Multiplex pathogen detection using a nanoplasmonic sensor for human papillomavirus

Abstract

This specification discloses a nanoplasmonic sensor for the molecular characterization evaluation of human papillomavirus (HPV) and diseases and disorders associated with HPV infection. In some embodiments, the nanoplasmonic sensor can also be used at the point of care. The nanoplasmonic sensor utilizes an optical phenomenon (localized surface plasmon resonance (LSPR)) that occurs between metal nanoparticles and a dielectric for the detection of viral nucleic acids. In some embodiments, the spectral peak shift is a function of the target sequence concentration.

Description

Technical Field

[0001] (Incorporation of Priority Applications by Reference) This application claims priority to U.S. Provisional Patent Application No. 63 / 352,985, filed Jun. 16, 2022. All applications with foreign or domestic priority claims are hereby incorporated by reference in their entirety.

[0002] (Reference to Sequence Listing) This application is being filed with a Sequence Listing submitted electronically in XML format. The Sequence Listing is provided as a file entitled NPATH.008WOSEQLISTING.xml created on Jun. 15, 2023, which is approximately 8,116 bytes in size. Information on the electronic format of the Sequence Listing is hereby incorporated by reference in its entirety.

[0003] The present disclosure relates to the field of molecular detection. Specifically, the present disclosure describes methods for the functionalization of nanoplasmonic sensors and functionalized nanoplasmonic sensors for the molecular characterization of human papillomavirus (HPV) and subsequent diseases and disorders associated with HPV infection in a subject.

Background Art

[0004] Cervical cancer is the fourth most common cancer in women and accounts for 7.5% of all cancer deaths in women. More than 80% of global cervical cancer cases occur in low- and middle-income countries, and almost all of these cases are caused by the human papillomavirus (HPV). Hundreds of HPV genotypes have been identified. Approximately 40 HPV genotypes affect the genital tract and are classified as either high-risk or low-risk. The current gold standard diagnosis is the Pap smear, which requires a cervical swab and analysis by an experienced cytologist. In developed countries, there are screening programs that identify and treat precancerous lesions early, preventing up to 80% of cervical cancers. In contrast, in low- and middle-income countries, only 5% of women receive a Pap smear test each year, and some women wait up to six months for results. Incorrect information, cultural sensitivity, partner concerns, and female prejudice are exacerbating the problems with testing. These discrepancies in test availability are thought to be the main cause of the high incidence and mortality rates of cervical cancer observed in low-income countries. Introducing more rapid and less invasive screening tests in resource-limited settings could significantly improve treatment rates and the patient outcomes in these regions.

[0005] Many existing and emerging diagnostic platforms use polymerase chain reaction (PCR) for the detection of genotype-specific HPV DNA in cervical swab samples. On average, these tests have higher sensitivity and specificity than other widely used techniques such as Pap smears or visual inspection with acetic acid, but are often too complex for routine screening in low-resource settings. If patients can be stratified as high-risk at the point of care using low-cost HPV genotyping technology, precancer and early cervical cancer may be treated in a single clinic visit. SUMMARY OF THE INVENTION

[0006] Disclosed herein is a nanoplasmonic sensor. In some embodiments, the nanoplasmonic sensor comprises an array of functionalized sensors, each of the functionalized sensors in the array comprising an array of nanostructures conjugated to a biological probe, the biological probe being configured to detect the presence of human papillomavirus. In some embodiments, at least one of the functionalized sensors in the array comprises a biological probe for detecting a segment of human papillomavirus that is different from other functionalized sensors. In some embodiments, the nanoplasmonic sensor is configured to simultaneously detect multiple strands, segments, particles, variants, and / or species of human papillomavirus. In some embodiments, each of the functionalized sensors in the array comprises a different biological probe. In some embodiments, the human papillomavirus is selected from the group consisting of HPV18, HPV16, hrHPV, type HPV16, type HPV18, type HPV31, type HPV33, type HPV35, type HPV39, type HPV45, type HPV51, type HPV52, type HPV56, type HPV58, type HPV59, type HPV66, type HPV68, and derivatives / mutants thereof. In some embodiments, the biological probe has a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. In some embodiments, the nanostructure contains gold. In some embodiments, the nanostructures in the array are regularly spaced apart at intervals of about 100 nm to about 1000 nm, and each nanostructure has a square shape with a side dimension of about 50 nm to about 400 nm. In some embodiments, the nanostructure has a thickness of about 20 nm to about 75 nm.

[0007] Also disclosed herein is a method for detecting the presence of one or more human papillomaviruses. In some embodiments, the method comprises: (1) exposing a bodily fluid sample of a patient suspected of having a human papillomavirus infection to any of the nanoplasmon sensors of the embodiments disclosed herein; (2) irradiating each of the functionalized sensors with light of a series of wavelengths; and (3) collecting absorbance, transmittance, or extinction data for each of the functionalized sensors. In some embodiments, the method further comprises comparing the collected absorbance, transmittance, or extinction data for each of the functionalized sensors to the respective baseline data of the functionalized sensors prior to exposure to the bodily fluid sample. In some embodiments, the comparing step reveals an optical peak shift when the human papillomavirus is detected. In some embodiments, the amount of the optical peak shift correlates with the concentration of the human papillomavirus in the bodily fluid sample. In some embodiments, the bodily fluid sample includes urine. In some embodiments, at least one of the functionalized sensors in the array comprises a different biological probe for detecting a segment of the human papillomavirus that is different from the other functionalized sensors. In some embodiments, the human papillomavirus is selected from the group consisting of HPV18, HPV16, hrHPV, HPV type 16, HPV type 18, HPV type 31, HPV type 33, HPV type 35, HPV type 39, HPV type 45, HPV type 51, HPV type 52, HPV type 56, HPV type 58, HPV type 59, HPV type 66, HPV type 68, and their derivatives / variants. In some embodiments, it has a sequence independently selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8. In some embodiments, each of the functionalized sensors in the array comprises a different biological probe. In some embodiments, multiple strands, segments, particles, variants, and / or species of the human papillomavirus are detected simultaneously. In some embodiments, the method is configured to be performed at the point of care.

