Influenza A antibodies in samples from convalescent individuals with a positive PCR test for SARS-CoV-2, according to some embodiments. [Figure 15B] 1 is a plot showing the effect of sample diluent on detection of SARS-CoV-2 and Influenza A antibodies in samples from convalescent individuals with a positive PCR test for SARS-CoV-2, according to some embodiments. [Figure 15C] 1 is a plot showing the effect of sample diluent on detection of SARS-CoV-2 and Influenza A antibodies in samples from convalescent individuals with a positive PCR test for SARS-CoV-2, according to some embodiments. [Figure 15D] 1 is a plot showing the effect of sample diluent on detection of SARS-CoV-2 and Influenza A antibodies in samples from convalescent individuals with a positive PCR test for SARS-CoV-2, according to some embodiments. [Figure 16A] 1 is a plot showing SARS-CoV-2 and influenza serology testing with multiplex hub and ZIVA according to some embodiments. [Figure 16B] 1 is a plot showing SARS-CoV-2 and influenza serology testing with multiplex hub and ZIVA according to some embodiments. [Figure 16C] 1 is a plot showing SARS-CoV-2 and influenza serology testing with multiplex hub and ZIVA according to some embodiments. [Figure 16D] 1 is a plot showing SARS-CoV-2 and influenza serology testing with multiplex hub and ZIVA according to some embodiments. [Figure 17A] 1 is a plot showing modeling of backside bonding according to some embodiments. [Figure 17B] 1 is a plot showing modeling of backside bonding according to some embodiments. [Figure 18A] 1 shows an exemplary experimental PIC, according to some embodiments. [Figure 18B] 1 shows an exemplary experimental PIC, according to some embodiments. [Figure 19] 1 is a plot showing a resonance signal measured from an output grating of a through-substrate grating array according to some embodiments. [Figure 20A] 1 is a plot showing measured power spectra from through-substrate coupling according to some embodiments. [Figure 20B] 1 is a plot showing measured power spectra from through-substrate coupling according to some embodiments. [Figure 20C] 1 is a plot showing measured power spectra from through-substrate coupling according to some embodiments. [Figure 20D] 1 is a plot showing measured power spectra from through-substrate coupling according to some embodiments. [Figure 20E] 1 is a plot showing measured power spectra from through-substrate coupling according to some embodiments. [Figure 20F] 1 is a plot showing measured power spectra from through-substrate coupling according to some embodiments. [Figure 20G] 1 is a plot showing measured power spectra from through-substrate coupling according to some embodiments. [Figure 21] 13 is a plot showing C-reactive protein biosensing data for through-substrate binding of a single sensor pair according to some embodiments. [Figure 22] 13 is a plot showing C-reactive protein biosensing data for through-substrate binding of a single sensor pair according to some embodiments. [Diagram 23] 1 is a plot showing backside coupling where a photonic chip is placed on a microscope slide cover slip, according to some embodiments. [Figure 24] 1 illustrates another example multiplexed photonic chip layout, according to some embodiments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] It should be understood that the drawings and description of the present invention are simplified to illustrate elements relevant to a clear understanding of the present invention, but omit many other elements found in photonic biosensing systems and methods for clarity. Those skilled in the art may recognize that other elements and / or steps are desirable and / or necessary in implementing the present invention. However, because such elements and steps are well known in the art and because they do not facilitate a better understanding of the present invention, descriptions of such elements and steps are not provided herein. The disclosure herein covers all such variations and modifications to such elements and methods known to those skilled in the art.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described.

[0044] As used herein, each of the following terms has the meaning associated with it in this section.

[0045] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0046] As used herein when referring to a measurable value, such as an amount, a temporal duration, and the like, "about" is intended to encompass variations of ±20%, ±10%, ±5%, ±1%, and ±0.1% from the specified value, as such variations are appropriate.

[0047] Ranges: Throughout this disclosure, various aspects of the invention can be expressed in a range format. Of course, the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Where appropriate, the description of a range should be considered to have all possible subranges specifically disclosed, as well as individual numerical values ​​within that range. For example, a description of a range such as 1-6 should be considered to have specifically disclosed narrower ranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as the individual numerical values ​​contained within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0048] Referring now in detail to the drawings, wherein like reference numerals indicate like parts or elements throughout the several views, in various embodiments, presented herein are multiplexed photonic biosensors and photonic integrated circuits (PICs).

[0049] Disclosed herein are low-cost photonic biosensor devices, systems, and methods that provide high performance for immunoassay diagnostics. Exemplary biosensors disclosed herein use silicon-based photonic integrated circuits ("PICs") in combination with a substrate that provides non-contact optical coupling and passive flow mechanisms. In some embodiments, one or more PICs are disposed on the substrate, allowing for detection of one or more analytes in a fluid sample. In some embodiments, multiple assays may be disposed on the substrate in a multiplex assay. In some embodiments, a single PIC can be configured to perform a multiplex assay. Additionally or alternatively, the substrate may also provide fluorescent and / or colorimetric detection in combination with the refractive light detection provided by the PIC, providing further analyte characterization capabilities.

[0050] The current state of the art for conventional heterogeneous immunoassay diagnostics is based on techniques such as immunofluorescence or chemiluminescence detection in either solid phase or magnetic particle form. Although the cost of reagents has been reduced over the years, the management of complex automated test procedures (including multiple process steps, precise sample and reagent additions, long / variable assay-specific incubation times, narrow incubation temperature tolerances, multiple / complex washing protocols, and the use of specialized signal generating reagents) has made the development costs of laboratory instrumentation prohibitive. Furthermore, this complexity leads to high service fees and high operational (in terms of labor, consumables, waste, and / or power usage) costs.

[0051] In contrast to conventional heterogeneous immunoassay diagnostics, the exemplary photonic biosensors disclosed herein provide a solution that eliminates many of the analyzer process steps. Instead, many analyzer steps, such as sample analysis, are designed into the biosensor itself. The exemplary photonic biosensors disclosed herein also reduce labor and operational costs, allowing instrumentation to be significantly reduced in size and complexity while providing relatively high throughput.

[0052] The ability to measure immunoassays utilizing label-free photonics significantly reduces the amount of reagents required. Additionally, the photonic biosensors of the present disclosure reduce the complexity of the reaction process (reduced instrument hardware) and provide reduced turnaround / analysis time (5-10 minutes). Additionally, as disclosed herein, the exemplary biosensors strategically provide for multiplexing of test panels. Additionally, other measurement modalities may be employed, including labeling strategies to enhance sensitivity, if desired.

[0053] Exemplary methods, devices, and systems provide a photonic biosensor that includes a microfluidic slide, cassette, membrane, fibrous substrate, or test card. The photonic biosensor also includes at least one photonic integrated circuit ("PIC") having a detection element coupled to a flow path provided on the slide, cassette, membrane, fibrous substrate, or test card. The exemplary slide, cassette, membrane, fibrous substrate, or test card also includes an area for receiving a sample, and the flow path passively draws the sample into contact with the detection element using wicking or capillary action (or other passive microfluidic transport structures). The slide, cassette, membrane, fibrous substrate, or test card further includes an optical port for optically coupling with a light source and a light detector of a laboratory analyzer, PoC device, or other analyte analysis device.

[0054] During use, in exemplary embodiments, a sample is applied to a receiving area surface of an exemplary slide, cassette, membrane, fibrous substrate, or test card. The flow path can include passive microfluidic transport that flows the applied sample to the detection zone and the wicking zone. The detection zone includes a silicon-based PIC with a functionalized detection element. In some embodiments, the detection element of the PIC is functionalized with one or more types of capture molecules. In some embodiments, light is applied to an input light port of the exemplary slide, cassette, membrane, fibrous substrate, or test card by an instrument light source, and the light is directed through the PIC and the detection element. In some embodiments, light is applied through the exemplary slide, cassette, membrane, fibrous substrate, or test card, and the light is directed through the PIC and the detection element. In some embodiments, an output light port of the exemplary slide, cassette, membrane, fibrous substrate, or test card receives the light after it passes through the detection element. In some embodiments, the output light is read directly through the exemplary slide, cassette, membrane, fibrous substrate, or test card. Contact between the functionalized sensing element and the fluid sample induces a change in refractive index that is detected by a photodetector or optical sensor of the instrument, the degree of change in refractive index being indicative of the presence of one or more analytes and / or the concentration of one or more analytes.

[0055] The use of passive fluid sample components and non-contact optical interfaces significantly reduces the cost of the exemplary biosensor slides, cassettes, membranes, fibrous substrates, or test cards compared to known biosensors with active fluid sample control and optical interfaces. Furthermore, the use of silicon-based PICs provides highly accurate immunoassay diagnostics while using the production scale available from conventional silicon foundry manufacturing. Furthermore, the disclosed biosensor slides or cassettes with integrated PIC(s) allow for further cost, size, and waste reduction through assay multiplexing (e.g., panel testing).

[0056] In light of the disclosure herein, and without limiting the disclosure in any way, in one aspect of the disclosure, which may be combined with any other aspect enumerated herein, unless otherwise specified, a photonic biosensor device includes a substrate having a sample application zone, a wicking zone, and a detection zone located between the sample application zone and the wicking zone. The substrate also includes a flow path fluidly coupling (e.g., in fluid communication) the sample application zone, the detection zone, and the wicking zone, an optical input port disposed in a section of the sample detection zone and configured for optical coupling to a light source, and an optical output port disposed in a section of the sample detection zone and configured for optical coupling to a light detector. The photonic biosensor device also includes a photonic integrated circuit connected to the substrate in a section of the sample detection zone. The photonic integrated circuit includes a first grating coupler aligned with the optical input port, at least two second grating couplers aligned with the optical output port, at least one waveguide between the first grating coupler and the second grating coupler, and at least one detection element disposed along the at least one waveguide and positioned to contact the fluid sample in the flow path at a section of the sample detection zone.

