Nanoplasmonic sensor

Abstract

The present disclosure relates to the field of molecular detection, and specifically describes a rational nanoplasmonic sensor design for molecular detection. A method for fabricating a functionalized nanoplasmonic sensing chip is also disclosed. The method includes providing a substrate comprising an array of sensors, each sensor comprising an array of nanostructures; attaching a microwell adapter on top of the substrate, thereby providing an array of microwells aligned therewith over the array of sensors; using an automated pipetting system to deliver a first batch of a reaction solution to one or more microwells on one or more sensors to form one or more of the functionalized sensors within the array of sensors; subsequently, using the automated pipetting system to remove the first batch of the reaction solution from the one or more microwells; and removing the microwell adapter from the substrate.

Description

Technical Field

[0001] (Incorporation of Priority Applications by Reference) This application claims priority to U.S. Provisional Patent Application No. 63 / 352,970, entitled "Nanoplasmonic Sensor," filed on June 16, 2022, and U.S. Provisional Patent Application No. 63 / 352,972, entitled "Functionalized Nanoplasmonic Sensor," filed on June 16, 2022. All applications with foreign or domestic priority claims are hereby incorporated by reference in their entirety.

[0002] This disclosure relates to the field of molecular detection. Specifically, this disclosure describes methods for functionalizing nanoplasmonic sensors and functionalized nanoplasmonic sensors.

Background Art

[0003] For biomedical research, clinical diagnostics, environmental testing, and other related fields, it is beneficial to have accurate, sensitive, specific, reproducible, and easy-to-use devices and systems for detecting analytes such as biomolecules and chemical substances. For example, a rapid, high-speed, and accurate test for detecting a specific analyte in a biological sample can be useful in a clinical diagnostic situation and can assist a physician in determining an optimal treatment regimen.

[0004] When metals are optically irradiated, they have a unique ability to support electromagnetic surface waves called surface plasmons. This property, along with its strong sensitivity to changes in refractive index, enables the use of metal nanostructures as highly sensitive transducers. In previous studies described by the inventors' group, an ensemble of randomly oriented nanostructures (i.e., colloidal nanorods dispersed on a chip) was used for sequence-specific nucleic acid sensing. These particle sensors have the advantage of rapid and device-free fabrication, but have the disadvantage of low sensitivity and figure of merit due to random particle dispersity.

Summary of the Invention

[0005] Disclosed herein is a plasmon resonance sensing device. In some embodiments, the plasmon resonance sensing device comprises: (1) an array of sensors, each sensor comprising an array of nanostructures regularly spaced apart with a spacing of about 100 nm to about 2000 nm between the nanostructures; (2) each of the nanostructures having a square shape with a side dimension of about 50 nm to about 400 nm; and (3) the nanostructures being conjugated with a biological probe configured to bind to an analyte. In some embodiments, the nanostructures have a thickness of about 20 nm to about 75 nm. In some embodiments, the nanostructures contain gold. In some embodiments, the biological probe is selected from the group consisting of peptide-nucleic acids, aptamers, antibodies, antibody fragments, complementary DNAs, and enzymes. In some embodiments, at least a first sensor within the array of sensors comprises nanostructures conjugated with a first biological probe, and at least a second sensor within the array of sensors comprises nanostructures conjugated with a second biological probe.

[0006] Also disclosed herein is a method for detecting an analyte in a sample. In some embodiments, the method comprises: (1) exposing at least one sensor in the plasmon resonance sensing device according to claim 1 to the sample, wherein the analyte in the sample binds to the at least one sensor; (2) irradiating the at least one sensor with light of a series of wavelengths; and (3) collecting absorbance, transmittance, or extinction data of the sensor. In some embodiments, the method further comprises heating the at least one sensor after exposing the at least one sensor to the sample. In some embodiments, the method further comprises comparing the collected absorbance, transmittance, or extinction data with the baseline data of the sensor before exposure to the sample. In some embodiments, an array of sensors in any of the plasmon resonance sensing devices of the present disclosure is exposed to the sample. In some embodiments, at least a first sensor in the sensor array comprises a nanostructure conjugated with a first biological probe, and at least a second sensor in the sensor array comprises a nanostructure conjugated with a second biological probe. In some embodiments, the first biological probe and the second biological probe are independently selected from the group consisting of protein-nucleic acid, aptamer, antibody, antibody fragment, complementary DNA, and enzyme. In some embodiments, the first biological probe is different from the second biological probe.

[0007] In some embodiments, the method further comprises flowing a plurality of functionalized particles over the at least one sensor after exposing the at least one sensor to the sample, wherein the plurality of functionalized particles are configured to bind to the analyte bound to the at least one sensor.

[0008] This specification also discloses a method of fabricating an array of nanostructures. In some embodiments, the method includes: (1) coating a conductive photoresist layer on a non-conductive substrate; (2) patterning the conductive photoresist layer via lithography to form a patterned substrate; (3) depositing an adhesion layer on the patterned substrate; and (4) depositing a metal layer on the adhesion layer. In some embodiments, the metal layer includes gold. In some embodiments, the metal layer has a thickness of from about 20 nm to about 75 nm. In some embodiments, the adhesion layer includes chromium. In some embodiments, the adhesion layer has a thickness of about 5 nm. In some embodiments, the nanostructures are regularly spaced apart at intervals of from about 100 nm to about 2000 nm, and each of the nanostructures has a square shape with a side dimension of from about 50 nm to about 400 nm.

[0009] Disclosed herein is a method of fabricating a functionalized nanoplasmonic sensing chip. In some embodiments, the method includes: (1) providing a substrate comprising an array of sensors, each sensor comprising an array of nanostructures; (2) attaching a microwell adapter on top of the substrate, thereby providing an array of microwells aligned therewith over the array of sensors; (3) forming one or more functionalized sensors within the array of sensors; and (4) removing the microwell adapter from the substrate. The step of forming one or more functionalized sensors within the array of sensors includes delivering a first batch of reaction solution to one or more microwells over one or more sensors and subsequently removing the first batch of reaction solution from the one or more microwells, the delivery and removal of the first batch of reaction solution being performed by an automated pipetting system. In some embodiments, the step of forming one or more functionalized sensors further includes delivering a second batch of reaction solution into the one or more microwells and subsequently removing the second batch of reaction solution from the one or more microwells, the delivery and removal of the second batch of reaction solution being performed by an automated pipetting system.

[0010] In some embodiments, the one or more functionalized sensors comprise one or more biological probes. In some embodiments, each of the one or more biological probes is independently selected from the group consisting of peptide-nucleic acids, oligonucleotides, aptamers, antibodies, antibody fragments, complementary DNAs, and enzymes. In some embodiments, the one or more biological probes are the same. In some embodiments, all of the one or more biological probes are different. In some embodiments, the first batch of reaction solution is delivered to the one or more microwells simultaneously.

[0011] Also disclosed herein is a functionalized nanoplasmonic sensing chip. In some embodiments, the functionalized nanoplasmonic sensing chip comprises an array of functionalized sensors. In some embodiments, each of the functionalized sensors in the array comprises an array of nanostructures conjugated to at least one biological probe configured to bind to at least one analyte. In some embodiments, the array of nanostructures is conjugated to two or more biological probes configured to bind to two or more analytes. In some embodiments, at least one biological probe is independently selected from the group consisting of peptide-nucleic acids, oligonucleotides, aptamers, antibodies, antibody fragments, complementary DNAs, and enzymes. In some embodiments, at least one of the functionalized sensors in the array comprises at least one biological probe different from the others. In some embodiments, each of the functionalized sensors in the array comprises at least one different biological probe. In some embodiments, the nanostructures contain gold. In some embodiments, the nanostructures in the array are regularly spaced apart at intervals of about 100 nm to about 2000 nm, and each nanostructure has a square shape with a side dimension of about 50 nm to about 400 nm. In some embodiments, the nanostructures have a thickness of about 20 nm to about 75 nm.