[0008] Another method for detecting the presence of one or more human papillomaviruses involves providing a sensor comprising one or more biological probes designed to detect one or more target nucleic acid sequences derived from one or more human papillomaviruses, exposing the sensor to a sample suspected of containing one or more human papillomaviruses, and collecting electrical, fluorescence, absorbance, transmittance, and / or extinction data from the sensor. In some embodiments, the one or more biological probes are selected using computer and / or bioinformatics methods. In some embodiments, the one or more biological probes include intentionally varying the degree of mismatch with the target nucleic acid. In some embodiments, the one or more biological probes are designed to bind to multiple target nucleic acid sequences. In some embodiments, one of the biological probes can bind to nucleic acids derived from multiple human papillomaviruses. In some embodiments, the one or more biological probes are designed to bind to a nucleic acid sequence specific to one high-risk HPV genotype.

[0009] It is to be understood that all combinations of the foregoing concepts and additional concepts discussed in detail below are intended to be part of the inventive subject matter disclosed herein and can be used to achieve the benefits and advantages described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The features of the embodiments of the present disclosure will become apparent by reference to the following detailed description and drawings. Like reference numbers correspond to like, but perhaps not identical, components. For brevity, reference numbers or features having the foregoing functions may or may not be described in connection with other drawings in which they appear.

[0011]

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DETAILED DESCRIPTION OF THE INVENTION

[0012] All patents, applications, published applications, and other publications referred to in this specification are hereby incorporated by reference in their entirety as if fully set forth herein. If a term or phrase is used in this specification in a manner that contradicts or is inconsistent with the definition set forth in a patent, application, published application, or other publication incorporated by reference herein, then the use herein shall prevail over the definition incorporated by reference.

[0013] Plasmon resonance sensing devices employing ordered arrays of nanostructures are described herein. The ordered array of nanostructures enables coupling into diffractive photon modes and can thus be used to improve sensor sensitivity. The dimensions and geometries of the nanostructures are tailored to provide high-quality signals and large optical shifts upon modeled analyte binding.

[0014] Disclosed herein is a nanoplasmonic biosensor for point-of-care molecular characterization of human papillomavirus. Some embodiments relate to a novel method for fabricating nanoplasmonic sensors for rapid (<15 minutes) and highly specific genotyping of human papillomavirus (HPV). The nanoplasmonic sensors of the present application utilize an extremely sensitive light conversion method based on light-matter interaction at the nanoscale, enabling the detection of target nucleic acids via hybridization events occurring at the surface of the sensor. The technology of the present disclosure uses an optical phenomenon (localized surface plasmon resonance (LSPR)) that occurs between metal nanoparticles and a dielectric for the detection of viral nucleic acids. LSPR is observed when the wavelength of the incident light is larger than the size of the conductive nanoparticles, presenting an opportunity for highly sensitive detection of specific nucleic acid sequences. In this study, gold nanoarrays are covalently functionalized with biological probes. The nanostructures result in a highly confined electric field in the LSPR mode, which functions as a sensitivity transducer to changes in the local dielectric environment (i.e., binding events). Upon hybridization to the nucleic acid target sequence, there is a continuous red shift in the spectral peak as a function of the target sequence concentration.

[0015] Any panel of this aspect was designed to enable genotyping of HPV16 and other high-risk HPVs. A negative control was also included in the panel for specificity. In a patient cohort of 50 patients from three clinical sites (three countries), there was >92% sensitivity and 100% specificity for single-genotype identification (HPV16) and pooled high-risk HPV genotyping from processed cervical swabs. Through careful in silico, a new panel of rationally designed peptide nucleic acid (PNA) probes was also fabricated, which included a group of five consensus probes with high inclusivity for other low-risk HPVs and minimal cross-reactivity. These results suggest a widely applicable method for manufacturing nanosensors and a powerful initial platform for HPV genotyping.

[0016] 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.

[0017] 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.

[0018] 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, about 25 nm to about 70 nm, about 30 nm to about 65 nm, about 35 nm to about 60 nm, about 30 nm to about 55 nm, or about 20 to about 75 nm.

[0019] The nanostructures include a metal. For example, the nanostructures may include gold, platinum, aluminum, silver, or copper. Preferably, the nanostructures include gold. In some embodiments, the nanostructures include a single metal. In some embodiments, the nanostructures include a mixture of metals.

[0020] In some embodiments, the nanostructures in the array are conjugated with 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 localized surface plasmon resonance. In some embodiments, the biological probe includes 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 includes 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 includes 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.

[0021] In some embodiments, at least a first sensor 101a within the array of sensors includes nanostructures 102 conjugated with a first biological probe. In some embodiments, at least a second sensor 101b within the array of sensors includes nanostructures conjugated with a second biological probe. In some embodiments, at least a third sensor within the array of sensors includes nanostructures conjugated with a third biological probe. In some embodiments, at least a fourth sensor within the array of sensors includes nanostructures conjugated with a fourth biological probe. In some embodiments, at least a fifth sensor within the array of sensors includes nanostructures conjugated with a fifth biological probe. In some embodiments, the "n" sensors within the array of sensors include nanostructures conjugated with "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 sensors may have an area of about 1 μm 2 to about 1 mm 2 . In some embodiments, the sensors may have an area of 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 The area of

[0022] ​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, plastic, or 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.

[0023] 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, vaginal swab, or 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.

[0024] 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.

[0025] A method for detecting or sensing an analyte further includes the step of irradiating light onto at least one sensor. In some embodiments, the method includes the step of irradiating at least one sensor with a series of wavelengths of light. 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 including 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 at 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.

[0026] 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 a 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.

[0027] In some embodiments, an array of sensors within any of the plasmon resonance sensing devices 100 of the present 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.