[0057] In another aspect of the present disclosure, which may be used in combination with any other aspect recited herein unless otherwise specified, at least one detection element includes at least one of a ring resonator, a double ring resonator, a cylindrical resonator, a spherical resonator, a spiral waveguide, a photonic crystal, and a Mach-Zehnder interferometer ("MZI").

[0058] In another aspect of the present disclosure, which may be used in combination with any other aspect recited herein unless otherwise specified, at least one waveguide comprises a silicon nitride waveguide.

[0059] In another aspect of the present disclosure, which may be used in combination with any other aspect recited herein unless otherwise specified, the photonic integrated circuit has a right angle prism or rectangular parallelepiped shape.

[0060] In another aspect of the present disclosure, which may be used in combination with any other aspect recited herein unless otherwise specified, a first grating coupler is provided on a first side of a face of a photonic integrated circuit, at least two second grating couplers are provided on opposing second sides of the same face of the photonic integrated circuit, and at least one detection element is positioned between the first side and the second side.

[0061] In another aspect of the present disclosure, which may be used in combination with any other aspect recited herein unless otherwise specified, the photonic integrated circuit has a length between 2 and 20 mm, a width between 0.25 and 10 mm, and a height between 0.1 and 5 mm.

[0062] In another embodiment of the present disclosure, which may be used in combination with any other embodiment recited herein unless otherwise specified, the substrate includes an enhancer zone or conjugate zone along the flow path between the detection zone and the sample addition zone, the enhancer zone or conjugate zone including at least one reagent for binding with the fluid sample.

[0063] In another aspect of the present disclosure, which may be used in combination with any other aspect recited herein unless otherwise specified, the light input port includes a first tunnel that penetrates the substrate and the light output port includes a second tunnel that penetrates the substrate.

[0064] In another aspect of the present disclosure, which may be used in combination with any other aspect recited herein unless otherwise specified, a first tunnel is located on a first side of the flow path within the section and a second tunnel is located on an opposing second side of the flow path within the section.

[0065] In another aspect of the disclosure, which may be used in combination with any other aspect recited herein unless otherwise specified, the substrate comprises at least one of a slide, a cassette, a membrane, a fibrous substrate, or a test card.

[0066] In another aspect of the disclosure, which may be used in combination with any other aspect recited herein unless otherwise specified, the light source and light detector are included within the read head of at least one of a laboratory analyzer or a point-of-care ("PoC") analyzer.

[0067] In another aspect of the present disclosure, which may be used in combination with any other aspect recited herein unless otherwise specified, at least a portion of the flow path includes micropillars or protrusions that are substantially perpendicular to the surface of the substrate and have a height, diameter, and reciprocal lattice spacing such that lateral capillary flow of the fluid sample is achieved.

[0068] In another embodiment of the present disclosure, which may be used in combination with any other embodiment recited herein unless otherwise specified, the height is between 1 and 1000 μm, the diameter is between 10 and 100 μm, and the reciprocal lattice spacing is between 5 and 100 μm.

[0069] In another aspect of the present disclosure, which may be used in combination with any other aspect recited herein unless otherwise specified, the detection zone is configured to provide at least one of fluorescent or colorimetric detection of one or more analytes within the fluid sample.

[0070] In another aspect of the present disclosure, which may be used in combination with any other aspect recited herein unless otherwise specified, the photonic integrated circuit is connected to the substrate using at least one of a UV-curable adhesive, physical lamination, or application of tape / glue.

[0071] In another aspect of the present disclosure, which may be used in combination with any other aspect recited herein unless otherwise specified, the optical input port is a first optical input port, the optical output port is a first optical output port, the section is a first section, the photonic integrated circuit is a first photonic integrated circuit, and the substrate further includes a second optical input port located in a second section of the sample detection zone and configured to optically couple to a light source, and a second optical output port located in the second section of the sample detection zone and configured to optically couple to a light detector.

[0072] In another aspect of the present disclosure, which may be used in combination with any other aspect recited herein unless otherwise specified, the device further includes a second photonic integrated circuit connected to the substrate at the second section, the second photonic integrated circuit including a first grating coupler aligned with the second optical input port, at least two second grating couplers aligned with the second optical output port, at least one waveguide between the first grating coupler and the second grating coupler, and at least one detection element disposed along the at least one waveguide and positioned to contact the fluid sample in the flow path at the second section.

[0073] In another aspect of the disclosure, which may be used in combination with any other aspect recited herein unless otherwise specified, at least one sensing element of the first photonic integrated circuit is configured for detection of a first analyte and at least one sensing element of the second photonic integrated circuit is configured for detection of a second analyte.

[0074] Therefore, in light of the present disclosure and the above aspects, it is an advantage of the present disclosure to provide a photonic biosensor with passive flow components and non-contact optical coupling.

[0075] It is another advantage of the present disclosure to provide a relatively inexpensive photonic biosensor for PoC and mainframe laboratory applications.

[0076] Another advantage of the present disclosure is to provide a photonic biosensor that performs multiplexed assays on a single slide, cassette, membrane, fibrous substrate, or test card.

[0077] Another advantage of the present disclosure is that it provides a photonic sensor with fluorescent or colorimetric detection on a single slide, cassette, membrane, fibrous substrate, or test card.

[0078] Further features and advantages will be described and will be apparent from the following detailed description and drawings. The features and advantages described herein are not all-inclusive, and in particular many further features and advantages will be apparent to those skilled in the art upon consideration of the drawings and description. Also, it is not necessary for any particular embodiment to have all of the advantages enumerated herein, and it is expressly intended to separately claim each advantageous embodiment. Furthermore, it should be noted that the language used herein has been selected primarily for purposes of readability and explanation, and not to limit the scope of the inventive subject matter.

[0079] Photonic-based diagnostic techniques promise to provide quantitative, multiplexed, and inexpensive platforms by leveraging the highly scalable processes developed for the manufacture of semiconductor microchips. In practice, however, the affordability of these platforms is limited by complex and expensive sample processing. A drawback of the relevant prior art is the inability to detect more than two or three analytes (i.e., proteins in a blood sample) at a time. The multiplexed system disclosed herein corrects these drawbacks.

[0080] 1A is a diagram of a photonic biosensor 100 according to an exemplary embodiment of the present disclosure. The exemplary biosensor 100 includes a substrate 102, which may include a slide, a cassette, a membrane, a fibrous substrate, or a test card. The substrate 102 may be constructed from glass, plastic, composites, cyclic olefin copolymers, polystyrene, polymethyl methacrylate ("PMMA"), nylon, polycarbonate, or combinations thereof. Additionally, the substrate 102 may be manufactured by hot embossing, micromolding, or any other molding or printing method.

[0081] The exemplary substrate includes a sample application zone 106, a wicking zone 108, and a detection zone 110. The zones 106-110 are fluidly coupled (e.g., in fluid communication) with one another via a flow path 112. The sample application zone 106 includes a metering port for receiving a fluid sample. The exemplary wicking zone 108 provides an area for flow control and / or waste collection. The wicking zone 108 provides an end of the flow path, and the sample application zone 106 provides an beginning. In some embodiments, the wicking zone 108 may be covered by a tape support to physically protect the fluid sample accumulated within the wicking zone 108. An inlet section leading to the wicking zone 108 may be configured to draw the fluid sample into the wicking zone 108 to prevent the fluid sample from flowing back through the detection zone 110.

[0082] The exemplary substrate 102 may include an optical enhancer zone (e.g., a conjugate zone) in some embodiments. The optical enhancer zone is located downstream from the sample addition zone 106. In some embodiments, the optical enhancer zone is located adjacent to the sample addition zone 106. Additionally, the optical enhancer zone is located upstream from the detection zone 110 and the wicking zone 108. The optical enhancer zone includes one or more reagents for binding with the fluid sample. In some embodiments, when the fluid sample flows through the optical enhancer zone, the fluid sample dissolves the fluorescently labeled conjugate.

[0083] The exemplary detection zone 110 includes one or more test zones configured to capture bound specific antigen / conjugate complexes. Different test zones may provide detection of sample analytes or different analytes. The concentration or presence of bound antigen / conjugate complexes in each test zone is measured using fluorescent or colorimetric detection. In some cases, after complete dissolution of the conjugates in the optical enhancer zone, the fluid sample acts as a wash fluid, moving unbound material to the wicking zone 108. After thorough washing with the fluid sample, the test zones of the detection zone 110 may be read by a fluorometer or other light analyzer. It should be understood that in some embodiments, the detection zone 110 may not be required. In these embodiments, the detection zone 110 is replaced by the flow path 112.

[0084] In some embodiments, at least some of the flow channel 112, the sample addition zone 106, the optical enhancer zone, the detection zone 110, and / or the wicking zone 108 may include a plurality of protrusions or micropillars. Exemplary protrusions or micropillars are substantially perpendicular to the surface of the substrate 102 and have a height, diameter, and reciprocal lattice spacing such that lateral capillary flow of the fluid sample is achieved. In some embodiments, the protrusions or micropillars have a height that is between 1 and 1000 μm, a diameter that is between 10 and 100 μm, and a reciprocal lattice spacing that is between 5 and 100 μm, preferably between 10 and 25 μm. In some cases, the base of the protrusion or micropillar may have a larger diameter compared to the top. In these cases, the diameter of the protrusion or micropillar may taper from the base to the top.