[0012] Also disclosed herein is a method for simultaneously detecting two or more analytes. In some embodiments, the method comprises exposing an array of functionalized sensors on a functionalized nanoplasmonic sensing chip of any of the embodiments of the present invention to a sample, irradiating each of the functionalized sensors with light of a series of wavelengths, and collecting absorbance, transmittance, and / or extinction data for each functionalized sensor. In some embodiments, the method further comprises comparing the collected absorbance, transmittance, or extinction data for each functionalized sensor with the baseline data for each functionalized sensor prior to exposure to the sample. In some embodiments, the method further comprises heating the array of functionalized sensors after exposing the array of functionalized sensors to the sample. In some embodiments, up to 50 analytes in the sample are detected.

[0013] It should be understood that all combinations of the concepts described above and additional concepts detailed below are intended to be part of the subject matter of the invention disclosed herein and can be used to achieve the benefits and advantages described herein.

Brief Description of the Drawings

[0014] The features of the embodiments of the present disclosure will become apparent by referring to the following detailed description and the drawings. Like reference numerals correspond to components that are similar, but perhaps not identical. For the sake of brevity, reference numerals or features having the above-described functions may or may not be described in connection with other drawings in which they appear.

[0015]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11A

Figure 11B

Figure 11C

Figure 11D

Figure 11E

Figure 11F

Figure 11G

Figure 11H

Figure 11I

Figure 12

DETAILED DESCRIPTION OF THE INVENTION

[0016] All patents, applications, published applications, and other publications referred to herein are hereby incorporated by reference in their entirety. If a term or phrase is used herein in a manner that contradicts or conflicts with the definition set forth in a patent, application, published application, or other publication incorporated by reference herein, the use herein shall prevail over the definition incorporated by reference herein.

[0017] A plasmon resonance sensing device employing a regular array of nanostructures is described herein. The regular array of nanostructures enables coupling into diffractive photon modes and can thus be used to improve sensor sensitivity. The dimensions and geometric shapes of the nanostructures are adjusted to provide high-quality signals and large optical shifts upon modeled analyte binding.

[0018] The present disclosure generally relates to plasmon resonance sensing devices employing regular arrays of nanostructures, and methods for detecting analytes using plasmon resonance sensing devices. Also disclosed herein are methods of fabricating nanostructure arrays, and the use of full-wave electromagnetic simulations coupled to experiments for the design of nanoplasmonic arrays for biosensing. The regular array of nanostructures enables coupling into diffractive photonic modes, thereby improving sensor sensitivity. The dimensions and geometric shapes of the nanostructures are tailored to provide high-quality signals and large optical shifts upon modeled analyte binding. The plasmon resonance sensing devices disclosed herein include rational design of sensor arrays for nanoplasmonic transfection of analyte binding. In some embodiments, the geometric shapes of nanoparticle arrays may be utilized to detect DNA sequences. The nanostructure array geometry design enables high-sensitivity and high-quality factor biosensing.

[0019] Plasmon resonance sensing device Disclosed herein is a plasmon resonance sensing device. As shown in FIGS. 1A and 1B, the plasmon resonance sensing device 100 includes an array of sensors 101. Each sensor 101 includes an array of regularly spaced nanostructures 102. In some embodiments, the nanostructures 102 are regularly spaced at intervals of about 100 nm, about 200 nm, about 300 nm, about 500 nm, about 750 nm, about 1000 nm, about 1200 nm, about 1500 nm, about 1800 nm, about 2000 nm, or any distance between about 100 nm and about 2000 nm between the nanostructures. In some embodiments, the array of nanostructures is regularly spaced at intervals of about 100 nm to about 2000 nm, about 100 nm to about 1800 nm, about 100 nm to about 1600 nm, about 100 nm to about 1400 nm, about 100 nm to about 1200 nm, about 100 nm to about 1000 nm, about 200 nm to about 900 nm, about 300 nm to about 800 nm, about 100 nm to about 400 nm, about 200 nm to about 500 nm, about 300 nm to about 600 nm, about 400 nm to about 700 nm, about 500 nm to about 800 nm, about 600 nm to about 900 nm, about 700 nm to about 1000 nm, about 500 nm to about 2000 nm, or about 500 nm to about 1500 nm between the nanostructures.

[0020] The nanostructures in the array may have various shapes. For example, the nanostructures may be rectangular, circular, triangular, star-shaped, pentagonal, parallelogram-shaped, diamond-shaped, or square-shaped. Preferably, each of the nanostructures in the array has a square shape. In some embodiments, each nanostructure has a side dimension of about 50 nm, about 75 nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm, about 300 nm, about 350 nm, or about 400 nm, or any integer between about 50 and about 400 nm. In some embodiments, the square has a side dimension in any range of about 50 nm to about 400 nm, about 100 nm to about 350 nm, 150 nm to about 300 nm, about 50 nm to about 150 nm, about 100 nm to about 200 nm, 150 nm to about 250 nm, about 200 nm to about 300 nm, about 250 nm to about 350 nm, or about 300 nm to about 400 nm, or about 50 nm to about 400 nm.

[0021] In some embodiments, the nanostructures in the array may have a thickness of about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, or any integer between about 20 and about 75 nm. In some embodiments, the nanostructures in the array may have a thickness in any range from about 20 nm to about 75 nm, from about 25 nm to about 70 nm, from about 30 nm to about 65 nm, from about 35 nm to about 60 nm, from about 30 nm to about 55 nm, or from about 20 to about 75 nm.

[0022] The nanostructures contain a metal. For example, the nanostructures may contain gold, platinum, aluminum, silver, or copper. Preferably, the nanostructures contain gold. In some embodiments, the nanostructures contain a single metal. In some embodiments, the nanostructures contain a mixture of metals.

[0023] In some embodiments, the nanostructures in the array are conjugated to a biological probe. The biological probe is configured to bind to an analyte. Binding of the analyte to the biological probe changes the surface properties of the nanostructures, thereby causing a change in the localized surface plasmon resonance. In some embodiments, the biological probe contains one or more of a protein, a peptide chain, an amino acid, an RNA chain, a DNA chain, and / or a nucleotide. In some embodiments, the biological probe contains one or more of a modified protein, a modified peptide, a modified amino acid, a modified RNA chain, a modified DNA chain, and / or a modified nucleotide. In some embodiments, the biological probe contains one or more of a peptide-nucleic acid, an aptamer, an antibody, an antibody fragment, a complementary DNA, and / or an enzyme. In some embodiments, the biological probe is selected from the group consisting of a peptide-nucleic acid, an aptamer, an antibody, an antibody fragment, a complementary DNA, and an enzyme.