[0028] In some embodiments, at least a first sensor 101a within the array of sensors includes nanostructures conjugated with a first biological probe, and at least a second sensor 101b within the array of sensors includes nanostructures conjugated with 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 include 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 first biological probe and the second biological probe independently include 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 first biological probe and the second biological probe independently include 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.

[0029] The detection of the analyte is based on an optical phenomenon (localized surface plasmon resonance (LSPR)) that occurs between a metal nanostructure and a 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.

[0030] In some embodiments, the sensor exposed to the sample, and thus the sensor having an analyte bound to a 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 made of 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.

[0031] 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.

[0032] 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.

[0033] For example, using photolithography, a portion of a photoresist layer where nanostructures are to be disposed / formed on a substrate may be removed, leaving a portion of the substrate that should not have nanostructures masked by the patterned photoresist layer. Thus, the patterned photoresist layer has removed portions that are similar in size, shape, and position to where the metal nanostructures are to be disposed. The portion of the substrate is exposed at the location where the nanostructures are 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.

[0034] The method further includes the step of lift-off of the patterned photoresist layer. When the patterned photoresist layer is lift-off, 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 nanostructures on the substrate disclosed herein.

[0035] 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.

[0036] Functionalization of Nanoplasmonic Sensing Chips Disclosed herein is a method for fabricating a functionalized nanoplasmonic sensing chip. The method includes providing a substrate with an array of sensors, attaching a microwell adapter on the substrate such that an array of microwells is on top of the array of sensors and is 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 a first batch of a reaction solution into one or more microwells above one or more sensors using an automated pipetting system, and then subsequently removing the first batch of the reaction solution from the one or more microwells using the automated pipetting system. 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, thus enabling delivery of two or more different reaction solutions to the array of microwells / sensors. The array of pipettes may also 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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 reaction solutions can be removed at different times to make the reaction time longer or shorter.

[0041] Figures 11A - 11B show two alternative views of a mold 3D printed for the fabricated polymer wells shown in Figures 11C - 11D. Other embodiments of the microwells are shown in Figures 11E - 11I.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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 sensor 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.

[0046] 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.

[0047] 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 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.

[0048] 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.

[0049] In some embodiments, the nanostructure includes a metal. In some alternatives, the nanostructure includes a single metal. In some alternatives, the nanostructure includes a mixture of metals. In some alternatives, the nanostructure may include gold, platinum, aluminum, silver, or copper. In some alternatives, the nanostructure includes gold. In some alternatives, the nanostructures in the array may be regularly spaced and have the geometric shapes described herein.

[0050] 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, up to 50, up to 80, or up to 100 analytes may be detected. The method includes exposing an array of functionalized sensors on a plasmon sensing chip of any of the alternatives disclosed herein to a sample. The functionalized sensors are configured to detect the presence of a specific 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 probe. 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.

[0051] 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.

[0052] 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.

[0053] 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 indicates 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.

[0054] 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.

[0055] In some embodiments, the plasmon resonance sensing device enables point-of-care (POC) detection of target analytes and POC diagnosis of diseases / conditions. In some embodiments, rapid results (less than about 15 minutes) can be provided.

[0056] Method for detecting HPV HPV may be detected using a sensor comprising a biological probe designed to target a nucleic acid sequence derived from an HPV pathogen. The method includes exposing the sensor to a sample that may contain a nucleic acid sequence derived from one or more HPV pathogens, and collecting electrical, fluorescence, absorbance, transmittance, and / or extinction data from the sensor. In some embodiments, the biological probe can be a peptide nucleic acid (PNA) probe or an oligonucleotide probe.

[0057] In some embodiments, the sensor may comprise one or more biological probes. In some embodiments, each of the biological probes may be designed to bind to a different target nucleic acid sequence. Thus, the sensor may be capable of simultaneously detecting multiple or various target nucleic acid sequences. For example, the sensor can detect the presence of any different HPV pathogen and confirm the HPV diagnosis of a patient. This means that HPV can be diagnosed regardless of which of the various HPV pathogens are present. In some embodiments, the sensor can detect and identify one or more specific HPV pathogens in a patient. This information can be useful for determining appropriate or most effective treatment options.

[0058] In some embodiments, a single biological probe can bind to nucleic acids derived from multiple HPV pathogens. In some embodiments, a single biological probe can bind to multiple nucleic acids derived from one high-risk HPV genotype. In some embodiments, the biological probe may be designed to bind to nucleic acid sequences derived from multiple HPV variants or mutations. As a result, a sensor comprising one or more biological probes may be capable of detecting multiple HPV species, strains, mutants, segments, particles or derivatives. In some embodiments, a sensor comprising one or more biological probes may be capable of identifying one or more HPV pathogens and derivatives.

[0059] The biological probe may be selected using computer and / or bioinformatics methods. These methods enable a rational selection of probe sequences that align on sequences known in the scientific literature. In some embodiments, computational techniques utilize custom python scripts, open access sequence databases, and thermodynamic modeling tools. In some embodiments, the biological probe includes intentionally varying the degree of mismatch with the target nucleic acid. These mismatches can provide additional degrees of freedom when measuring the presence of the target nucleic acid.

[0060] In some embodiments, the biological probes described herein have sequences independently selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.

[0061] The sensor may have a physical property that changes when one or more target nucleic acid sequences bind to the biological probe associated with the sensor. The change in the physical property can be detected by changes in electrical, fluorescence, absorbance, transmittance, and / or extinction measurements. Some non-limiting examples of sensors may include electrochemical sensors, fluorescence-based sensors, resistive sensors, and optical sensors.