[0085] Exemplary substrates are further described in U.S. Pat. Nos. 10,073,091, 9,689,870, 9,389,228, 9,285,361, 8,895,293, 8,821,812, 8,409,523, and 8,025,854, each of which is incorporated by reference herein in its entirety.

[0086] In some embodiments, the PIC 104 may be used in combination with one or more detector sections in the detection zone 110. The detector sections may be configured for colorimetric / digital detection and / or fluorescent detection. The sample addition zone 106 is configured to receive serum / plasma, whole blood, or other fluids for analysis by the PIC 104 and the detector sections. The optical enhancer zone may provide one or more capture options, including conjugate capture and / or mass enhancer capture. Additionally, the wicking zone 108 may include one or more features, such as fluid control and / or end-of-test detection. In some embodiments, the wicking zone 108 may include a porous material to increase fluid flow rate.

[0087] As previously described in connection with FIG. 1A, the flow channel 112 may provide fluid flow using micropillars. The micropillars may be located along the flow channel 112 in the sample application zone 106, the wicking zone 108, the detection zone 110, the optical enhancer zone, the washing zone, and / or in the spaces between these zones. In other embodiments, capillary flow may be achieved without the use of micropillars. For example, the flow channel 112 and / or zones 106, 108, and / or 110 may be achieved using texturing / surface patterning. Alternatively, capillary flow may be achieved using porous media (e.g., "paper in poly," fiber material, or yarn / fabric bundles). In other embodiments, capillary flow may be provided using thin film coatings and / or various coated extension layers and channel beads. Coatings to provide wettable / hydrophilic surfaces to flow channel 112 and / or zones 106, 108, and / or 110 include oxygen plasma treatment, neutral atom beam bombardment, gas cluster ion beam bombardment, surface silanization, and the like.

[0088] 1B is a diagram of an exemplary micropillar layout of the fluid flow path 112 of the substrate 102 for the biosensor 100 of FIG. 1A, according to an exemplary embodiment of the present disclosure. The micropillars 1202 are disposed within the fluid flow path 112, including locations along the fluid flow path 112 that align with the functionalized sensing elements 212 of the PIC 104. The micropillars 1202 may comprise cylinders having a diameter of 50 μm. As shown, the micropillars 1202 are disposed in a hexagonal array such that the micropillars 1202 are spaced 100 μm apart. The spacing and size of the micropillars 1202 provide capillary flow along the fluid flow path 112.

[0089] In other embodiments, the micropillars have a rectangular shape. In these other embodiments, the micropillars may be arranged in rows with a spacing between adjacent micropillars of 50 μm to 150 μm. Additionally, each adjacent row may be offset from one another. The offset may correspond to the gaps between adjacent rows, such that the micropillars of one row are aligned with the gaps between the micropillars of an adjacent row.

[0090] In some embodiments, alignment channels 1102 and 1104 are formed in substrate 102. Alignment channels 1102 and 1104 may include through holes or apertures and are located adjacent to ports 202 and 204, respectively.

[0091] In an example, the photonic reader includes alignment pins. After the biosensor 100 is moved to a designated position within the experimental instrumentation, the photonic reader and / or substrate 102 are moved so that the alignment pins pass through the alignment channels 1102 and 1104. This provides for fast alignment. In some embodiments, after the coarse alignment, the photonic reader and / or substrate 102 may have fine adjustments made to the alignment to ensure that the light source and light detector are optically aligned with the optical input port 202 and the optical output port 204.

[0092] It should be noted that while FIGS. 1A, 1B, 2A, and 2B show one input port 202 and one output port 204, in other embodiments, the substrate 102 may have more than one input port, no input ports, more than one output port, and / or no output ports. In some embodiments, a larger output port allows for the use of multiple channels in the PIC 104 for multiplexing. In some embodiments, the use of multiple ports allows for the use of multiple channels in the PIC 104 for multiplexing. For example, the substrate may have a single input port 202 and multiple output ports 204. In this example, the PIC 104 has multiple channels corresponding to the number of output ports 204. Light received through the input port 202 is split along separate channels to provide different types of optical analysis. Additionally or alternatively, the substrate 102 may include multiple input ports 202 and multiple output ports 204 (and / or multiple alignment channel pairs) to accommodate multiple PICs 104 positioned at different locations along the flow path 112.

[0093] 1A also shows the exemplary biosensor 100 in approximate dimensions, having a length of about 22 mm and a width of about 15 mm. In this example, the sample application zone 106 has a width of 5.7 mm and a length of 21.2 mm. The flow path has a width of 1.16 mm and the wicking zone 108 has a diameter of 7.6 mm. It is understood that the biosensor 100 may have alternative dimensions based on the design and end use.

[0094] In some embodiments, alignment channels 1102 and 1104 and ports 202 and 204 may be omitted. In these alternative embodiments, the optical coupling is provided directly to the waveguide 210 of the PIC 104. In an example, an input fiber connected to a light source is configured to align with a side of the PIC 104 for direct optical coupling with the waveguide 210. An output fiber is positioned on the opposite side of the PIC 104 to receive the light.

[0095] The exemplary substrate 102 of FIG. 1A also includes an optical port for non-contact optical coupling with a read head of a laboratory or PoC analyzer. FIGS. 2A and 2B show cutaway views of the substrate 102 at a section 200 of the sample detection zone 110 including an optical input port 202 and an optical output port 204, according to an exemplary embodiment of the present disclosure. The input port 202 is configured to optically couple to a light source, and the output port 204 is configured to optically couple to a light detector. To provide non-contact optical alignment, the substrate 102 is positioned within the analyzer instrument such that the light source is directly aligned with the optical input port 202. Similarly, positioning of the substrate 102 within the analyzer instrument aligns the output port 204 with the light detector. This non-contact coupling eliminates the need for complex optical coupling with the PIC 104.

[0096] The optical input port 202 includes a first tunnel that passes through the substrate 102, and the optical output port 202 includes a separate second tunnel that passes through the substrate 102. Although the tunnels are shown as cylindrical, the tunnels may have other contours, e.g., rectangular, triangular, etc. In the illustrated embodiment, the input port 202 is shown as being on one side of the flow channel 112, and the output port 204 is shown as being on the opposite side of the flow channel 112. In other embodiments, the ports 202 and 204 may be on the same side of the flow channel 112.

[0097] 2A and 2B also show close-up views of PIC 104. Figure 2A shows a view of PIC 104 before it is connected to substrate 102. Figure 2B shows a view of PIC 104 after it has been placed on substrate 102. The example PIC 104 includes a first grating coupler 206 that is aligned with optical input port 202. PIC 104 also includes a second grating coupler 208 that is aligned with optical output port 204. Grating couplers 206 and 208 have shapes that match the circular contours of their respective optical ports 202 and 204.

[0098] Grating couplers 206 and 208 include periodic etched structures that diffract light in specific directions. In the illustrated example, grating coupler 206 diffracts light from a vertical direction through optical input port 202 to a horizontal direction through PIC 104. Additionally, grating coupler 208 diffracts light from a horizontal direction from PIC 104 to a vertical direction through optical output port 204. In other embodiments, the grating couplers may be replaced with mirrors or reflective coatings that direct light between ports 202 and 204 and PIC 104.

[0099] The example PIC 104 also includes at least one waveguide 210 between the first grating coupler 206 and the second grating coupler 208. The PIC 104 further includes at least one sensing element 212 disposed along the at least one waveguide 210. The sensing element 212 is positioned to contact the fluid sample within the flow path 112. It should be understood that the sensing element 212 and / or the PIC 104 generally do not block the fluid passage along the flow path 112. Instead, a small space is provided between the floor of the flow path 112 and the sensing element 212 to allow the fluid sample to pass through. In some embodiments, the small space is between 10 μm and 5000 μm.

[0100] 2A, the substrate 102 may include recessed sections 220 and 222 around the ports 202 and 204 to receive corresponding sides of the PIC 104. The recessed sections 220 and 222 allow the PIC 104 to be fixedly coupled to the substrate 102. In some embodiments, the PIC 104 is fixed to the substrate 102 at the recessed sections 220 and 222 using at least one of a UV-curable adhesive, physical lamination, or tape / glue application.

[0101] In one embodiment, the PIC 104 is configured for surface-side coupling, where the PIC 104 is coupled to a top surface of the substrate 102 opposite the fiber bundle 120 .

[0102] In one embodiment, the PIC 104 is configured for backside coupling, where the PIC 104 is coupled to the bottom surface of the substrate 102 between the substrate 102 and the fiber bundle 120 .

[0103] In some embodiments, the photonic biosensor device 100 includes a sample addition zone 106 in fluid communication with a wicking zone 108 and a sample detection zone 110, the sample detection zone 110 being between the sample addition zone 106 and the wicking zone 108. In some embodiments, the biosensor device 100 further includes at least one optical input port 202 disposed within the sample detection zone 110, the optical input port 202 configured to optically couple to a light source. In some embodiments, the biosensor device 100 further includes at least one optical output port 204 disposed within the sample detection zone 110, the optical output port 202 configured to optically couple to a light detector via a fiber bundle 120. In some embodiments, the biosensor device 100 further includes at least one photonic integrated circuit (PIC) 104 disposed directly on the substrate 102. Further details of the PIC 104 are described below.

[0104] In some embodiments, the fiber bundle 104 includes a plurality of individual fibers. In some embodiments, the individual fibers of the fiber bundle 104 include multimode fibers and / or single mode fibers. In some embodiments, the individual fibers of the fiber bundle 104 include one single mode fiber and at least one multimode fiber. In some embodiments, the individual fibers of the fiber bundle 104 are positioned in a hexagonal close-packed configuration. In some embodiments, the individual fibers of the fiber bundle 104 are positioned in a square close-packed configuration.