[0024] In some embodiments, at least a first sensor 101a within the array of sensors includes nanostructures 102 conjugated to a first biological probe. In some embodiments, at least a second sensor 101b within the array of sensors includes nanostructures conjugated to a second biological probe. In some embodiments, at least a third sensor within the array of sensors includes nanostructures conjugated to a third biological probe. In some embodiments, at least a fourth sensor within the array of sensors includes nanostructures conjugated to a fourth biological probe. In some embodiments, at least a fifth sensor within the array of sensors includes nanostructures conjugated to a fifth biological probe. In some embodiments, the "n" sensors within the array of sensors include nanostructures conjugated to "n" biological probes, where "n" is any number from 1 to 2000. In some embodiments, 6 or 12 sensors may be present in the array of sensors on the array substrate 103. In some embodiments, the sensor may be about 1 μm 2 to about 1 mm 2 in area. In some embodiments, the sensor may be about 10 μm 2 to about 1 mm 2 , about 50 μm 2 to about 1 mm 2 , about 100 μm 2 to about 1 mm 2 , about 200 μm 2 to about 1 mm 2 , about 400 μm 2 to about 1 mm 2 , or about 500 μm 2 to about 1 mm 2 in area.

[0025] The substrate 103 may be a dielectric or non-conductive substrate. In some embodiments, the substrate 103 is transparent and the sensor is exposed to incident light through the substrate 103. For example, the substrate 103 may be a glass substrate, a plastic substrate, or a polymer substrate. In some embodiments, the substrate 103 may be a polymer substrate or a plastic substrate. The substrate and the sensor array on the substrate may be integrated with a microfluidic module to provide means for introducing or exposing a sample to the sensor.

[0026] Analyte Detection Disclosed herein is a method for detecting an analyte in a sample. In some embodiments, the method includes exposing at least one sensor 101 within a plasmon resonance sensing device 100 of any of the embodiments disclosed herein to the sample. The sample may or may not contain the target analyte. The plasmon resonance sensing device 100 can be utilized to detect the presence of an analyte (i.e., the target analyte). In some embodiments, the method includes exposing at least two sensors within a plasmon resonance sensing device 100 of any of the embodiments disclosed herein to the sample. In some embodiments, the method includes exposing at least three sensors, at least four sensors, at least five sensors, or at least six sensors within a plasmon resonance sensing device 100 of any of the embodiments disclosed herein to the sample. In some embodiments, the method includes exposing "n" sensors within a plasmon resonance sensing device of any of the embodiments disclosed herein to the sample, where "n" is any number from 1 to 2000. In some embodiments, an array of sensors is exposed to the sample. The sample may include a body fluid such as blood, plasma, mucus, serum, urine, or saliva. Mucus can be collected via a cervical swab, a vaginal swab, or a nasal swab. When at least one sensor 101 is exposed to the sample, the biological probe within each sensor will selectively bind to an analyte configured such that the biological probe binds thereto.

[0027] Optionally, at least one sensor may be subjected to a heating step after exposure to the sample. In some embodiments, at least one sensor is heated to up to about 85 °C or to any temperature between 25 °C and 85 °C. In some embodiments, at least one sensor may be exposed to heat before, during, or after a subsequent step. In some embodiments, at least one sensor may be exposed to heat before, during, or after measurement.

[0028] A method for detecting or sensing an analyte further comprises the step of irradiating light onto at least one sensor. In some embodiments, the method comprises irradiating at least one sensor with light of a series of wavelengths. In some embodiments, the light may be emitted from a light source within an apparatus for analyte detection. The light source may be configured to emit a series of wavelengths for irradiating the sensor. In some embodiments, a plasmon sensing chip comprising the sensor may be inserted into an apparatus for analyte detection. The apparatus is configured to collect the optical spectrum of the light emitted onto the sensor, transmitted through the sensor, absorbed by the sensor, or reflected from the sensor, of a series of wavelengths. For example, the apparatus can perform absorbance / transmittance measurements. In some embodiments, the measurement is performed at wavelengths in the range of 500 - 1000 nm.

[0029] The method further includes the step of collecting data from a sensor. In some embodiments, the method includes the step of collecting absorbance data from a sensor. In some embodiments, the method includes the step of collecting transmittance data from a sensor. In some embodiments, the method includes the step of collecting extinction data from a sensor. In some embodiments, the method includes the step of collecting absorbance, transmittance, and / or extinction data of the sensor. In some embodiments, the method further includes the step of comparing the collected data with the baseline data of the sensor before sample exposure. In some embodiments, the method further includes the step of comparing at least one of the collected absorbance, transmittance, and / or extinction data with the baseline data of the sensor before sample exposure. For example, the absorbance / transmittance measurement of the functionalized sensor is performed before exposure to the sample. Identify the peak absorbance wavelength of the functionalized sensor (before binding to the target analyte). When the target analyte is present in the sample and binds to the probe on the functionalized sensor, the absorbance / transmittance of the sensor is measured again after exposure to the sample, and a shift in the peak absorbance can be observed. The shift represents the detection signal.

[0030] In some embodiments, an array of sensors within any of the plasmon resonance sensing devices 100 of this embodiment is exposed to a sample. In some embodiments, at least a first sensor 101a within the array of sensors 101 includes nanostructures conjugated to a first biological probe. In some embodiments, at least a second sensor 101b within the array of sensors 101 includes nanostructures conjugated to a second biological probe. In some embodiments, at least a third sensor within the array of sensors includes nanostructures conjugated to a third biological probe. In some embodiments, at least a fourth sensor within the array of sensors includes nanostructures conjugated to a fourth biological probe. In some embodiments, at least a fifth sensor within the array of sensors includes nanostructures conjugated to a fifth biological probe. In some embodiments, the "n" sensors within the array of sensors include nanostructures conjugated to "n" biological probes, where "n" is any number from 1 to 2000. The biological probes conjugated to different sensors may be the same or different. In some embodiments, each sensor in the array can be conjugated to different biological probes for multiplex sensing capabilities. In this configuration, multiple analytes can be detected simultaneously.

[0031] In some embodiments, at least a first sensor 101a within the array of sensors comprises nanostructures conjugated to a first biological probe, and at least a second sensor 101b within the array of sensors comprises nanostructures conjugated to a second biological probe. In some embodiments, a first set of sensors within the sensor array is functionalized with a first biological probe, and a second set of sensors within the sensor array is functionalized with a second biological probe. In some embodiments, the first biological probe and the second biological probe are different. In some embodiments, the first biological probe and the second biological probe are the same. In some embodiments, the first biological probe and the second biological probe independently comprise one or more of a protein, a peptide chain, an amino acid, an RNA strand, a DNA strand, and / or a nucleotide. In some embodiments, the first biological probe and the second biological probe independently comprise one or more of a modified protein, a modified peptide, a modified amino acid, a modified RNA strand, a modified DNA strand, and / or a modified nucleotide. In some embodiments, the first biological probe and the second biological probe independently comprise one or more of a peptide-nucleic acid, an aptamer, an antibody, an antibody fragment, a complementary DNA, and / or an enzyme. In some embodiments, the first biological probe and the second biological probe are independently selected from the group consisting of a peptide-nucleic acid, an aptamer, an antibody, an antibody fragment, a complementary DNA, and an enzyme.

[0032] The detection of the analyte is based on an optical phenomenon (localized surface plasmon resonance (LSPR)) that occurs between the metal nanostructure and the dielectric. LSPR is observed when the wavelength of the incident light is larger than the size of the conductive nanostructure. The nanostructure creates a highly confined electric field of the LSPR mode, which functions as a sensitivity transducer to changes in the local dielectric environment (binding event). The nanostructure can be conjugated / covalently functionalized with a probe that can bind to the target analyte. When binding to the target analyte, a red shift of the spectral peak can be observed. In some embodiments, the amount of the red shift can be observed as a function of the target analyte concentration. In some embodiments, the sensor detects transmittance, reflectance, and / or absorbance in a certain wavelength range.