[0062] Nanoplasmonic Sensor for Pathogen / HPV Detection A nanoplasmonic sensor is an example of a sensor that can be functionalized with a biological probe for detecting human papillomavirus pathogens. In some embodiments, the nanoplasmonic sensor comprises an array of functionalized sensors, each of the functionalized sensors in the array comprising an array of nanostructures conjugated to a biological probe / capture ligand such as a peptide nucleic acid (PNA) probe or an oligonucleotide probe. In some embodiments, the biological probe is configured to detect the presence of a pathogen associated with the human papillomavirus (HPV). In some embodiments, the biological probe is configured to detect the presence of the human papillomavirus. In some embodiments, the biological probe is configured to detect the presence of the human papillomavirus pathogen using a specific marker associated with a given pathogen. In some embodiments, the specific marker is derived from the human papillomavirus. In some embodiments, the specific marker is derived from the subject's response to infection by the human papillomavirus. In some embodiments, the nanoplasmonic sensor is configured to simultaneously detect multiple strains, segments, particles, variants, and / or species of the human papillomavirus.

[0063] In some embodiments, the plurality of functionalized sensors in the array can detect the human papillomavirus in a sample. In some embodiments, at least two of the functionalized sensors in the array comprise the same biological probe for detecting the human papillomavirus. In some embodiments, at least two of the functionalized sensors in the array comprise the same biological probe for detecting the same marker of the human papillomavirus. In some embodiments, all of the functionalized sensors in the array comprise the same biological probe for detecting the human papillomavirus. In some embodiments, all of the functionalized sensors in the array comprise the same biological probe for detecting the same marker of the human papillomavirus.

[0064] In some embodiments, at least one of the functionalized sensors in the array comprises a different biological probe for detecting a strain, segment, particle, variant, and / or species of human papillomavirus that is different from other functionalized sensors. In some embodiments, at least one of the functionalized sensors in the array comprises a different biological probe for detecting a marker for human papillomavirus that is different from other functionalized sensors. In some embodiments, different markers may be used to detect the same human papillomavirus. In some embodiments, different markers may be used to detect different human papillomaviruses. In some embodiments, different markers may be used to detect different strains, segments, particles, variants, and / or species of human papillomavirus. In some embodiments, all of the functionalized sensors in the array comprise different biological probes from each other for detecting different strains, segments, particles, variants, and / or species of human papillomavirus. In some embodiments, the nanoplasmonic sensors are configured to simultaneously detect multiple strains, segments, particles, variants, and / or species of human papillomavirus. In some embodiments, each of the functionalized sensors in the array comprises a different biological probe.

[0065] In some embodiments, the functionalized sensor may be functionalized with a negative control biological probe. The negative control biological probe may be designed to be complementary to a synthetic sequence of DNA / RNA that does not naturally occur. The negative control functionalized sensor is expected to always return a negative result.

[0066] In some embodiments, the functionalized sensor may be functionalized with a positive control biological probe. The positive biological probe is complementary to a synthetic sequence of DNA. A low concentration of that DNA sequence may be spiked into the sample early in the reaction. This shows whether sample preparation and fluid handling were able to get a known concentration of target DNA to the sensor and indicates that the assay operation was successful.

[0067] It is understood that the human papillomavirus can be any virus or viral component related to the virus family of the human papillomavirus. Non-limiting examples of HPV include HPV18, HPV16, hrHPV, HPV type 16, HPV type 18, HPV type 31, HPV type 33, HPV type 35, HPV type 39, HPV type 45, HPV type 51, HPV type 52, HPV type 56, HPV type 58, HPV type 59, HPV type 66, HPV type 68, and any derivative strains / mutants thereof.

[0068] It is understood that a biological probe can comprise any peptide and / or nucleic acid sequence capable of binding / associating with a segment of the human papillomavirus. In some embodiments, the sequence is complementary to a sequence present on / in the human papillomavirus. In some embodiments, the sequence directly recognizes the human papillomavirus. In some embodiments, the biological probe comprises 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 biological probe comprises 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 probe comprises at least one of a peptide-nucleic acid, an aptamer, an antibody, an antibody fragment, a complementary DNA, and / or an enzyme. In some embodiments, the 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. In some embodiments, the biological probe may be a PNA probe or an oligonucleotide probe having a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. The probe sequences are listed in Table 5.

[0069] The nanostructure array is as disclosed herein. The nanostructure contains a metal. In some embodiments, the nanostructure contains a single metal. In some embodiments, the nanostructure contains a mixture of metals. In some embodiments, the nanostructure contains silver. In some embodiments, the nanostructure contains copper. In some embodiments, the nanostructure contains gold. The nanostructures in the sensor can be functionalized with biological probes using the automated pipetting systems and methods described herein.

[0070] Also disclosed herein is a method for detecting the presence of one or more human papillomaviruses. The method includes exposing a sample to any of the nanoplasmon sensors of the embodiments disclosed herein, irradiating each of the functionalized sensors with light of a series of wavelengths, and collecting absorbance, transmittance, or extinction data for each of the functionalized sensors. In some embodiments, the sample is a body fluid sample from a patient suspected of having a human papillomavirus infection. In some embodiments, the body fluid sample is urine, blood, sweat, saliva, plasma, and / or mucus. In some embodiments, the body fluid sample is mucus, cells, and cell debris that can be collected using a cervical swab or vaginal swab. In some embodiments, the light for illuminating the functionalized sensor 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. For example, the series of wavelengths includes wavelengths in the range of 500 - 1000 nm.

[0071] In some embodiments, the method further includes comparing the absorbance, transmittance, and / or extinction data of each functionalized sensor collected to the baseline data of each functionalized sensor prior to exposure to the sample. In some embodiments, the comparing step reveals an optical peak shift when at least one human papillomavirus is detected. The baseline data of the functionalized sensor includes the absorbance / transmittance measurements of the functionalized sensor taken prior to exposure to the sample. Identify the peak absorbance wavelength of the functionalized sensor (before binding to the target analyte). The absorbance / transmittance of the sensor is measured again after exposure to the sample, and a shift in peak absorbance can be observed when a target analyte such as human papillomavirus or human papillomavirus particles is present in the sample and binds to the probe on the functionalized sensor. The shift represents the detection signal. In some embodiments, the amount of the optical peak shift correlates with the concentration of the pathogen in the sample. In some embodiments, the amount of the optical peak shift correlates with the concentration of human papillomavirus or human papillomavirus particles in the body fluid sample.