[0105] In some embodiments, device 100 is configured to detect three or more measurands simultaneously.In some embodiments, device 100 is configured to detect three or more analytes simultaneously.

[0106] In some embodiments, the substrate 102 comprises at least one of a slide, a cassette, a membrane, a fibrous substrate, or a test card. In some embodiments, the light source and the light detector are included within a read head of at least one of a laboratory analyzer or a point-of-care ("PoC") analyzer.

[0107] In some embodiments, the device 100 further comprises at least one of a flow path, a paper path, or a membrane path fluidly coupling the sample application zone 106, the detection zone 110, and the wicking zone 108. In some embodiments, the flow path comprises micro-pillars or protrusions that are substantially perpendicular to the surface of the substrate, the micro-pillars or protrusions having a height between about 1 μm and 1000 μm, a diameter between about 10 μm and 100 μm, and a reciprocal lattice spacing between the micro-pillars between about 5 μm and 100 μm such that lateral capillary flow of the fluid sample is achieved.

[0108] In some embodiments, the detection zone 110 is configured to provide at least one of fluorescence, refractive index shift, Raman signal, absorbance signal, plasmon shift, or colorimetric detection of one or more analytes in the fluid sample. In some embodiments, the PIC 104 is connected to the substrate 102 using at least one of a UV-curable adhesive, a physical lamination, a lamina, or a tape / glue application.

[0109] 3 illustrates an exemplary PIC 104. In the illustrated example, the PIC 104 includes at least one first (input) grating coupler 206, at least two second (output) grating couplers 208, at least one waveguide 210 between the first grating coupler 206 and the second grating coupler 208, and at least one detector element 212 disposed within the at least one waveguide 210. In some embodiments, the at least one first (input) grating coupler 206 is aligned with the optical input port 202. In some embodiments, the at least two second (output) grating couplers 208 are aligned with the optical output port 204.

[0110] In some embodiments, the at least one detection element 212 can include at least one capture molecule. In some embodiments, the capture molecule is configured to capture an analyte, such as a protein, an antibody, a peptide, a nucleic acid, and any other suitable substance. In some embodiments, the at least one detection element 212 includes at least one of a ring resonator, a double ring resonator, a cylindrical resonator, a spherical resonator, a spiral waveguide, a photonic crystal, a Mach-Zehnder interferometer ("MZI"), and any other suitable design or combination thereof.

[0111] In some embodiments, the at least one waveguide 210 comprises a silicon nitride waveguide. In some embodiments, the at least one waveguide 210 is split into multiple branches from the first grating coupler 206 to at least two second grating couplers 208. In some embodiments, the at least one sensing element 212 is positioned on one of the multiple branches. In some embodiments, the at least one sensing element 212 is positioned to contact the fluid sample within the sample detection zone 110.

[0112] In some embodiments, the PIC 104 includes a right angle prism or a rectangular parallelepiped shape. In some embodiments, the PIC 104 has a length between 2-20 mm (e.g., 4 mm), a width between 0.25-10 mm (e.g., 1 mm), and a height between 0.1-5 mm. In some embodiments, an array of PICs 104 may be connected to the substrate 102 for multiplexing applications. In some embodiments, the PIC 104 may include 0-100, 1-20, 2-10, 2 or more, 5 or more, 8 or more, or any suitable number of detector elements 212. In some embodiments, a PIC 104 with zero detector elements 212 may be included in the array. In some embodiments, the PIC 104 may be used as a reference for calibration and / or adjustment of the light.

[0113] In some embodiments, at least one detection element 212 has an extinction ratio under the aqueous cladding of greater than 5 dB. In some embodiments, each detection element 212 has a unique extinction ratio. In some embodiments, the spectral footprints of individual detection elements 212 sharing the same bus waveguide can be made distinguishable by altering the quality factor and / or extinction ratio, thereby allowing identification of control / experimental rings. In some embodiments, deliberate reduction in quality factor and / or extinction ratio broadens the resonance dip in the spectrum (or reduces the depth of the resonance dip), effectively labeling the ring of interest based on the peak shape.

[0114] FIG. 4 shows a ray diagram of an exemplary multiplexed hub optical system of biosensor 100. Light emerging from output grating 208 of PIC 104 passes through a first surface of hub output lens L1S1, reflects 90 degrees off the angled inner surface of the hub, and exits at a second surface L1S2 into free space as a collimated beam. The beam then reflects 90 degrees to lens L2, which focuses the beam onto a facet of fiber bundle 120. Contrary to the usual implementation of fiber bundles, in this case each fiber is intended to capture light from a single source in an array of discrete sources. The fibers of fiber bundle 120 can be in any suitable arrangement, including hexagonal close-packed and / or square close-packed.

[0115] In some aspects of the invention, software executing the instructions provided herein is stored on a non-transitory computer readable medium, which when executed on a processor performs some or all of the steps of the invention.

[0116] Aspects of the invention relate to algorithms implemented in computer software. Although certain embodiments may be described as being written in a particular programming language and executing on a particular operating system or computing platform, it should be understood that the systems and methods of the invention are not limited to any particular computing language, platform, or combination thereof. Software executing the algorithms described herein may be written, compiled, or interpreted in any programming language known in the art, including, but not limited to, C, C++, C#, Objective-C, Java, JavaScript, MATLAB, Python, PHP, Perl, Ruby, or Visual Basic. Furthermore, it should be understood that elements of the invention may be executed on any acceptable computing platform, including, but not limited to, a server, a cloud instance, a workstation, a thin client, a mobile device, an embedded microcontroller, a television, or any other suitable computing device known in the art.

[0117] Some of the present invention are described as software running on a computing device. Although the software described herein may be disclosed as running on one particular computing device (e.g., a dedicated server or workstation), it is understood in the art that software is portable in nature, and that most software running on a dedicated server may also be run, for the purposes of the present invention, on any of a wide range of devices, including desktop devices, mobile devices, laptops, tablets, smartphones, watches, wearable electronics or other wireless digital / cellular telephones, televisions, cloud instances, embedded microcontrollers, thin client devices, or any other suitable computing devices known in the art.

[0118] Similarly, portions of the invention are described as communicating over various wireless or wired computer networks. For purposes of the present invention, the terms "network," "networked," and "networking" are understood to encompass wired Ethernet, fiber optic connections, wireless connections including any of the various 802.11 standards, cellular WAN infrastructures such as 3G, 4G / LTE, or 5G networks, Bluetooth, Bluetooth Low Energy (BLE), or Zigbee communications links, or any other manner in which one electronic device can communicate with another electronic device. In some embodiments, elements of the networked portion of the invention may be implemented via a Virtual Private Network (VPN).

[0119] 5 and the following discussion are intended to provide a brief, general description of a suitable computing environment in which the invention may be implemented. Although the invention has been described above in the general context of program modules executed in conjunction with application programs running on an operating system on a computer, those skilled in the art will recognize that the invention may also be implemented in combination with other program modules.

[0120] Generally, program modules include routines, programs, components, data structures, and other types of structures that perform particular tasks or implement particular abstract data types. Moreover, those skilled in the art will appreciate that the present invention may be practiced with other computer system configurations including handheld devices, microprocessor systems, microprocessor-based or programmable consumer electronics, minicomputers, mainframe computers, and the like. The present invention may also be practiced in distributed computing environments where tasks are performed by remote processing devices that are linked through a communications network. In a distributed computing environment, program modules may be located in both local and remote memory storage devices.

[0121] FIG. 5 illustrates an exemplary computer architecture of a computer 500 for implementing various embodiments of the present invention. The computer architecture illustrated in FIG. 5 illustrates a conventional personal computer including a central processing unit 550 ("CPU"), a system memory 505 including random access memory 510 ("RAM") and read only memory ("ROM") 515, and a system bus 535 coupling the system memory 505 to the CPU 550. A basic input / output system, including basic routines that help to transfer information between elements within the computer, such as during start-up, is stored in the ROM 515. The computer 500 further includes an operating system 525, applications / programs 530, and a storage device 520 for storing data.

[0122] Storage device 520 is connected to CPU 550 through a storage controller (not shown) that is connected to bus 535. Storage device 520 and its associated computer-readable media provide non-volatile storage for computer 500. Descriptions of computer-readable media contained herein refer to storage devices such as hard disks or CD-ROM drives, but those skilled in the art will appreciate that computer-readable media may be any available media that can be accessed by computer 500.

[0123] By way of example, and not limitation, computer readable media may include computer storage media. Computer storage media includes volatile and nonvolatile, removable and fixed media implemented in any method or technology for storage of information such as computer readable instructions, data structures, program modules, or other data. Computer storage media includes, but is not limited to, RAM, ROM, EPROM, EEPROM, flash memory or other solid-state memory technology, CD-ROM, DVD or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other medium that can be used to store the desired information and that can be accessed by a computer.

[0124] According to various embodiments of the present invention, computer 500 may operate in a networked environment using logical connections to remote computers through a network 540, such as a TCP / IP network such as the Internet or an intranet. Computer 500 may connect to network 540 through a network interface unit 545 connected to bus 535. Of course, network interface unit 545 may also be utilized to connect to other types of networks and remote computer systems.

[0125] The computer 500 may also include an input / output controller 555 for receiving and processing input from a number of input / output devices 560, including a keyboard, mouse, touch screen, camera, microphone, controller, joystick, or other type of input device. Similarly, the input / output controller 555 may provide output to a display screen, printer, speaker, or other type of output device. The computer 500 may be connected to the input / output devices 560 via a wired connection, including but not limited to optical fiber, Ethernet, or copper wire, or via wireless means, including but not limited to Bluetooth, Near Field Communication (NFC), infrared, or other suitable wired or wireless connection.