[0033] In some embodiments, the sensor exposed to the sample, and thus the sensor having the analyte bound to the selective biological probe on the sensor, can be further exposed to functionalized particles configured to bind to the sensor having the analyte bound to the biological probe. The functionalized particles can be nanoparticles or microparticles. In some embodiments, the particles can be metal, polymer, glass, or any material having a high refractive index (e.g., a refractive index of about 1.5 or greater). When the functionalized particles are bound to the sensor, there is a possibility of improving both the sensitivity and specificity of the sensor. Without being bound by theory, the improvement in sensitivity may be due to the fact that the functionalized particles increase the change in refractive index at the sensor surface in the presence of the analyte. The additional binding of the functionalized particles to the sensor can improve the sensor signal through a larger peak shift in the optical measurement. The improvement in specificity may be due to the fact that two selective binding events are required (i.e., the first analyte must bind to the sensor, and then the functionalized particle must bind to the analyte bound to the sensor). In some embodiments, the functionalized particles are functionalized to bind to the analyte bound to the biological probe.

[0034] In some embodiments, the spectrum of a sensor comprising an array of functionalized nanostructures can be obtained prior to exposure to a sample. This may provide baseline data for the determination and analysis of analyte binding events.

[0035] Fabrication of Nanostructures Also disclosed herein is a method of fabricating an array of nanostructures. The method includes coating a photoresist layer on a substrate, patterning the photoresist, and depositing a metal layer on the patterned photoresist layer. In some embodiments, the substrate may be non-conductive, and the modified method may provide improved results. The method includes coating a conductive photoresist layer on a non-conductive substrate, patterning the conductive photoresist layer via photolithography, depositing an adhesion layer on the patterned conductive photoresist layer, and depositing a metal layer on the adhesion layer. In some embodiments, patterning the conductive photoresist layer includes exposing the photoresist layer to an electron beam to generate a desired pattern. In some embodiments, the pattern should match the dimensions of the nanostructures and the spacing between the nanostructures. In some embodiments, the method may involve lithography techniques such as electron beam lithography, UV photolithography, or nanoimprint lithography. In some embodiments, roll-to-roll manufacturing may be employed to fabricate the sensor array.

[0036] For example, using photolithography, a portion of the photoresist layer where the nanostructure is to be disposed / formed on the substrate may be removed, leaving a portion of the substrate that should have no nanostructures masked by the patterned photoresist layer. Thus, the patterned photoresist layer has a removed portion similar in size, shape, and position to the location where the metal nanostructure is to be disposed. The portion of the substrate is exposed at the location where the nanostructure is to be formed. When a metal layer is then disposed on the patterned photoresist layer, a portion of the metal layer is disposed on the exposed portion of the substrate, and a portion of the metal layer is disposed on the remaining photoresist masking the substrate.

[0037] The method further includes the step of lift-off of the patterned photoresist layer. When the patterned photoresist layer is lift-off, the portions of the adhesion layer and the metal layer disposed on the remaining patterned photoresist layer are also removed, leaving the portion of the adhesion layer in contact with the substrate and the portion of the metal layer on that portion of the adhesion layer. In some embodiments, the adhesion layer includes chromium. In some embodiments, the adhesion layer has a thickness of about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 8 nm, about 9 nm, or any thickness from about 2 to about 9 nm. In some embodiments, the adhesion layer has a thickness of about 5 nm. In some embodiments, the metal layer includes a single metal. In some embodiments, the metal layer includes a mixture of metals. In some embodiments, the metal layer includes gold, silver, aluminum, platinum, or copper. In some embodiments, the metal layer includes gold. The thickness of the metal layer is the same as the thickness of the nanostructure on the substrate disclosed herein.

[0038] The method disclosed herein provides an array of sensors comprising an array of regularly spaced nanostructures. The shape, dimensions, and spacing of the nanostructures fabricated by such a method are the same as those disclosed herein.

[0039] Functionalization of Nanoplasmonic Sensing Chips Disclosed herein is a method of fabricating a functionalized nanoplasmonic sensing chip. The method includes providing a substrate having an array of sensors, attaching a microwell adapter on the substrate such that an array of microwells is over the array of sensors and aligned with each sensor, and forming one or more functionalized sensors within the array of sensors. The step of forming one or more functionalized sensors includes delivering, using an automated pipetting system, a first batch of a reaction solution into one or more microwells over one or more sensors, and then subsequently removing, using the automated pipetting system, the first batch of the reaction solution from the one or more microwells. The automated pipetting system includes an array of pipettes capable of loading one or more reaction solutions. In some embodiments, the array of pipettes may be loaded with two or more different reaction solutions, and thus delivery of two or more different reaction solutions to the array of microwells / sensors is enabled. Also, the array of pipettes may be used to remove the reaction solution from some or all of the microwells / sensors after the reaction. The array of pipettes can deliver or remove the reaction solution from a particular microwell / sensor or a particular group of microwells / sensors. In some embodiments, each reaction solution may include one or more reagents for modifying an array of nanostructures within the sensor. In some embodiments, each reaction solution may include one or more biological probes.

[0040] In some embodiments, a multi-step reaction may be utilized to functionalize the sensors. Thus, the step of forming one or more functionalized sensors may further include delivering a second batch of a reaction solution into one or more microwells and subsequently removing the second batch of the reaction solution from the one or more microwells, the delivery and removal of the second batch of the reaction solution being performed by the automated pipetting system.

[0041] In some embodiments, the first batch of reaction solution comprises two or more different reaction solutions. In some embodiments, the second batch of reaction solution may also comprise two or more different reaction solutions. In some embodiments, the reaction solution may comprise different biological probes. Thus, an array of functionalized sensors may comprise two or more different biological probes. For example, some of the functionalized sensors in the array may comprise a particular biological probe, while other functionalized sensors comprise different biological probes. In some embodiments, each of the functionalized sensors may comprise a different biological probe. In some embodiments, the reaction solution may comprise one or more biological sensors. Thus, each functionalized sensor may comprise one or more biological probes. The one or more biological probes can conjugate to an array of nanostructures within each sensor. In some embodiments, the sensor may comprise one, two, three, four, or more biological probes configured to bind to one or more analytes.

[0042] The method then further comprises removing the microwell adapter from the substrate. In some embodiments, one or more sensors are functionalized with biological probes while the first batch of reaction solution within one or more microwells is in contact with the sensors. In some embodiments, one or more sensors are functionalized with biological probes after two or more reaction steps. In some embodiments, the sensors (e.g., one or more sensors) each comprise an array of nanostructures disclosed herein.

[0043] In some embodiments, the automated pipetting system can be configured to deliver different reaction solutions to a plurality of microwells in order to functionalize a plurality of sensors within the array. In some embodiments, a plurality of reaction solutions are delivered to different sensors within the array, thereby functionalizing the plurality of sensors substantially simultaneously. In some embodiments, the automated pipetting system may be configured to remove different reaction solutions from the plurality of microwells. In some embodiments, the plurality of reaction solutions are removed substantially simultaneously from different sensors within the array. In other embodiments, some of the reaction solutions can be removed at different times to make the reaction time longer or shorter.