[0072] In some embodiments, two or more of the functionalized sensors may comprise the same biological probe. In some embodiments, at least one of the functionalized sensors may comprise different biological probes. In some embodiments, each of the functionalized sensors may comprise different biological probes. In some embodiments, if one or more of the functionalized sensors in the array comprise different biological probes, multiple strains or species of human papillomavirus or human papillomavirus particles can be detected simultaneously (i.e., using the same nanoplasmonic sensor / test kit). In some embodiments, the method may be performed at the point of care (i.e., in a physician's office, clinic, hospital, long-term care facility, or patient's home, etc.).

[0073] 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 belongs, unless otherwise clearly indicated.

[0074] 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.

[0075] The terms "comprising", "including", "containing", and various forms thereof are synonymous with each other and are meant to be equally broad. Further, unless explicitly stated to the contrary, an example that comprises, includes, or has one or more elements with a particular property may include additional elements, whether or not the additional elements have that property.

[0076] The term "nanostructure", as used herein, has its standard scientific meaning and thus refers to any structure that is approximately molecular size to approximately microscopic size. Nanostructures include nanomaterials, and nanomaterials can be any material in which a single unit is on the order of about 1 nm to about 200 nm in size. Examples of nanostructures include 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, nanochips, nanowires, and nanostructured films. It is understood that nanostructures can have various geometric shapes and properties based on the components of the nanostructure.

[0077] The term "analyte" refers to the substance or chemical component of interest. For example, an analyte can include biological or chemical substances that can be detected by a sensing device and that can be of interest for diagnosing a disease or condition.

[0078] All patents and other publications cited throughout this application, including references, issued patents, published patent applications, and co-pending patent applications, are hereby expressly incorporated by reference herein 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 for 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 for 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 date or content of these documents.

[0079] The description of embodiments of the present disclosure is not intended to be exhaustive or to limit the present disclosure to the precise form 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 also 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. Additionally, in view of considerations of biological functional equivalence, some modifications can be made to the protein structure without affecting the type or amount of biological or chemical action. These and other modifications 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.

[0080] Any particular element of the foregoing embodiments can be combined with or replaced by elements in other embodiments. Further, although the advantages associated with particular embodiments of the present disclosure have been described in the context of these embodiments, other embodiments may exhibit such advantages, and not all embodiments necessarily exhibit such advantages so as to fall within the scope of the present disclosure.

Example

[0081] The techniques described herein are further illustrated by the following examples, which should in no way be construed as imposing further limitations.

[0082] 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 geometries T1 - T3 are nanorods, and the test geometries T4 - T10 are coupled nanoarrays.

Table 1

[0083] 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 of defined refractive index was modeled on the nanostructure. Extinction and transmittance curves were returned in the wavelength range of 400-1200 nm.

[0084] Example 2: Definition of Simulation Settings and Figure of Merit To study the sensitivity to changes in plasmon resonance shape and refractive index, the initial simulation included a bulk refractive index sensitivity analysis. In the first iteration, gold nanorods with wide spacings designed to represent a regular nanoarray were tested.

[0085] 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 considering the peak shift (s) and the resonance width (full width at half maximum - FWHM), as shown in FIG. 3. The figure of merit was defined as the shift over the full width at half maximum, enabling direct comparison between different geometric shapes. A larger figure of merit indicates better sensing performance due to (1) a larger peak shift for the same refractive index change and (2) an easier identification of the peak shift due to a narrower resonance curve. This analysis was repeated for all geometric shapes considered.

[0086] 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) was much more pronounced 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.

[0087] Example 4: Fabrication of the Nanosenor 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.

[0088] First, a thin layer of conductive photoresist was spin-coated onto a transparent quartz wafer and then patterned by an electron beam (JEOL E-beam microscope). Then, a thin (about 5 nm) chromium adhesion layer was thermally evaporated onto the patterned substrate, followed by evaporation of a thicker (about 40 - 50 nm) pure gold layer. After performing chemical lift-off to form a 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.

[0089] Example 5: Simulation of Selected Geometries Bulk refraction simulations were performed for sample geometries T8 - T10 described in Table 1. The transmittance through the samples was measured using an optical reading device. The wavelength range was set from 450 nm to 950 nm. For seamless integration with the reading device, the individual sensors of the sensing device were fabricated to have a nanostructure array with an area of 1 mm 2 so as to be perfectly 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 FIGS. 5A / B, 6A / B, and 7A / B respectively, showing both the peak shape and the refractive index peak shift. The performance indices calculated for T8 - T10 were 12.8, 6.7, and 10.7 respectively. Further, the refractive index sensitivities for each of these geometries are shown in FIGS. 5B, 6B, and 7B. All sensitivities are compared with a 220p sample of 140 nm × 40 nm labeled "uncoupled nanorods". A steeper gradient indicates better sensing performance. Sample geometry T10 exhibits the best performance due to its high performance index (10.7) and its relatively high refractive index sensitivity (267 nm / RIU).

[0090] Example 6: Comparison between Simulation and Experiment Nanostructure array samples 1 - 5 were fabricated with the nanostructure dimensions shown in Table 2. The transmittance of each sample was measured experimentally (shown in FIG. 8) and compared with the peak shape from the simulation (shown in FIG. 9). Excellent agreement was found between the experimental data, including the peak shape and resonance position, and the simulation data.

Table 2

[0091] The present disclosure also presents a methodology for the rational design of regularly spaced nanoparticle arrays for plasmon sensing. The applicant tested 5 - 7 geometric shapes through both simulation and experimental analysis, and finally selected 145 nm x 145 nm. Through both simulation and experimental analysis, a nanoarray geometry showing high - amplitude resonance and refractive index sensitivity may be selected for the fabrication of plasmonic resonance sensing devices.