[0126] As briefly mentioned above, a number of program modules and data files may be stored in the storage device 520 and / or RAM 510 of the computer 500, which includes an operating system 525 suitable for controlling the operation of a networked computer. The storage device 520 and RAM 510 may also store one or more applications / programs 530. In particular, the storage device 520 and RAM 510 may store applications / programs 530 for providing various functions to a user. For example, the applications / programs 530 may include many types of programs, such as word processing applications, spreadsheet applications, desktop publishing applications, database applications, gaming applications, Internet browsing applications, email applications, messaging applications, and the like. According to an embodiment of the present invention, the applications / programs 530 include multi-function software applications for providing word processing functionality, slide presentation functionality, spreadsheet functionality, database functionality, and the like.

[0127] In some embodiments, computer 500 may include various sensors 565 for monitoring the environment around and within computer 500. These sensors 565 may include global positioning system (GPS) sensors, photosensitive sensors, gyroscopes, magnetometers, thermometers, proximity sensors, accelerometers, microphones, biometric sensors, barometers, humidity sensors, radiation sensors, or any other suitable sensors.

[0128] The systems, processes, and methods described herein may be utilized in any desired application, as will be appreciated by those of skill in the art. For example, the systems, processes, and methods described herein may be utilized as single-use, multiplexed assays in measuring the presence or level of analytes, such as proteins, antibodies, peptides, nucleic acids, antigens, viruses, metabolites, and any other suitable substances.

[0129] In one example, antibody responses for clinical diagnosis of individuals to various diseases such as SARS-CoV-2 and influenza antigens. Additionally, the foregoing systems, processes and methods described herein can be utilized with any suitable system, such as those described in U.S. Patent Application No. 17 / 585,914, which is incorporated herein by reference in its entirety.

[0130] Experimental Example The invention will now be described with reference to the following examples, which are provided for illustrative purposes only, and the invention should not be construed as being limited in any way to these examples, but rather as embracing any and all variations that become evident as a result of the teachings provided herein.

[0131] Without further elaboration, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the methods set forth in the claims. The following examples therefore specifically illustrate exemplary embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

[0132] As the third year of the COVID-19 pandemic approaches, more than 230 million cases have been reported, with over 4.6 million deaths (Dong et al., 2020). Despite vaccine availability, new SARS-CoV-2 variants continue to emerge, triggering new waves of infection among unvaccinated people and through vaccine breakthroughs. New diagnostics capable of rapidly measuring serum antibodies against SARS-CoV-2 have been announced. However, as new variants, as well as extraordinary spikes of RSV and influenza, surface, inexpensive multiplex diagnostics are needed to assess an individual's immunity. To address the need for multiplex detection, a new approach was needed for both the sensor itself and the optical I / O system (the "hub").

[0133] The first generation hub allowed coupling to a single input and a single output grating. This limited the multiplexing capabilities of the design due to the difficulty of interpreting the spectrum with more than two sensing rings per bus waveguide. Constrained by the 1 × 4 mm PIC geometry and the width of the micropillar channels, it was found possible to fit eight rings with four bus waveguides onto the multiplexed PIC. By using a four-way splitter, the optical hub and PIC inputs could be left unchanged. However, a different design was required for the output to capture the output light from the four individual gratings. This was best achieved by using a fiber bundle that resulted in a reproducible configuration of close-packed output fibers. The output grating could then be positioned to match this configuration, and a new lens design imaged the grating onto the bundle with the appropriate magnification.

[0134] SARS-CoV-2 has been shown to depend on the coronavirus surface spike glycoprotein (S protein) interacting with the receptor angiotensin-converting enzyme 2 (ACE2) for cell entry (Hoffmann et al., 2020; Zhou et al., 2020). Recent structural studies have demonstrated that the S protein interfaces with ACE2 via the receptor binding domain (RBD) (Wrapp et al., 2020; Yan et al., 2020). Furthermore, the presence of antibodies against the N protein has been shown to distinguish individuals who have acquired immunity through infection from those who have acquired immunity through vaccination. Herein, we demonstrate the detection of antibodies against several SARS-CoV-2 antigens (RDB, S1+S2, N) as well as influenza A H3N2 (A / Switzerland / 9715293) in human samples.

[0135] Recombinantly expressed (baculovirus) SARS-CoV-2 antigens (RBD, S1+S2, N) and hemagglutinin of influenza A H3N2 (A / Switzerland / 9715293) from Sino Biological, Inc. (Wayne, PA) were used. Anti-fluorescein (anti-FITC) antibody used as a non-specific binding control was obtained from Rockland Immunochemicals (Limerick, PA). The diluent for antibody / antigen printing was modified (i.e., potassium-free) phosphate-buffered saline (mPBS). Assay wash buffer (AWB) used to dilute serum samples consisted of mPBS with 3 mM EDTA and 0.01% Tween®-20. All serum samples were diluted 1 to 5 in AWB. Pooled normal human serum (PNHS) was purchased (Innovative Research, Novi, MI) and diluted 1:5 in AWB for use as a blocker of nonspecific binding and as a refractive index matching fluid, and was prepared for assaying each serum sample on the instrument. ACS reagent grade toluene (Fisher) was distilled over sodium metal immediately prior to use.

[0136] Serum samples were obtained from convalescent COVID-19 patients. All subjects had been free of active disease for at least 14 days. Samples were obtained from vaccinated subjects. All subjects were at least 18 years old at the time of blood collection and provided informed consent.

[0137] After collection, whole blood samples were allowed to clot for 30 minutes. Samples were then spun at 1200×g for 5 minutes and serum was pipetted into 15 mL conical tubes and spun again for 10 minutes to remove any remaining cellular material. Serum was then aliquoted and stored at −80° C. until use.

[0138] A silicon nitride ring resonator was designed with an upper water-soluble cladding for use in biosensing. The ring resonator investigated in this work had a silicon nitride waveguide with a width of 1.5 μm and a height of 220 nm, supporting a single transverse electric (TE) polarization mode. Modeling was performed using the finite difference (FD) method in OptoDesigner, a component of the Synopsys photonic design suite.

[0139] A detailed description of the layer stack and modeling of the microring resonators has been reported previously (Cognetti et al., 2021). To fit the eight rings within the 800 micron width of the PIC, the ring diameter was reduced to 164 microns and the coupling gap was reduced to 375 nm to compensate for the increased bending losses and maintain critical coupling. The sensor PIC was designed to have two rings per bus waveguide with slightly different diameters to give resonant signals at two different wavelengths. Each PIC contained eight exposed rings for sensing. In the experiments detailed below, four rings were designated for the experiment and four for the reference.

[0140] The multiplexed PIC was a 1x4mm PIC design featuring a single input grating, a 4-way MMI splitter, eight microring resonators (two per bus waveguide), and four output gratings. Fiducial marks on the PIC surface were provided for use by an automated alignment system.

[0141] The design of the grating coupler has been described in detail previously (Cognetti et al., 2021). On the output side of the PIC, four gratings were positioned to output light to the four top and bottom fibers of a custom 7-fiber bundle, described in further detail below. This fiber bundle configuration can reasonably accommodate up to 14-plex assays. This fiber bundle is not limited to functioning solely as an output, one or more of the fibers in the fiber bundle may also be used as simultaneous inputs.

[0142] The photonic sensors were fabricated in a custom run format using a 300 mm AIM Photonics manufacturing line (Fahrenkopf et al., 2019) modifying the standard AIM passive multi-project wafer (MPW) process and layer stack.

[0143] After fabrication, the wafer was diced by an outside vendor (GDSI) and placed back on the dicing tape. Prior to functionalization, the sensor PIC was removed from the dicing tape and first cleaned in a 1:1 mixture of methanol and concentrated hydrochloric acid for 15 min, then rinsed 4 × 30 s in Nanopure water and dried with nitrogen. The PIC was then placed in a chemical vapor deposition (CVD) oven (Yield Engineering Systems, Fremont, CA) and a monolayer of (3-glycidyloxypropyl)trimethoxysilane (GOPS; Gelest, Inc., Morrisville, PA) was deposited on its surface.

[0144] Antigen and control antibodies were covalently attached to the functionalized surface by spotting directly on the rings using a sciFLEXARRAYER SX piezoelectric microarrayer (Scienion AG, Berlin, Germany) and precisely localizing the rings using the manufacturer's Find Target Reference Points (FTRP) machine vision protocol. Control rings were spotted with anti-FITC antibody at 550 μg / mL in mPBS (pH 5.8), and test rings were spotted with SARS-CoV-2 receptor binding domain (RBD) peptide, S1+S2 extracellular domain, N protein, or influenza A H3N2 (A / Switzerland / 9715293) hemagglutinin at 400 μg / mL in mPBS (pH 7.2). The configuration of the PICs functionalized with only RBD and anti-FITC is shown in Figure 8A. The configuration of the PICs functionalized with all probes is presented in Figure 8B. All rings received approximately 3 nL of antibody / antigen solution. The chips were kept at 75% humidity for 30 min and then overspotted with an equal volume of stabilizer solution (StabilGuard Immunoassay Stabilizer, Surmodics IVD Inc., Eden Prairie, MN). An image of the PIC after applying StabilGuard is shown in Figure 8C. Twenty minutes after spotting the ring with stabilizer, the PICs were removed from the arrayer and kept in a desiccator for at least 4 h and until use.