[0044] Figures 11A-11B show two alternative views of the 3D printed mold for the fabricated polymer wells shown in Figures 11C-11D. Other embodiments of the microwells are shown in Figures 11E-11I.

[0045] In some embodiments, additional pretreatment steps can be performed before delivering any reaction solution. The pretreatment steps may include cleaning the nanostructured surface, wetting the nanostructured surface, or activating the nanostructures for subsequent reaction / functionalization. In some embodiments, the method may include delivering an activation solution to at least a portion of the microwells on the sensors within the array using an automated pipetting system, and then removing the activation solution before delivering the reaction solution.

[0046] The methods disclosed herein provide at least one functionalized sensor comprising at least one biological probe. In some alternatives, the first functionalized sensor comprises a first array of nanostructures conjugated to a first biological probe. In some alternatives, the second functionalized sensor comprises a second array of nanostructures conjugated to a second biological probe. In some alternatives, additional sensors comprising nanostructure arrays may be conjugated to additional biological probes up to the number of sensors in the sensor array. For example, an “n” number of sensors in an array of sensors includes nanostructures conjugated to “n” biological probes, where n is any number from 1 to 2000. In some embodiments, n may be any number from 1 to 1000, 1 to 500, 1 to 100, or 1 to 25.

[0047] Each of the biological probes is independently selected from the group consisting of peptide-nucleic acid (PNA), oligonucleotide, aptamer, antibody, antibody fragment, complementary DNA, and enzyme. In some alternatives, the first biological probe and the second biological probe are independently selected from the group consisting of peptide-nucleic acid, aptamer, antibody, antibody fragment, complementary DNA, and enzyme. In some alternatives, the first biological probe and the second biological probe are different. In some alternatives, the first biological probe and the second biological probe are the same. In some embodiments, each sensor may be functionalized with a different biological probe. In some embodiments, some of the sensors in the array may be functionalized with different biological probes. In some embodiments, all of the sensors in the array may be functionalized with the same biological probe.

[0048] In some embodiments, the reaction solution is delivered to all the microwells simultaneously. In some alternatives, subsequently, the reaction solution is removed from the microwells simultaneously. In some alternatives, the reaction solution is removed from the microwells at different times to accommodate different reaction times for functionalizing the sensors with various biological probes. In some embodiments, the reaction solution may also be delivered to different microwells at different times. In some alternatives, a first reaction solution and a second reaction solution are delivered to a first microwell and a second microwell simultaneously, and then the first reaction solution and the second reaction solution are removed from the first microwell and the second microwell. In some embodiments, the delivery and removal of the reaction solution may be performed by an automated pipetting system. In some embodiments, the automated pipetting system may be configured to remove different reaction solutions at different times. In some embodiments, the automated pipetting system may be configured to deliver different reaction solutions at different times.

[0049] In some embodiments, the nanostructure contains a metal. In some alternatives, the nanostructure contains a single metal. In some alternatives, the nanostructure contains a mixture of metals. In some alternatives, the nanostructure contains gold, platinum, aluminum, silver, or copper. In some alternatives, the nanostructure contains gold.

[0050] Functionalized Plasmonic Sensing Chip Disclosed is a functionalized plasmon sensing chip comprising an array of functionalized sensors. In some embodiments, each of the functionalized sensors in the array comprises an array of nanostructures conjugated to at least one biological probe. In some embodiments, the array of nanostructures is conjugated to two or more biological probes configured to bind to two or more analytes. The biological probe is configured to bind to at least one analyte. In some embodiments, at least one biological probe independently comprises a peptide-nucleic acid, an oligonucleotide, an aptamer, an antibody, an antibody fragment, a complementary DNA, and / or an enzyme. In some embodiments, the biological probe is independently selected from the group consisting of a peptide-nucleic acid, an oligonucleotide, an aptamer, an antibody, an antibody fragment, a complementary DNA, and an enzyme. In some embodiments, all of the functionalized sensors in the array comprise the same biological probe. In some alternatives, at least one of the functionalized sensors within the array comprises at least one biological probe that is different from other biological probes. For example, some of the functionalized sensors in the array may comprise a particular biological probe, while other functionalized sensors comprise different biological probes. In some embodiments, each of the functionalized sensors in the array comprises at least one different biological probe. One or more biological probes can be conjugated to the array of nanostructures within each sensor. In some embodiments, the functionalized sensor may comprise one, two, three, four, or more biological probes configured to bind to one or more analytes.

[0051] In some embodiments, the functionalized plasmonic sensor chip may include from 1 to 100 (and any number in between) different biological probes. For example, the functionalized plasmonic sensor chip may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 24, 30, 36, 40, 48, 50, 54, 60, 70, 80, 90, or 100 different biological probes. In some embodiments, each functionalized sensor within the functionalized plasmonic sensor chip may include a different biological probe. In some embodiments, the array of nanostructures within each sensor may be conjugated to one or more biological probes, and the one or more biological probes may be different.

[0052] In some embodiments, the nanostructures include a metal. In some alternatives, the nanostructures include a single metal. In some alternatives, the nanostructures include a mixture of metals. In some alternatives, the nanostructures may include gold, platinum, aluminum, silver, or copper. In some alternatives, the nanostructures include gold. In some alternatives, the nanostructures in the array may be regularly spaced and have the geometric shapes described herein.

[0053] Multianalyte Detection Methods for simultaneously detecting two or more analytes are also described. In some alternatives, the method may detect 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 24, 30, 36, 40, 48, 50, 54, 60, 70, 80, 90, and / or 100 analytes. In some embodiments, up to 50 analytes are detected. In some embodiments, up to 24, 50, 80, or 100 analytes may be detected. The method includes exposing an array of functionalized sensors on any of the plasmon sensing chips of the alternatives disclosed herein to a sample. The functionalized sensors are configured to detect the presence of a particular target analyte. In some embodiments, the functionalized sensors may be configured to identify or detect various markers, subtypes, strains, genotypes, and / or variants of a biological species. When the functionalized sensors are exposed to the sample, one or more target analytes, if present, bind to the corresponding biological probes. The binding event causes a change in the local dielectric environment of the sensor. The sample may include a body fluid such as blood, urine, or saliva. In some embodiments, the sample may be drained or removed from the functionalized sensors after the exposure step.

[0054] Optionally, the array of functionalized sensors may be subjected to a heating step after exposure to the sample. In some embodiments, the array of functionalized sensors is heated to up to about 85 °C or to any temperature between 25 °C and 85 °C. In some embodiments, the array of functionalized sensors may be exposed to heat before, during, or after a subsequent step. In some embodiments, the array of functionalized sensors may be exposed to heat before, during, or after measurement.

[0055] This method further includes the steps of irradiating a functionalized sensor with light of a series of wavelengths and collecting absorbance, transmittance, and / or extinction data from the functionalized sensor. The light may be emitted from a light source within the device for analyte detection. The light source may be configured to emit a series of wavelengths for irradiating the sensor. In some embodiments, a plasmon sensing chip including the functionalized sensor may be inserted into the device for analyte detection. The device is configured to emit light of a series of wavelengths onto the functionalized sensor and collect the optical spectrum of the light transmitted through, absorbed by, or reflected from the sensor.