[0092] Example 7: Functionalization of Nanostructures 1 mm 2 A 2×6 array of sensors (12 sensors in total) was functionalized with peptide - nucleic acid (PNA) probes. Each sensor contains an array of 145 nm×145 nm gold nanostructures 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).

[0093] The micro - well structure on the sensing array enables individual fluid delivery to each sensing spot and allows multiplexing of up to 12 targets on a single sensing chip. For this purpose, a mold was designed using Solidwaorks CAD, enabling the fabrication of a polymer micro - well array that aligns with the coordinates of the sensors (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 mm 3 )). The pillars were arranged to match the coordinates of the sensor array on the glass substrate. Then, the master mold was fabricated using SLA 3D printing as shown in Figures 11A and 11B.

[0094] The microwell array device was fabricated in a 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. As shown in FIGS. 11C and 11D, the cured PDMS microwell array was removed from the master mold. The polymer microwell array was fixed onto the sensor array using a washable adhesive, enabling a removable bond for reuse of the sensor. The entire system was attached to a standard 75×25 mm microfluidic chip and then was ready for molecular detection.

[0095] Example 8: Automated robotic functionalization of the sensor The prepared 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, e.g., 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 PNA probe dispersed in Tris-EDTA buffer (pH 7.0) for 30 - 45 minutes. Transmission spectra were collected before and after conjugation to characterize the success of PNA conjugation.

[0096] 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 manner. 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, having 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

[0097] 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 crosslinking 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 crosslinking 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, 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

[0098] Pipetting robot systems have been widely used for fluid filling applications, but as far as the applicant knows, 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 successive dispensing and aspiration steps on the sensor.

[0099] Example 9: Plasmon Sensor for HPV Screening The plasmon sensor can be used to perform HPV genotyping directly from a processed cervical swab sample without amplification and to stratify high-risk strains. The principle of operation is based on localized surface plasmon resonance (LSPR). LSPR utilizes the unique characteristics of metal nanoparticles. When the particles are excited by incident light, they vibrate collectively, and this collective vibration is very sensitive to changes in the bulk and local refractive indices. This plasmon phenomenon results in the resonance peak wavelength of the nanosensor, which shifts in response to changes in the refractive index near the sensing substrate. In this specification, the nanoparticle array is covalently functionalized with peptide nucleic acid (PNA) probes complementary to at least a portion of the target DNA. The bound probes selectively bind to the target HPV DNA, enabling highly sensitive and quantitative transduction after hybridization of the target DNA to the PNA probes and the sensing substrate. Collectively, these data present a rationalized method for the functionalization and testing of plasmonic nanoarray substrates for DNA detection.

[0100] All sensing experiments performed on HPV used a 2×6 array of 1 mm2 nanosensors (a total of 12 sensors). Each sensor consisted of 145 nm gold nanocubes with regular spacing. The selected nanoarray substrate contained 12 1 mm2 nanosensor arrays. To individually functionalize the nanosensor arrays to be target-specific, a polydimethylsiloxane (PDMS) polymer microwell array was fabricated. This microwell array was aligned with the substrate such that each nanosensor could be accessed through a single well. This approach created repeatable and programmable coordinates for an Integra ASSIST PLUS pipetting robot.

[0101] The mold for casting the PDMS microwells was 12 2 mm×2 mm×5 mm (20 mm 3) pillars in Solidworks. The pillars were arranged to match the coordinates of the nanosensor array on the glass substrate. The master mold was fabricated using SLA 3D printing. The micro-well array device was manufactured 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 at 80 °C in a convection oven for about 1.5 hours. The cured PDMS micro-well array was removed from the master mold and fixed to the nanosensor substrate using a washable adhesive stick, enabling a removable bond.

[0102] To covalently functionalize the nanosensors with the selected PNA probes, the gold nanostructures on the glass substrate were 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 conjugation to characterize the success of PNA conjugation.

[0103] The above-described nanosensor 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 is designed as described herein and can be 3D printed and 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 suction deck of the robot. To start the machine, a Voyager electronic 125 μL 8-channel pipette was loaded onto the robot. A suite of 6 programs was developed to aspirate, dispense, and remove the chips in an automated fashion.

[0104] Example 10: Development of HPV Probes An in silico method was used to design PNA probes for the evaluation of the nanoplasmonic properties of human papillomavirus (HPV) and related HPV infections in subjects. Two serotype-specific PNA probes for HPV16 and HPV18 and five additional probes encompassing all 14 hrHPV serotypes (HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) were designed.

[0105] Genes that are conserved within the desired group of target pathogens but are sufficiently different from their nearest neighbors were determined by a literature search for species identification and a visual inspection of the alignment for species identification. The reference target gene was subjected to BLAST against 5000 records in the nucleotide database to generate an XML file containing the complete results of the alignment of homologous sequences (coverage / identity > 80%). The XML file containing the alignment records was parsed into python using the Biopython module. The identical sequence records were grouped to indicate the number of iterations and parsed into a Fasta file. The Fasta file was used to realign the sequence records for further analysis.

[0106] The array alignment was visually inspected to identify potential positions for probe placement. The PNA probes were designed such that the Tm of the PNA-DNA duplex was approximately 80 °C. The length of the probes was kept <25 nt. The Tm of the PNA-DNA hybrid was determined using the PNA Bio Tool. The purine composition was kept <50% to avoid precipitation of the PNA probes. Sequences producing stable homodimers and hairpins (Tm > 30 °C) were avoided.

[0107] Once the probe sequences were determined, the analytical inclusivity of a given probe was evaluated using multiple databases. All probes were tested against the NCBI nucleotide database to retrieve complete records of high-scoring pairs (HSPs). Parameters including accession numbers, identities, coverages, numbers of mismatches, mismatch bases, and positions were retrieved using custom scripts. The same results were grouped, and a single representative record and the number of records replicating the parameters were marked. Further, based on the targets, additional databases were used to further verify inclusivity / cross-reactivity using the same analytical criteria. HPV targets were tested against the HPV representative genomic database to determine serotype inclusivity and cross-reactivity. The HPV probes were also evaluated against a representative genomic database of prokaryotes to confirm no cross-reactivity with prokaryotic pathogens.