[0145] Figure 8A shows the functionalization of a "singleplex" experiment with replicates: four rings were printed with anti-FITC as a negative control and four rings were printed with RBD antigen. Figure 8B shows the functionalization of a "multiplex" experiment with four anti-FITC replicates and RBD, S1+S2, N, and FluA antigens printed on one ring each. Figure 8C shows the image of the PIC after printing each capture probe and overspotting with StabilGuard.

[0146] The PIC was integrated with an inexpensive microfluidic card designed to provide a passive flow of sample liquid to the photonic chip for analysis. To precisely control analyte delivery, the microfluidic card requires a sample introduction zone, a channel for directing the fluid flow, a detection zone where the PIC chip comes into contact with the fluid, and a wicking zone that acts as a fluid sink and promotes flow by capillary or evaporation. An image of the PIC positioned in the detection zone is shown in Figure 6A.

[0147] Polystyrene micropillar fluidic cards were first treated with oxygen plasma for 10 min to increase the hydrophilicity of the fluidic channels (Plasmod Plasma System, Nordson Plasma Systems, Concord, CA). 57 μm thick double-sided adhesive tape (467MP, 3M, St. Paul, MN) was patterned using a laser cutter (Full Spectrum Laser, Hobby Series 20 × 12) to interface the fluidic card with the photonic chip. The adhesive covered the entire micropillar channel leaving small windows for the fiber optic signal to access the photonic grating and for the ring resonator sensor to interface with the sample flowing through the channel (Figure 6B). In addition, a large entrance hole allowed pipette access for sample addition to the sample introduction zone. The patterned adhesive tape was added to the fluidic card and a custom alignment device was used to place a piece of filter paper (Q1, Whatman, Little Chalfont, UK) between the micropillar outlet channel and the adhesive to facilitate continuous flow after the channel was filled. After applying adhesive to the fluidic card, the photonic chip was manually aligned to the channels and optical access ports aided by a custom jig.

[0148] Figure 6A shows the PIC attached to the fully assembled micropillar card by adhesive tape and a Whatman wicking pad. Sample is applied to the sample zone and flows under the PIC into the wicking zone reservoir. The Whatman wicking pad allows for running assays with larger sample volumes than current micropillar reservoirs allow. Figure 6B shows how the multiplexed PIC is aligned to the microfluidic channels of the micropillar card as well as the input and output optical pass-throughs. The PIC is affixed to the card with adhesive tape that includes gratings and microring pass-throughs. The microring pass-throughs extend slightly beyond the width of the sensing trench and microfluidic channel to prevent leakage.

[0149] The assembled assay consumables were aligned to the light source, which contains custom multiplex optics (Syntec Optics, Rochester, NY) that allow light to be coupled from below the micropillar cards to and from the photonic grating couplers. A side view of the multiplex optical hub test fixture is shown in Figure 7. The output optics design is configured to allow interfacing with four gratings instead of one. Input light arrives via a single-mode (SM) fiber on the right and travels through the input optics of the hub to the PIC. A ray diagram of the modified output optics design is shown in Figure 4.

[0150] In some embodiments, alignment of the multiplex hub to the PIC requires an infrared microscope positioned directly above the hub lens system in the path of the IR beam. Because silicon PICs are transparent to infrared light, the IR beam scatters off the trench and grating features on the PIC as it passes to the camera. The IR beam is aligned to the input grating of the PIC.

[0151] A modified output lens system is designed to image the four output gratings of the multiplexed PIC (Figure 10) into the top two and bottom two fibers of the custom multimode fiber bundle shown in Figure 9. Figure 9 shows an end-on view of the custom 7-fiber bundle with hexagonal close packing. The two top and bottom multimode fibers collect light from the four output gratings of the PIC. Figure 10 shows an IR micrograph of the four output gratings of the PIC with light being coupled through the input. The output gratings of the multiplexed PIC correspond to the illuminated gratings in the IR micrograph.

[0152] A schematic diagram of the multiplexed optical hub device is presented in Figure 11. A tunable laser source (Keysight 81606A) is directed by a polarization controller (Thorlabs FPC561 with SMF-28FC / PC connector) to obtain linearly polarized light TE-oriented with respect to the silicon nitride waveguides. The light is directed by the input of the optical hub and focused onto the input grating of the PIC. The output light from the four PIC output gratings is collected by the optical hub and directed to the four channels of an optical power meter (Keysight N7745A) via a custom fiber bundle (IDIL Optics) of multimode fibers (Thorlabs FP200ERT). Alignment of the PICs to the optical hub is facilitated by a dual-camera VIS / IR microscope. A 5x IR objective (Mitutoyo Plan Apo NIR46-402) with on-axis illumination directs light through a long-pass dichroic mirror (Thorlabs DMLP950R) to either an IR camera (WiDy InGaAs650) or a VIS CMOS camera (Thorlabs DCC1645C). Proper alignment is confirmed by IR micrographs and resonance spectra.

[0153] The tunable laser and optical power meter were connected to a computer via a general purpose interface bus (GPIB) and controlled by the Keysight Photonic Application Suite (N7700A) Insertion Loss software. Measurements were performed by repeated wavelength scans (6 nm scans) in the vicinity of the resonance signals from the control and probe rings. The resonance red shift is proportional to the binding of material to the ring surface. The specific shift due to capture of the target analyte is calculated by subtracting the red shift of the control ring from that of the probe ring using the data analysis protocol described below.

[0154] After initial assembly of the multiplex hub, proper alignment of the individual components was verified to generate a spectrum. The input grating of the PIC was aligned to the input wavefront, and the z-axis alignment was optimized by maximizing the light emitted by the output grating. An IR micrograph of the light emitted by these four gratings is presented in Figure 12A. The output alignment was then verified by coupling light back through the multimode fiber of the output fiber bundle. This aids in the initial alignment of the hub by observing the overlap between the output light from the gratings and the light emitted from the fiber bundle. The initial configuration was rotated 11 degrees and translated approximately 200 microns further away from the input grating than necessary, as shown in Figure 12B. Inspection of the fiber bundle revealed that the rotational misalignment occurred as a result of the fibers not being properly aligned with the connector keys (Figure 12C). The issue was resolved by replacing the fiber bundle with a correctly manufactured one.

[0155] Figure 12A shows an IR micrograph of light emanating from the output grating of a multiplexed PIC with the input grating aligned to the hub. The dashed line indicates the perimeter of the PIC. Figure 12B shows the light routed back through the output multimode fibers of the fiber bundle, revealing their alignment relative to the PIC (black outline). Here, the fiber bundle is clearly misaligned along the bundle axis. Figure 12C shows inspection of the fiber bundle revealing that it is misaligned by 11 degrees with respect to the key of the APC connector.

[0156] To reduce the 200 micron translational misalignment, it was necessary to remove the collar of the FC ferrule connector, providing an additional goniomic degree of freedom. The separation between the input and output wavefronts was then reduced by carefully tightening the bottom two screws of the input bulkhead against the optical hub. The spectrum obtained from a properly aligned hub is presented in Figure 13. The resonances of the eight individual sensing rings are obtained by four spectra with equal peak intensities. Figure 13 shows a spectrum scanned from 1520 to 1580 nm of a multiplexed PIC aligned to the hub. All four output channels (blue, red, green, cyan) were captured equally by the hub. The arched shape of the spectrum reflects the wavelength dependence of the grating, optimized for 1550 nm light.

[0157] After aligning the instrument, 6 nm spectra were acquired consecutively with 1 pm resolution, generally centered at 1550 nm, with each spectral sweep taking approximately 6 seconds. All spectra were automatically saved for analysis. Once the spectra were acquired after alignment, the experimenter added the samples sequentially as follows: For the data reported in Figure 14, first, 15 μL of 10% BSA in assay wash buffer (AWB) was added. This step served three purposes: first, to wash away the stabilizer (StabilGuard) and expose the antigen-functionalized rings, second, to equilibrate the peaks from each ring to an environment with a bulk refractive index similar to that of a human serum sample, and third, to block nonspecific binding sites. Once the spectrum stabilized, indicating removal of the StabilGuard, the serum sample to be measured was added. The sample was diluted 1 to 5 in 10% BSA in AWB. For all other experiments, the procedure was the same as indicated, except that the 10% BSA in the AWB diluent was replaced with AWB alone.

[0158] Collected spectra were automatically processed through a custom pipeline in MATLAB (MathWorks, Natick, MA) and a previously described Python script (Cognetti et al., 2021). Output spectra per channel were collected simultaneously and stored in the same file. Minor modifications were made to the pipeline to accommodate multiple output channels. Briefly, spectral features including peak positions, peak heights, quality factors, chi-squared values, and peak fitting parameters were extracted by fitting the data with Lorentzian functions. Output spectra per waveguide were stored separately as individual columns in the same file to allow for independent analysis.

[0159] Typical spectra from sensing experiments are presented in Figure 14. The measured quality factors are typically 5 × 10 4and half of that observed in previous experiments utilizing singleplex hubs. This was expected, since the diameter of the multiplex rings had to be reduced to fit eight rings within the same 1 × 4 mm PIC footprint. The benefit of reducing the ring diameter is a corresponding increase in the free spectral range (FSR) between successive resonance peaks. The FSR of the multiplex hub ring was 2.517 nm at a vacuum wavelength of 1555 nm, compared to 2.122 nm for the singleplex ring. With an extinction ratio of over 20 dB under the aqueous cladding, these rings were successfully designed to enable sensing.