[0056] In some embodiments, this method further includes the step of comparing the collected data with the baseline data of the sensor before sample exposure. The baseline data of the functionalized sensor can be collected using the device for analyte detection described above. In some embodiments, the baseline data can be collected before exposing the sensor to the sample. In some embodiments, the baseline data is provided for a sensor functionalized with a specific biological probe. A shift in the spectral peak after sample exposure indicates the binding of the target analyte to the biological probe and thus the presence of the target analyte in the sample. In some embodiments, the amount of spectral peak shift may further be interpreted as providing a quantitative or semi - quantitative measurement of the concentration of the target analyte in the sample.

[0057] In some embodiments where the sensors in the array are functionalized with different biological probes, when the array is exposed to a sample, various target analytes may bind to the corresponding sensors. By irradiating the sensor array with light of a series of wavelengths, the optical spectrum of each sensor can be collected and compared with the baseline data. It becomes possible to detect and identify different target analytes with just one exposure of the sensing device chip.

[0058] In some embodiments, the plasmon resonance sensing device enables point-of-care (POC) detection of a target analyte and POC diagnosis of a disease / condition. In some embodiments, rapid results (e.g., less than about 15 minutes) can be provided.

[0059] Definitions 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 disclosure pertains, unless otherwise clearly indicated.

[0060] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an array” can include a plurality of such arrays and the like.

[0061] The terms “comprising,” “including,” “containing,” and various forms of these terms are synonymous with each other and are meant to be equally broad. Further, examples that comprise, include, or have one or more elements with a particular property can include additional elements, whether or not the additional elements have that property, unless the contrary is explicitly stated.

[0062] As used herein, the term "nanostructure" has its standard scientific meaning and thus refers to any structure that is approximately molecular-sized to approximately microscopic-sized. Nanostructures include nanomaterials, which may be any material where the individual unit is on the order of about 1 nm to about 200 nm in size. Nanostructures include, but are not limited to, nanoparticles, nanorods, nanosquares, nanocubes, gradient multilayer nanofilm (GML nanofilm), icosahedral twins, nanocages, magnetic nanochains, nanocomposites, nanofibers, nanofibers, nanoflowers, nanofoams, nanoholes, nanomeshes, nanopillars, nanopin films, nanoplatelets, nanoribbons, nanorings, nanobipyramids, irregular nanoparticles, nanosheets, nanoshells, nanotips, nanowires, and nanostructured films. It is understood that nanostructures can have various geometric shapes and properties based on the components of the nanostructure.

[0063] The term "analyte" refers to the substance or chemical component of interest. For example, an analyte may be a biological or chemical substance that can be detected by a sensing device and may be of interest for diagnosing a disease or condition.

[0064] All patents and other publications, including references, issued patents, published patent applications, and co-pending patent applications, are hereby incorporated by reference into this specification for the purpose of describing and disclosing, for example, the methodologies described in such publications that may be used in connection with the technologies described herein. These publications are provided only as of their disclosure prior to the filing date of this application. In this regard, it should not be construed that the inventors admit any right to antedate such disclosure by virtue of prior invention or any other reason. All statements as to the date or content of these documents are based on the information available to the applicant and do not admit the accuracy of the dates or content of these documents.

[0065] The description of embodiments of the present disclosure is not intended to be exhaustive or to limit the present disclosure to the precise forms disclosed. Specific embodiments and examples of the present disclosure are described herein for illustrative purposes, but as will be recognized by those of ordinary skill in the art, various equivalent modifications are possible within the scope of the present disclosure. For example, method steps or functions are presented in a given order, but alternative embodiments may perform the functions in a different order or the functions may be performed substantially simultaneously. The teachings of the present disclosure provided herein can also be applied to other procedures or methods as needed. It is also possible to combine the various embodiments described herein to provide further embodiments. Aspects of the present disclosure can be modified to use the compositions, functions, and concepts of the above references and applications to provide further embodiments of the present disclosure if necessary. Further, considering biological functional equivalency, some changes can be made to the protein structure without affecting the type or amount of biological or chemical action. These and other changes can be made to the present disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0066] Any particular element of any of the foregoing embodiments can be combined with or replaced by an element in other embodiments. Further, while the advantages associated with particular embodiments of the present disclosure have been described in the context of those embodiments, other embodiments can also exhibit such advantages, and it is not necessary for all embodiments to exhibit such advantages in order to fall within the scope of the present disclosure.

Examples

[0067] The techniques described herein are further illustrated by the following examples, which should in no way be construed as further limiting.

[0068] Example 1: Electromagnetic Simulation Several geometric shapes for simulation and testing included several nanorods and several coupled nanoarrays. The nanorods were designed to reflect randomly oriented colloidal nanorods dispersed on a glass slide. The coupled nanoarrays were designed to generate surface lattice resonances. The seven geometric shapes for the fabrication dose test are as shown in Table 1 and FIGS. 2A and 2B. FIG. 2A shows a grid with dimensions labeled for the length (l), width (w), thickness (t), and spacing / periodicity (p) of the nanorods. FIG. 2B is a map of the arrangement of the nanorod arrays within the sensor unit. As shown in Table 1, the test geometric shapes T1-T3 are nanorods, and the test geometric shapes T4-T10 are coupled nanoarrays.

Table 1

[0069] Full-wave electromagnetic simulations were performed using Lumerical photonic simulation software. As shown in FIGS. 2A-2B, for each of the geometric shapes T1-T7, periodic boundary conditions were applied in the x and y dimensions. In the bulk sensing experiment, the refractive index of the surrounding medium was varied. For the PNA-DNA binding experiment, a conformal shell layer with a defined refractive index was modeled on the nanostructure. Extinction and transmittance curves were returned in the wavelength range of 400-1200 nm.

[0070] Example 2: Definition of Simulation Settings and Performance Indices To study the sensitivity of the plasmon resonance shape and refractive index changes, the initial simulation included a bulk refractive index sensitivity analysis. In the first iteration, gold nanorods with wide spacing designed to represent a regular nanoarray were tested.

[0071] Resonance was modeled in air, water, and glycerol (increasing refractive index), and the peak position was calculated for each of the extinction curves. This enabled the development of a figure of merit (FOM) for the sensor that takes into account the peak shift(s) and resonance width (full width at half maximum - FWHM), as shown in Figure 3. The figure of merit was defined as the shift over the full width at half maximum, enabling a direct comparison between different geometric shapes. A larger figure of merit indicates superior sensing performance due to (1) a larger peak shift for the same refractive index change and (2) easier discrimination of the peak shift due to the narrow resonance curve. This analysis was repeated for all geometric shapes considered.

[0072] Example 3: PNA - DNA Binding Simulation Another way to simulate these nanostructures involves simulating peptide nucleic acid (PNA) probes bound to DNA and a conformal layer having the same refractive index as the expected refractive index of the PNA probes. It was observed that the shift of PNA + DNA binding with respect to the surface lattice geometry (shown in Figure 4B) is much more prominent than the shift with respect to the dispersed nanorod geometry (shown in Figure 4A). These simulations point out the expected shifts associated with DNA biosensing for each geometry.

[0073] Example 4: Fabrication of Nanoparticle Sensors Electron beam lithography is a common method for patterning accurate nanoscale features on a substrate. Typically, such patterns are processed on a silicon wafer that is optically opaque and highly conductive. For the transmission mode operation of the sensor, the nanostructures were configured to be located on a transparent quartz wafer. A protocol for nanoscale patterning on a transparent non - conductive surface was developed.