[0108] Serotype-specific PNA probes for HPV16 were designed. As shown in Table 5, a new set of PNA probes, including those highly specific for HPV18, and five additional probes encompassing all 14 hrHPV serotypes (HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) were designed.

Table 5

[0109] Due to the high heterogeneity among HPV serotypes, further in silico analysis was performed to minimize the thermodynamic penalties resulting from any possible mismatches. All probe sequences were subjected to in silico analysis against representative HPV genomes available in the Papillomavirus Episteme (PaVE) database of the National Institute of Allergy and Infectious Diseases (NIAID) to determine specificity and cross-reactivity (Figure 13). The probes were also evaluated against sequence records available in the nucleotide database and representative genomes of prokaryotes to confirm inclusivity and exclude cross-reactivity with other non-specific pathogens.

Table 6

Table 7(1)

Table 7(2)

Table 8(1)

Table 8(2)

[0110] Example 11: In Vitro HPV Genotyping Using a Nanoplasmonic Sensor The sensor was functionalized with the PNA sequences selected from Table 9. Specifically, in addition to the negative control PNA, both HPV16-specific (SEQ ID NO: 1) and HPV consensus (SEQ ID NO: 8) PNAs were used.

Table 9

[0111] The functionalized sensor was tested with synthetic complementary oligos at known high concentrations (10,000 - 100,000 copies / mL). A large measurable peak shift (>3 nm) was observed, demonstrating that the sensor was ready to test patient samples at unknown concentrations.

[0112] Example 12: Methodology for Sample Preparation and Processing of Clinical Samples Fifty discarded, anonymized patient samples were collected from the Center for Clinical Genomics and Advanced Technology at Dartmouth-Hitchcock Medical Center. All samples were ThinPrep cervical swab samples that underwent nucleic acid extraction procedures. Samples were collected from the Dartmouth-Hitchcock catchment area and also through outreach efforts at the Center for Global Oncology at the Norris Cotton Cancer Center of Dartmouth-Hitchcock Medical Center. In total, cervical swab samples were collected from New Hampshire, Kosovo, and Honduras. Nucleic acid extraction was performed on an automated EZ1 bacteria card / tissue or using an Atila nucleic acid extraction kit. All gold standard genotyping was performed using the Roche Cobas® HPV test. This commercially available test can distinguish between HPV16, HPV18, and other pooled hrHPV (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68). Samples processed as "other hrHPV" were reflected in the Atila Multiplex High-Risk HPV test for genotyping. The total nucleic acid concentration (human and HPV) of the extracted samples was provided to the Nanopath team and ranged from 0.56 to 74.8 ng / μL. All samples were anonymized and the team was blinded to the results until after sample processing and data analysis.

[0113] Fifty discarded, anonymized patient samples were stored at -80 °C. Prior to sample analysis, the microarray was immobilized onto the nanosensor array, and the combined array was immobilized onto a 75×25 mm microchip. Transmission measurements were performed through the dry chip to confirm proper alignment and signals from each sensing spot. The nano sensing spots were then functionalized. Each sample was exposed to a negative control probe, an HPV16 probe, and an HPV consensus probe. A total of four patient samples were tested using a fully functionalized chip with twelve nano sensing arrays. The samples were thawed and 8 μL was pipetted onto each sensing spot. Immediately upon sample delivery, after a 5-minute incubation, the transmission spectrum was collected again.

[0114] All transmission spectra were collected using a custom readout instrument from Nanopath coupled to our integrated user interface. The assembly includes a linear stage, a light source, a spectrometer, and a focusing lens component. The readout instrument moves the slide to a specified coordinate position and measures the transmission at wavelengths from 500 nm to 1000 nm through the sample. For each sample, paired measurements are made through the nanoarray and through a background position. This data is then analyzed within the integrated user interface.

[0115] The normalized transmission spectrum was calculated as the ratio of the signal to the background at every wavelength. The extinction was then calculated as the negative natural logarithm of the normalized transmission. These extinction spectra were smoothed using Lowess smoothing (10% smoothing) in MATLAB® and then the resonance peak wavelength was calculated. The resonance peak wavelength was determined by a centroid calculation using numerical integration at the wavelength boundaries 750 nm to 975 nm. The spectral shift was calculated by subtracting the sample resonance peak positions before and after sample incubation. The UI returns the positive / negative of each sample defined as a spectral shift of 1 nm or more.

[0116] Example 13: Genotyping of HPV in Clinical Samples Using Nanoplasmonic Sensors Using two probes, Array ID number 1 and Array ID number 8, 50 processed ThinPrep cervical swab samples were analyzed using a sensor, a reader, and a UI. The experiment was conducted as a blind test for the genotype of each sample until after sample processing and analysis. Each 12-plex sensor chip was functionalized with three selected probes (HPV16, HPV consensus, and negative control) to process four patient samples each. All samples were processed as described above and as described in Tables 10 and 11, and a peak shift of 1 nm or more was defined as a positive result.

Table 10

Table 11

[0117] Despite sample handling and variations in total DNA concentration, the results showed exceptional sensitivity and specificity. In particular, the HPV16 probe showed 100% (32 / 32) specificity and 93% (14 / 15) sensitivity for the detection of HPV in samples. Other high-risk HPV probes showed 100% (34 / 34) specificity and 92% (12 / 13) sensitivity for the detection of HPV in samples. There were no false positives identified in this study (i.e., 100% specificity), and the sensitivity exceeded 92%. The comprehensive breakdown of patient samples, genotypes, and raw results is as shown in Tables 12 and 13.