[0160] FIG. 14 is a plot showing the antibody sensing spectral shift for anti-RBD measurements. Each ring has a corresponding resonant wavelength, where the resonant wavelengths are seen as troughs in the transmitted power. The peak on the left corresponds to the RBD peptide functionalized ring, and the peak on the right corresponds to the anti-FITC functionalized ring. One RBD and one anti-FITC peak are not present in the spectrum because the rings were damaged during the trench etching process and did not provide sufficient resonant signal to noise ratio. Upon addition of a human serum sample diluted 1:5 in 10% BSA / AWB, the left peak shifts as the antibody binds to the ring (mean: 317 pm, standard error: 38 pm), while the anti-FITC ring shifts much less due to nonspecific interactions with serum proteins (mean 22 pm, standard error: 26 pm). The net shift corrected for nonspecific binding by subtracting the negative control shift is 295 pm.

[0161] Convalescent serum samples from individuals who tested positive by PCR for SARS-CoV-2 were then diluted 1:5 in either 10% BSA / AWB or AWB alone and the serological results were compared (Figures 15A-D). Figure 15A shows the raw shift for the 10% BSA / AWB dilutions, Figure 15B shows the raw shift for the AWB dilutions, Figure 15C shows the anti-FITC corrected shift for the 10% BSA / AWB dilutions, and Figure 15D shows the anti-FITC corrected shift for the AWB dilutions. Comparing Figures 15A and 15B, there is clearly an increase in the magnitude of response in all rings, including the anti-FITC control, when using AWB alone. This can be understood as a bulk shift due to the larger difference in total protein concentration between AWB and a 1:5 dilution of serum in AWB compared to 10% BSA / AWB and a 1:5 dilution of serum in 10% BSA / AWB. 10% BSA is already a very high protein concentration, and dilution of serum has less impact on total background protein concentration than AWB alone. Comparing the anti-FITC corrected shifts, which account for background and nonspecific binding, in Figures 15C and 15D, the top two responders for SARS-CoV-2 antigens were N and RBD. This is consistent across dilutions and is expected, as a large N response is typically seen in samples from individuals with positive PCR tests. However, the shift observed with AWB as a diluent was 3 times greater in magnitude than with 10% BSA / AWB. Similarly, the response to S1+S2, which would be expected in a convalescent sample, is present with AWB and absent with 10% BSA / AWB. Finally, this sample is known to be positive for antibodies to influenza A H3N2 hemagglutinin (A / Switzerland / 9715293) (FluA). In 10% BSA / AWB, the assay fails to capture the expected positive FluA response, while in AWB, the strongest responder (Figure 5.10(d)). This comparison shows that the AWB dilutions perform better, with greater contrast between responders and controls, the desired outcome.

[0162] Next, the serological results of samples from convalescent individuals were compared to serological results of samples from vaccinated individuals who did not test positive for SARSCoV-2. These results were cross-validated with results from ZIVA, a commercially available high-throughput arrayed imaging reflectometry platform. Based on previous performance, the diluent used for these samples was AWB. Results from Multiplex Hub and ZIVA are presented in Figures 16A-16D. Figures 16A-16D show serological results from Multiplex Hub and ZIVA (arrayed imaging reflectometry) of samples from two individuals: (1) a convalescent individual who tested positive for SARS-CoV-2 PCR and was not vaccinated (convalescent), and (2) an individual who did not test positive for PCR and received a second dose of the vaccine one month prior to providing the sample (vaccinated). RBD, SARS-CoV-2 receptor binding domain; N protein, SARS-CoV-2 nuclear peptide; S1+S2, SARS-CoV-2 S1+S2 extracellular domain; FluA, Influenza A H3N2 hemagglutinin (A / Switzerland / 9715293); FITC: anti-FITC negative control. Figure 16A shows multiplex hub results for convalescent samples, Figure 16B shows multiplex hub results for vaccinated samples, Figure 16C shows ZIVA results for convalescent samples, and Figure 16D shows ZIVA results for vaccinated samples.

[0163] As before, in the convalescent sample, the response to Influenza A was most prominent, with a shift of 502 pm, followed by the N peptide at 318 pm. The strong N protein response is consistent with expectations for individuals who have recovered from infection. The RBD response was 203 pm, and the S1+S2 response was less than half as strong at 74 pm. The standard error of the anti-FITC negative control response was 10 pm. Overall, the relative responses to SARS-CoV-2 peptides obtained from the multiplex hub compared well with the results from ZIVA. On the other hand, the FluA response from the multiplex hub was significant, and the moderate response with ZIVA was judged negative in this sample, but positive in three subsequent samples from the same individual (data not shown). In the vaccinated sample, the response to RBD was most prominent, with a shift of 145 pm, followed by S1+S2 at 110 pm, while the response to the N protein was negative (Figure 16B). The standard error of the anti-FITC negative control response was 8 pm. This pattern is one of the key differentiators between convalescent and vaccinated samples. Vaccinated but never infected individuals have not been exposed to the N protein. This individual was known to have never been exposed to influenza A (A / Switzerland / 9715293), so the apparent negative result is expected. When compared to ZIVA, these results were identical in outcome, though not in relative magnitude (Figure 16D). The measured responses for RBD and S1+S2 were 19071 and 1072 relative reflectance units, respectively, and both were scored as positive. Meanwhile, responses to the N protein and influenza A were both negative.

[0164] When performing the assay with human serum samples, the choice of sample diluent can play an important role in both assay performance and reproducibility. Here, SARS-CoV-2 / Influenza A serological results showed a marked improvement when AWB was used alone, instead of 10% BSA / AWB. However, more samples need to be performed to establish the effect of AWB on assay reproducibility. Further studies of alternative diluents may continue to improve both the performance and reproducibility of this multiplex assay with serum samples.

[0165] As the pandemic becomes endemic, multiplex serological assays can play an important role. As novel variants continue to emerge in the population, it will be important to understand the cross-immunity profile of individuals against novel variants. Such studies may aid in the selection of variants to be included in future booster vaccines. Here, we used the multiplex capability of this assay to distinguish naturally immune from vaccinated individuals by the presence of responses against the N protein. The assay further distinguished between individuals with and without immunity to a particular influenza A strain.

[0166] A critical issue for photonic biosensors is how light is coupled into the photonic integrated circuit (PIC). One possibility is on-chip light sources, but these require expensive manufacturing processes. A second is to use edge coupling, but this requires either alignment with an external fiber array or direct attachment of optical fibers, a very expensive and slow process. A third possibility is to use grating couplers. Grating couplers allow light to be coupled into the PIC from different angles above the chip (depending on how the coupler is designed), but the situation becomes complicated when the biosensor is used for human samples. As human samples usually contain aqueous solutions, a gasket must be used to isolate the sample from the area of ​​the chip where the grating coupler is. This increases the complexity of the measurement system and also significantly increases the cost of the sensor, since the cost is directly related to the area of ​​the chip. We have developed an approach to address these issues.

[0167] To solve this problem, the fact that the substrate silicon of the PIC is transparent to infrared light was taken into account. Therefore, it was decided to determine whether it was possible to construct a grating that is the exact opposite of the current teachings for the design of grating couplers. This would allow light to be coupled from the back side of the chip through the substrate. The data presented below shows that back-side coupling is feasible, albeit with lower efficiency, using a grating coupler designed for "normal" front-side coupling. As this method is particularly useful for photonic biosensors, preliminary data is provided that demonstrate the successful use of back-side coupling in this context. Simulations of a grating coupler designed for optimized back-side coupling are also presented.

[0168] A typical photonic grating layer is patterned above a substrate and light is coupled from an optical fiber or free space optics positioned above the grating. A number of approaches have been reported to improve grating efficiency by minimizing the interaction between the incident light and the substrate, such as the through-substrate concept, which couples the light into the grating and / or additional backside structures to improve efficiency by shaping the beam for interaction with the grating.

[0169] Modeling of back-side coupling for a typical optical grating geometry is shown in FIG. 17A, where a state-of-the-art photonic grating is modeled for coupling light from above the surface of the substrate into a waveguide. When illuminated from below the substrate, 5.9% is absorbed by the grating, 66.4% is transmitted, and 27.6% is reflected. FIG. 17B shows the improvement in performance of a grating optimized for back-side through-substrate coupling, where the through-substrate grating is modeled indoors. When illuminated from below the substrate, 65.8% of the light is absorbed by the grating, 18.1% is transmitted, and 15.9% is reflected. This is an 11x improvement over state-of-the-art grating couplers.

[0170] Preliminary tests were then performed as shown in Figures 18A and 18B. A PIC was designed and fabricated to integrate the biosensing grating and ring resonator on the backside as shown in the layout in Figure 18A. A vertically oriented fiber array was used underneath the PIC to couple light to and from the grating (Figure 18B). Resonance spectra collected from each of the seven output gratings are presented in Figures 20A-G. Figure 19 shows the expanded single free spectral range of both rings on a single bus waveguide.

[0171] Figure 18A shows the layout of an example multiplexed photonic chip. A single input grating 206 and seven output through-substrate gratings 208 were arranged in a 127 micron pitch array. Guided light is split equally by a 7-way photonic splitter and interacts with seven banks of micro-ring resonators 212, on which covalently attached probe molecules capture specific target analytes. Figure 18B shows a fiber array containing SMF-28 fiber on a 127 micron pitch v-groove substrate, with laser light passing through the underside of the silicon chip to couple to and from the gratings.

[0172] Figure 19 shows the measured resonance signal from output grating 5 of a through-substrate grating array, with two consecutive resonances from two different rings on the same bus waveguide. Figures 20A-G show the measured output spectra from through-substrate coupling to output gratings 1-7, respectively.

[0173] Figure 21 shows C-reactive protein biosensing data for through-substrate binding of a single sensor pair. The blue trace is the initial control sample spectrum and the green trace is the sample spiked with 500ng / mL C-reactive protein. The spectral notch on the left represents the negative control sensor and the notch on the right represents the experimental detection sensor device. A shift of approximately 90pm due to binding is observed.