[0074] First, a thin layer of conductive photoresist was spin-coated onto a transparent quartz wafer and then patterned using an electron beam (JEOL E-beam microscope). Subsequently, a thin (approx. 5 nm) chromium adhesion layer was thermally evaporated onto the patterned substrate, followed by the deposition of a thicker (approx. 40 - 50 nm) pure gold layer. After performing chemical lift-off to form the nanostructure array, the substrate was diced for testing. The first sample fabricated with this pattern was a dose matrix test to evaluate the power of the electron beam. After identifying this parameter, all future processes were carried out under the same conditions.

[0075] Example 5: Simulation of Selected Geometric Shapes Bulk refractive simulations were performed for the sample geometries T8 - T10 listed in Table 1. The transmittance through the samples was measured using an optical readout device. The wavelength range was set from 450 nm to 950 nm. For seamless integration with the readout device, the individual sensors of the sensing device were fabricated to have a nanostructure array with an area of 1 mm 2 to be fully aligned with the light source spot size and minimize signal loss. The results for the three surface lattice resonance geometries (T8 - T10) are as shown in Figures 5A / B, 6A / B, and 7A / B respectively. Both the peak shape and the refractive index peak shift are shown. The figure of merit calculated for T8 - T10 was 12.8, 6.7, and 10.7 respectively. Additionally, the refractive index sensitivity for each of these geometries is shown in Figures 5B, 6B, and 7B. All sensitivities are compared to a 220p sample of 140 nm × 40 nm labeled "unbonded nanorod". A steeper gradient indicates better sensing performance. Sample geometry T10 exhibits the best performance due to its high figure of merit (10.7) and its relatively high refractive index sensitivity (267 nm / RIU).

[0076] Example 6: Comparison of Simulation and Experiment The nano-structure array samples 1 to 5 were fabricated with the nano-structure dimensions shown in Table 2. The transmittance of each sample was experimentally measured (shown in Figure 8) and compared with the peak shape from the simulation (shown in Figure 9). A remarkable agreement was found between the experimental data and the simulation data including the peak shape and resonance position.

Table 2

[0077] The present disclosure also presents a methodology for the rational design of regularly spaced nano-particle arrays for plasmon sensing. The applicant tested 5 to 7 geometric shapes through both simulation and experimental analysis and finally selected 145 nm x 145 nm. Through both simulation and experimental analysis, a nano-array geometry showing high-amplitude resonance and refractive index sensitivity may be selected for the fabrication of plasmonic resonance sensing devices.

[0078] Example 7: Functionalization of Nano-structures 1 mm 2 An array of 2 x 6 sensors (a total of 12 sensors) of the sensor was functionalized with peptide-nucleic acid (PNA) probes. Each sensor includes an array of 145 nm × 145 nm gold nano-structures with regular spacing. To individually functionalize the sensor array to be target-specific, a polydimethylsiloxane (PDMS) polymer micro-well array was fabricated. This micro-well array was aligned with the substrate such that each sensor could be accessed through a single micro-well. This approach created repeatable and programmable coordinates for an automated pipetting system (e.g., Integra ASSIST PLUS pipetting robot).

[0079] The micro-well structure on the sensing array enables individual fluid delivery to each sensing spot, allowing multiplexing of up to 12 targets on a single sensing chip. For this purpose, a mold was designed using Solidwaorks CAD, enabling the production of a polymer micro-well array that aligns with the coordinates of the sensor (Figure 10). The mold for casting the PDMS micro-wells was designed in Solidworks consisting of 12 pillars of 2 mm × 2 mm × 5 mm (20 mm3). The pillars were arranged to match the coordinates of the sensor array on the glass substrate. Subsequently, a master mold was fabricated using SLA 3D printing as shown in Figures 11A and 11B.

[0080] The micro-well array device was fabricated in the mold using PDMS soft lithography. Sylgard 184 silicone elastomer, base and curing agent (Dow Corning, Midland, Michigan) were mixed at a weight ratio of 10:1. Next, the PDMS prepolymer was cast onto the master mold and cured in a convection oven at 80 °C for approximately 1.5 hours. The cured PDMS micro-well array was removed from the master mold as shown in Figures 11C and 11D. The polymer micro-well array was fixed onto the sensor array using a washable adhesive, enabling a removable bond for reuse of the sensor. The entire system was mounted onto a standard 75 × 25 mm microfluidic chip and then ready for molecular detection.

[0081] Example 8: Automatic robotic functionalization of the sensor The fabricated plasmonic sensing chip was integrated with an automated pipetting system (e.g., Integra ASSIST Plus) for surface functionalization. To covalently functionalize the selected biological probe, such as a PNA probe, onto the gold nanostructures on the glass substrate, the gold nanostructures were first incubated with 1 mg / mL of dithiobis(succinimidyl propionate) (DSP) dissolved in dimethyl sulfoxide (DMSO) for 20 minutes. This crosslinking molecule activated the gold surface and enabled the binding of free amines on the PNA. Next, the sensor array was contacted with 1 mg / mL of the PNA probe dispersed in Tris-EDTA buffer (pH 7.0) for 30 - 45 minutes. Transmission spectra were collected before and after the conjugation to characterize the successful PNA conjugation.

[0082] The above-described sensor functionalization process was automated using an Integra ASSIST PLUS pipetting robot. To effectively place the devices on the deck of the Integra ASSIST PLUS pipetting robot, a custom 4-slot microscope slide holder / adapter of the size of a standard 96-well plate was designed and fabricated (3D printed). This adapter can be easily integrated with the robotic deck of the liquid handler. The 96-well plates were pre-filled with the functionalization reagents and placed on the aspiration deck of the robot. To start the machine, a Voyager electronic 125 μL 8-channel pipette was loaded onto the robot. A series of six programs were developed to aspirate, dispense, and remove the chips in an automated fashion. These custom programs enable the multiplexed functionalization of 12 PNAs on the sensor array. Table 4 shows the six programs for the automated functionalization of the sensors using the pipetting robot. The programs indicate the pipette tip position, 96-well plate position, aspiration volume, and dispense volume at each step. Figure 12A is a photograph of the Integra ASSIST PLUS pipetting robot 1200, which has a pipette holder 1201 on the left, a tip box 1202, a 96-well plate holder 1203, and a custom tip adapter 1204. Figure 12B shows the tip box 1202 aligned under the pipette holder 1201. Figure 12C shows the 96-well plate 1203 and the adapter 1204 during functionalization.

Table 3

[0083] First, dispense Tris-EDTA (TE) buffer and remove it from the chip surface to clean the surface and ensure the sealing of the microwell array onto the sensing substrate. Next, DSP, a divalent cross-linking molecule, is introduced onto the chip surface and readily adsorbs onto the gold surface within 15 - 20 minutes. The presence of active NHS groups enables cross-linking to proteins (i.e., PNA). An example of a linker for attaching the capture ligand / biological probe (such as PNA) is shown in Table 5. Finally, aspirate the DSP and directly dispense the PNA probe onto the sensing surface and bind it to the free amines on the nanostructure. After aspirating the excess PNA solution, the chip is covalently functionalized with PNA and made ready for sample testing.

Table 4

[0084] Pipetting robot systems have been widely used for fluid filling applications, but as far as the applicant is aware, this is the first time such a system has been employed for covalently attaching a molecular capture probe to a solid sensor. The applicant achieves this through sequential dispensing and aspiration steps on the sensor.