Table 12

Table 13

[0118] The above-described first clinical sample test demonstrates the potential to genotype individual high-risk HPV sequences (i.e., HPV16) and the ability to broadly identify a pool of hrHPV. One issue identified in the literature is the ability to broadly capture all 12 high-risk HPVs with a small number of probes. Most existing approaches use up to 12 individual probes to ensure adequate coverage of all high-risk genotypes. Through careful probe design, a set of 5 consensus primers with inclusivity beyond 14 well-known high-risk genotypes was designed (Table 5). These probes were also designed to be thermodynamically favorable PNA probes with low purine content and high melting temperature. To the applicant's knowledge, these are the best probes for consensus high-risk HPV genotyping due to their broad inclusivity. There is minimal cross-reactivity between these probes and other low-risk HPV genotypes (Figure 13), thus reducing the risk of false negatives in hrHPV stratification.

[0119] 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, but 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 interpreted 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 nanoplasmon sensor comprising an array of functionalized sensors, Each of the functionalized sensors in the array comprises an array of nanostructures conjugated to a biological probe, The aforementioned biological probe is configured to detect the presence of human papillomavirus (HPV), The aforementioned biological probe is a nanoplasmon sensor capable of binding to nucleic acids derived from multiple high-risk HPV genotypes.

2. The nanoplasmon sensor according to claim 1, wherein the biological probe is a peptide nucleic acid probe or an oligonucleotide probe.

3. The nanoplasmon sensor according to claim 1, wherein at least one of the functionalized sensors in the array comprises a different biological probe for detecting a segment or species of human papillomavirus, different from the other functionalized sensors.

4. The nanoplasmon sensor according to claim 3, wherein the nanoplasmon sensor is configured to simultaneously detect multiple strains, segments, particles, variants, and / or species of the human papillomavirus.

5. The nanoplasmon sensor according to claim 3, wherein each of the functionalized sensors in the array comprises a different biological probe.

6. The nanoplasmon sensor according to claim 1, wherein the biological probe has a sequence selected from the group consisting of sequence ID number 1, sequence ID number 2, sequence ID number 3, sequence ID number 4, sequence ID number 5, sequence ID number 6, sequence ID number 7, and sequence ID number 8.

7. The nanoplasmon sensor according to claim 1, wherein the human papillomavirus is selected from a group selected from the group consisting of HPV18, HPV16, hrHPV, HPV16 type, HPV18 type, HPV31 type, HPV33 type, HPV35 type, HPV39 type, HPV45 type, HPV51 type, HPV52 type, HPV56 type, HPV58 type, HPV59 type, HPV66 type, HPV68 type, and derivative / mutant strains thereof.

8. The nanostructure comprises gold, as described in any one of claims 1 to 7, for the nanoplasmon sensor.

9. The nanoplasmon sensor according to claim 1, wherein 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 side dimensions of about 50 nm to about 400 nm.

10. The nanoplasmon sensor according to claim 9, wherein the nanostructure has a thickness of about 20 nm to about 75 nm.

11. A method for detecting the presence of one or more human papillomaviruses, The steps include: exposing a bodily fluid sample from a patient suspected of having a human papillomavirus infection to the nanoplasmon sensor described in claim 1; A step of irradiating each of the functionalized sensors with light of a series of wavelengths, A step of collecting absorbance, transmittance, or extinction data from each functionalized sensor, Methods that include...

12. The method according to claim 11, further comprising the step of heating the nanoplasmon sensor after exposing it to the bodily fluid sample.

13. The method according to claim 11, wherein the bodily fluid sample is first subjected to a thermal, mechanical, chemical, or biological treatment so as to dissolve the human papillomavirus capsid before being exposed to the nanoplasmon sensor.

14. The method according to claim 11, further comprising the step of comparing the absorbance, transmittance, or extinction data collected from each functionalized sensor with the respective baseline data of the functionalized sensor before exposure to the bodily fluid sample.

15. The method according to claim 14, wherein the comparison step reveals an optical peak shift when at least one human papillomavirus is detected.

16. The method according to claim 15, wherein the amount of the optical peak shift correlates with the concentration of the human papillomavirus in the bodily fluid sample.

17. The method according to claim 11, wherein the bodily fluid sample includes mucus.

18. The method according to claim 11, wherein at least one of the functionalized sensors in the array comprises a different biological probe for detecting a different human papillomavirus from the other functionalized sensors.

19. The method according to claim 18, wherein the human papillomavirus is selected from the group consisting of HPV18, HPV16, hrHPV, HPV16 type, HPV18 type, HPV31 type, HPV33 type, HPV35 type, HPV39 type, HPV45 type, HPV51 type, HPV52 type, HPV56 type, HPV58 type, HPV59 type, HPV66 type, HPV68 type, and derivative strains / mutants thereof.

20. The method according to claim 18, for simultaneously detecting multiple strains, segments, particles, mutants, and / or species of the human papillomavirus.

21. The method according to any one of claims 11 to 20, wherein the biological probe has a sequence independently selected from the group consisting of sequence ID number 1, sequence ID number 2, sequence ID number 3, sequence ID number 4, sequence ID number 5, sequence ID number 6, sequence ID number 7, and sequence ID number 8.

22. The method according to claim 11, wherein each of the functionalized sensors in the array comprises a different biological probe.

23. The method according to claim 12, wherein the method is configured to be performed at a point of care.

24. A method for detecting the presence of one or more human papillomaviruses, A sensor comprising one or more biological probes designed to detect one or more target nucleic acid sequences derived from one or more human papillomaviruses, wherein one of the biological probes is capable of binding to nucleic acids derived from multiple high-risk HPV genotypes; The steps include: exposing the sensor to a sample suspected to contain one or more human papillomaviruses; A step of collecting electrical, fluorescence, absorbance, transmittance, and / or extinction data from the sensor, Methods that include...

25. The method according to claim 24, wherein one or more biological probes are selected using a computer and / or bioinformatics method.

26. The method according to claim 24, wherein the one or more biological probes are configured to intentionally vary the degree of mismatch with one or more target nucleic acid sequences.

27. The method according to any one of claims 24 to 26, wherein the one or more biological probes are designed to bind to a plurality of target nucleic acid sequences.

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