[0174] Figure 22 shows C-reactive protein biosensing data for through-substrate binding of a single sensor pair. The blue trace is the initial control sample spectrum and the green trace is the sample spiked with 100ng / mL C-reactive protein. The spectral notch on the left represents the negative control sensor and the notch on the right represents the experimental detection sensor device. A shift of approximately 24pm due to binding is observed.

[0175] Figure 23 shows backside coupling where the photonic chip was placed on a microscope slide cover slip. Light was coupled into the input grating through the cover slip and the backside of the chip and extracted through the output grating. Six photonic sensor pairs were exposed to nanopore water.

[0176] The following publications are hereby incorporated by reference in their entireties:

[0177] Cognetti, JS, Steiner, DJ, Abedin, M., Bryan, MR, Shanahan, C., Tokranova, N., Young, E., M. Klose, A., Zavriyev, A., Judy, N., et al. (2021). Disposable photonics for cost-effective clinical bioassays: application to COVID-19 antibody testing. Lab on a Chip 21,2913-2921.

[0178] Dong, E., Du, H., and Gardner, L. (2020). An interactive web-based dashboard to track COVID-19 in real time. The Lancet Infectious Diseases 20,533-534.

[0179] Fahrenkopf,NM,McDonough,C.,Leake,GL,Su,Z.,Timurdogan,E.,and Coolbaugh,DD(2019).The AIM Photonics MPW:A Highly Accessible Cutting Edge Technology for Rapid Prototyping of Photonic Integrated Circuits.IEEE Journal of Selected Topics in Quantum Electronics25,1-6.

[0180] Hoffmann,M.,Kleine-Weber,H.,Schroeder,S.,Kruger,N.,Herrler,T.,Erichsen,S.,Schiergens,T.S.,Herrler,G.,Wu,N.-H.,Nitsche,A.,et al.(2020).SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor.Cell.

[0181] Wrapp,D.,Wang,N.,Corbett,K.S.,Goldsmith,J.A.,Hsieh,C.-L.,Abiona,O.,Graham,B.S.,and McLellan,J.S.(2020).Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation.Science367,1260-1263.

[0182] Yan,R.,Zhang,Y.,Li,Y.,Xia,L.,Guo,Y.,and Zhou,Q.(2020).Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2.Science367,1444-1448.

[0183] Zhou,P.,Yang,X.-L.,Wang,X.-G.,Hu,B.,Zhang,L.,Zhang,W.,Si,H.-R.,Zhu,Y.,Li,B.,Huang,C.-L.,et al.(2020).A pneumonia outbreak associated with a new coronavirus of probable bat origin.Nature579,270-273.

[0184] U.S. Patent No. 10,073,091, "Lateral Flow Assay Device"

[0185] U.S. Patent No. 9,689,870, "Assay Device Having Multiple Reagent Cells"

[0186] U.S. Patent No. 9,389,228, "Lateral Flow Assay Devices for use in Clinical Diagnostic Apparatus and Configuration of Clinical Diagnostic Apparatus for Same"

[0187] U.S. Patent No. 9,285,361, "Method for the Analysis of Circulating Antibodies"

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[0194] U.S. Patent No. 9,239,432, "Photonics grating coupler and method of manufacture"

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[0197] U.S. Patent No. 9,128,241, "Integrated Photonics Waveguide Grating Coupler"

[0198] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated by reference in their entirety. Although the present invention has been disclosed with reference to specific embodiments, it will be apparent to those skilled in the art that other embodiments and variations of the invention may be devised without departing from the true spirit and scope of the invention.

Claims

1. A photonic biosensor device, A sample addition zone that is in fluid communication with the wicking zone and the sample detection zone, wherein the sample detection zone is located between the sample addition zone and the wicking zone, and A photonic integrated circuit (PIC) which is directly placed on a substrate and optically coupled to a light source and a photodetector via a fiber bundle, At least one first grating coupler, At least two second grating couplers, At least one waveguide between the first grating coupler and the second grating coupler, At least one detection element disposed within the at least one waveguide, The at least one photonic integrated circuit, including The apparatus comprising the above.

2. The apparatus according to claim 1, wherein the PIC is optically coupled through the substrate.

3. At least one optical input port located within the sample detection zone, wherein the optical input port is configured to be optically coupled to the light source, At least one optical output port located within the sample detection zone, wherein the optical output port is configured to be optically coupled to the photodetector via the fiber bundle, It further includes, The apparatus according to claim 1, wherein at least one first grating coupler is aligned with the optical input port, and at least two second grating couplers are aligned with the optical output port.

4. The apparatus according to claim 1, wherein the fiber bundle comprises a plurality of individual fibers, and each of the at least one second grating coupler is mapped to an individual fiber of the fiber bundle.

5. The apparatus according to claim 1, wherein at least one of the individual fibers of the fiber bundle includes a multimode fiber.

6. The apparatus according to claim 1, wherein at least one of the individual fibers of the fiber bundle includes a single-mode fiber.

7. The apparatus according to claim 1, wherein the individual fibers of the fiber bundle are positioned within a hexagonal close-packed configuration.

8. The apparatus according to claim 1, wherein the individual fibers of the fiber bundle are positioned within a square close-packed configuration.

9. The apparatus according to claim 1, wherein the PIC is configured for surface-side bonding, and the PIC is bonded to the upper surface of the substrate opposite to the fiber bundle.

10. The apparatus according to claim 1, wherein the PIC is configured for back-side bonding, and the PIC is bonded to the bottom surface of the substrate between the substrate and the fiber bundle.

11. The apparatus according to claim 1, wherein the apparatus is configured to simultaneously detect three or more analytes.

12. The apparatus according to claim 1, wherein the substrate includes at least one of a cassette, a slide, a film, a fibrous substrate, or a test card.

13. The apparatus according to claim 1, wherein the light source and the photodetector are contained within the reading head of at least one of a laboratory analyzer or a point-of-care ("PoC") analyzer.

14. The apparatus according to claim 1, further comprising at least one of a flow channel, a paper channel, or a membrane channel that fluidly connects the sample addition zone, the detection zone, and the wicking zone.

15. The apparatus according to claim 1, wherein the flow path includes micropillars or projections substantially perpendicular to the surface of the substrate, the micropillars or projections having a height between about 1 μm and 1000 μm, a diameter between about 10 μm and 100 μm, and a reciprocal grid spacing between the micropillars between about 5 μm and 100 μm, so as to achieve lateral capillary flow of the fluid sample.

16. The apparatus according to claim 1, wherein the detection zone is configured to provide at least one of the following: fluorescence, refractive index shift, Raman signal, absorbance signal, plasmon shift, or colorimetric detection of one or more analytes in the fluid sample.

17. The apparatus according to claim 1, wherein the photonic integrated circuit is connected to the substrate using at least one of a UV-curing adhesive, physical lamination, lamination, or tape / glue application.

18. A photonic integrated circuit (PIC), At least one first grating coupler, At least two second grating couplers, At least one waveguide between the first grating coupler and the second grating coupler, At least one detection element disposed within the at least one waveguide, The PIC includes the PIC.

19. The PIC according to claim 18, wherein the at least one detection element includes at least one capture molecule.

20. The apparatus according to claim 18, wherein at least one first grating coupler is aligned with an optical input port, and at least two second grating couplers are aligned with an optical output port.

21. The PIC according to claim 18, wherein the at least one detection element includes at least one of a ring resonator, a double-ring resonator, a cylindrical resonator, a spherical resonator, a spiral waveguide, a Vernier filter, a photonic crystal, and a Mach-Zehnder interferometer ("MZI").

22. The PIC according to claim 18, wherein the at least one waveguide includes a silicon nitride waveguide.

23. The PIC according to claim 18, wherein the photonic integrated circuit has a right-angle prism or rectangular parallelepiped shape with a length between 2 and 20 mm, a width between 0.25 and 10 mm, and a height between 0.1 and 5 mm.

24. The PIC according to claim 18, wherein the at least one waveguide is divided into a plurality of branch sections from the first grating coupler to the at least two second grating couplers.

25. The PIC according to claim 18, wherein the at least one detection element is positioned on one of the plurality of branching portions.

26. The PIC according to claim 18, wherein the at least one detection element has an extinction ratio greater than 5 dB under a water-soluble cladding.

27. The PIC according to claim 18, wherein each detection element has a unique extinction ratio.

28. It is a substrate, A sample addition zone that is in fluid communication with the wicking zone and the sample detection zone, wherein the sample detection zone is located between the sample addition zone and the wicking zone, and A photonic integrated circuit (PIC) is disposed directly on the upper or lower surface of the substrate and is optically coupled to a light source and a photodetector via a fiber bundle, At least one first grating coupler, At least one second grating coupler, At least one waveguide between the first grating coupler and the second grating coupler, At least one detection element disposed within the at least one waveguide, The at least one photonic integrated circuit, including The substrate comprising the above.

29. The substrate according to claim 28, wherein the at least one detection element is positioned to contact a fluid sample within the sample detection zone.

30. The substrate according to claim 28, wherein the PIC is photocoupled through the substrate.

31. At least one optical input port located within the sample detection zone, wherein the optical input port is configured to be optically coupled to the light source, At least one optical output port located within the sample detection zone, wherein the optical output port is configured to be optically coupled to the photodetector via the fiber bundle, It further includes, The substrate according to claim 28, wherein at least one first grating coupler is aligned with the optical input port, and at least two second grating couplers are aligned with the optical output port.

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