[0085] The scope of the present disclosure is not intended to be limited by the specific disclosure of the examples in this section or elsewhere in this specification, and may be defined by the claims as presented in this section or elsewhere in this specification, or as may be presented in the future. The language of the claims should be construed broadly based on the language employed in the claims and not limited to the examples described in this specification or the examples during the prosecution of this application, and these examples should be construed as non-exclusive.

Claims

1. A plasmon resonance sensing device comprising an array of sensors, Each sensor is equipped with an array of nanomaterials that are regularly spaced apart with intervals of approximately 100 nm to 2000 nm between them. Each of the aforementioned nanomaterials has a square shape with side dimensions of approximately 50 nm to approximately 400 nm. The nanomaterial is conjugated with a biological probe configured to bind to an analyte in a plasmon resonance sensing device.

2. The plasmon resonance sensing device according to claim 1, wherein the nanomaterial has a thickness of about 20 nm to about 75 nm.

3. The plasmon resonance sensing device according to claim 1, wherein the nanomaterial contains gold.

4. The plasmon resonance sensing device according to claim 1, wherein the biological probe is selected from the group consisting of peptide-nucleic acids, aptamers, antibodies, antibody fragments, complementary DNA, and enzymes.

5. A plasmon resonance sensing device according to any one of claims 1 to 4, wherein at least a first sensor in the array of sensors comprises a nanomaterial conjugated with a first biological probe, and at least a second sensor in the array of sensors comprises a nanomaterial conjugated with a second biological probe.

6. A method for detecting analytes in a sample, A step of exposing at least one sensor in the plasmon resonance sensing device according to claim 1 to the sample, wherein the analyte in the sample is coupled to the at least one sensor, The steps include: exposing the at least one sensor to the sample, and then heating the at least one sensor; A step of irradiating at least one sensor with light of a series of wavelengths, The steps include collecting absorbance, transmittance, or extinction data from the aforementioned sensor, Methods that include...

7. The method according to claim 6, further comprising the step of exposing the at least one sensor to the sample, and then flowing a plurality of functionalized particles over the at least one sensor, wherein the plurality of functionalized particles are configured to bind to the analyte bonded to the at least one sensor.

8. The method according to claim 6 or 7, further comprising the step of comparing the collected absorbance, transmittance, or extinction data with baseline data of the sensor before exposure to the sample.

9. The method according to claim 6, wherein the array of sensors in the plasmon resonance sensing device according to claim 1 is exposed to the sample.

10. The method according to claim 9, wherein at least a first sensor in the array of sensors comprises a nanomaterial conjugated with a first biological probe, and at least a second sensor in the array of sensors comprises a nanomaterial conjugated with a second biological probe.

11. The method according to claim 10, wherein the first biological probe and the second biological probe are independently selected from the group consisting of proteins, nucleic acids, aptamers, antibodies, antibody fragments, complementary DNA, and enzymes.

12. The method according to claim 11, wherein the first biological probe and the second biological probe are different.

13. A method for fabricating an array of nanomaterials, The steps include coating a conductive photoresist layer onto a non-conductive substrate, The steps include patterning the conductive photoresist layer via lithography to form a patterned substrate, The steps include depositing an adhesive layer on the patterned substrate, The steps include depositing a metal layer on the aforementioned adhesive layer, Methods that include...

14. The method according to claim 13, wherein the metal layer contains gold.

15. The method according to claim 13, wherein the metal layer has a thickness of about 20 nm to about 75 nm.

16. The method according to claim 13, wherein the adhesive layer contains chromium.

17. The method according to claim 13, wherein the adhesive layer has a thickness of about 5 nm.

18. The method according to any one of claims 13 to 17, wherein the nanomaterials are arranged regularly spaced apart at intervals of about 100 nm to about 2000 nm, and each of the nanomaterials has a square shape with side dimensions of about 50 nm to about 400 nm.

19. A method for fabricating a functionalized nanoplasmon sensing chip, The steps include providing a substrate equipped with a sensor array, The steps include: attaching a microwell adapter to the top of the substrate, thereby providing an array of microwells aligned with the sensor array; A step of forming one or more functionalized sensors within the array of the sensors, Delivering a first batch of the reaction solution to one or more microwells on one or more sensors, Next, the process includes removing the first batch of the reaction solution from one or more microwells, The delivery and removal of the first batch of the reaction solution are performed by an automated pipetting system, step by step. The steps include removing the microwell adapter from the substrate, Methods that include...

20. The method according to claim 19, wherein the first batch of reaction solutions comprises two or more different reaction solutions.

21. The method according to claim 19, wherein the step of forming one or more functionalized sensors further comprises delivering a second batch of reaction solution into the one or more microwells, and subsequently removing the second batch of reaction solution from the one or more microwells, wherein the delivery and removal of the second batch of reaction solution is performed by the automated pipetting system.

22. The method according to claim 21, wherein the second batch of reaction solution comprises two or more different reaction solutions.

23. The method according to claim 19, wherein the one or more functionalized sensors comprises one or more biological probes.

24. The method according to claim 23, wherein each of the one or more biological probes is independently selected from the group consisting of peptide-nucleic acids, oligonucleotides, aptamers, antibodies, antibody fragments, complementary DNA, and enzymes.

25. The method according to claim 23, wherein the one or more biological probes are the same.

26. The method according to claim 23, wherein all of the one or more biological probes are different.

27. The method according to any one of claims 19 to 26, wherein the first batch of the reaction solution is delivered simultaneously into one or more microwells.

28. A functionalized nanoplasmon sensing chip comprising an array of functionalized sensors, wherein each of the functionalized sensors in the array comprises an array of nanomaterials conjugated to at least one biological probe configured to bind to at least one analyte.

29. The functionalized nanoplasmon sensing chip according to claim 28, wherein the array of nanomaterials is conjugated to two or more biological probes configured to bind to two or more analytes.

30. The functionalized nanoplasmon sensing chip according to claim 28, wherein the at least one biological probe is independently selected from the group consisting of peptide-nucleic acids, oligonucleotides, aptamers, antibodies, antibody fragments, complementary DNA, and enzymes.

31. The functionalized nanoplasmon sensing chip according to claim 28, wherein at least one of the functionalized sensors in the array comprises at least one biological probe distinct from the others.

32. The functionalized nanoplasmon sensing chip according to claim 31, wherein each of the functionalized sensors in the array comprises at least one different biological probe.

33. The array or nanomaterial comprises gold, as described in any one of claims 28 to 32, for the functionalized nanoplasmon sensing chip.

34. The functionalized nanoplasmon sensing chip according to claim 28, wherein the nanomaterials in the array are arranged regularly spaced apart at intervals of about 100 nm to about 2000 nm, and each nanomaterial has a square shape with side dimensions of about 50 nm to about 400 nm.

35. The functionalized nanoplasmon sensing chip according to claim 34, wherein the nanomaterial has a thickness of about 20 nm to about 75 nm.

36. A method for simultaneously detecting two or more analytes in a sample, The steps of exposing the array of functionalized sensors on the functionalized nanoplasmon sensing chip according to claim 32 to the sample, The steps include heating the array of the functionalized sensors, A step of irradiating each of the functionalized sensors with light of a series of wavelengths, A step of collecting absorbance, transmittance, and / or extinction data from each functionalized sensor, Methods that include...

37. The method according to claim 36, further comprising the step of comparing the absorbance, transmittance, and / or extinction data collected from each functionalized sensor with the respective baseline data of the functionalized sensor before exposure to the sample.

38. The method according to claim 36 or 37, wherein up to 50 analytes are detected in the sample.

Images