Homogenous deposition of colloidal nanoparticles

Abstract

Disclosed herein includes a method for high-throughput, controlled deposition of low-density nanoparticle ensembles onto a substrate. The nanoparticle ensembles may further be functionalized with biological probes for molecular detection, such as molecular plasmonic sensing. Sensing devices containing low-density nanoparticle ensembles functionalized with biological probes and methods of nucleic acid detection are also described.

Description

NPATH.014WO PATENT HOMOGENOUS DEPOSITION OF COLLOIDAL NANOPARTICLES BACKGROUND Field of the Invention

[0001] This disclosure is related to the field of molecular detection. Specifically, the disclosure describes a method for making nanoparticle ensembles on a substrate and a sensing device that includes an array of nanoparticle ensembles. Description of the Related Art

[0002] For biomedical research, clinical diagnostics, environmental testing, and other related fields, it is beneficial to have apparatuses and systems for detecting analytes, such as biomolecules and chemical substances with high accuracy, sensitivity, specificity, reproducibility, and ease of use. For example, having fast, rapid, and accurate tests for detecting certain analytes in a biological sample may aid in clinical diagnosis contexts, and assist physicians in determining optimal treatment regimens.

[0003] Metals have the unique ability to support electromagnetic surface waves, called surface plasmons, when optically illuminated. This property, and its strong sensitivity to changes in refractive index, allows for the use of metal nanostructures as highly sensitive transducers for molecular detection. Deposition of metallic nanoparticles on a substrate surface (such as colloidal nanorods dispersed on chip) can be a low cost and high throughput method for fabrication of sensor arrays, but it can be challenging to produce a sensor unit with homogeneous distribution of nanoparticles. While bulk coverage of nanoparticles on entire substrate surface can mitigate the less-than-optimal distribution of nanoparticles, bulk coverage results in (1) a significant increase in available binding sites, which would greatly decrease detection sensitivity and (2) the use of a larger quantity of nanoparticles, which would result in higher cost. Therefore, there is a need for sensors that have a high detection sensitivity and can be produced by a low-cost, high-throughput fabrication method, which allows scale up for manufacturing. SUMMARY

[0004] Disclosed herein is a method of making nanoparticle ensembles. In some embodiments, the nanoparticle is selected from the group comprising gold, platinum, palladium,aluminum, silver, metal coated iron oxide, copper, and cesium oxide. In some embodiments, prior to deposition, the nanoparticles are conjugated to a biological probe, wherein the biological probe is selected from the group consisting of a peptide-nucleic acid, an oligonucleotide, an aptamer, an antibody, an antibody fragment, a complimentary DNA or RNA, a complementary oligonucleotide, a modified oligonucleotide, a locked nucleic acid, an enzyme, and a small molecule. In some embodiments, the nanoparticle solution is deposited on the chemically functionalized substrate at an optical density of 0.02 to 20. In some embodiments, the nanoparticles are capped. In some embodiments, the nanoparticles are capped with a cap selected from a group consisting of citrate, cetyltrimethylammonium bromide (CTAB), Polyethylene glycol (PEG), Polyvinylpyrrolidone (PVP), Polyvinyl alcohol (PVA), Bovine serum albumin (BSA), Ethylene diamine tetra acetic acid (EDTA), and a combination thereof. In some embodiments, the capped nanoparticles are further functionalized with a biological probe, wherein the biological probe is selected from the group consisting of a peptide-nucleic acid, an oligonucleotide, an aptamer, an antibody, an antibody fragment, a complementary DNA or RNA, a complementary oligonucleotide, a modified oligonucleotide, a locked nucleic acid, an enzyme, or a small molecule. In some embodiments, the spot size of the nanoparticle solution deposited on the chemically functionalized substrate is 0.01 mm to 50 mm in diameter.

[0005] Also disclosed herein is a sensing device comprising an array of nanoparticle ensembles on a substrate, wherein each of the nanoparticle ensembles comprises a homogenous distribution of nanoparticles, and the nanoparticle ensembles have a nanoparticle optical density ranging from 0.02 to 20. In some embodiments, the nanoparticles are capped. In some embodiments, the nanoparticles are capped with a cap selected from a group consisting of citrate, cetyltrimethylammonium bromide (CTAB), Polyethylene glycol (PEG), Polyvinylpyrrolidone (PVP), Polyvinyl alcohol (PVA), Bovine serum albumin (BSA), Ethylene diamine tetra acetic acid (EDTA), and a combination thereof. In some embodiments, the capped nanoparticles are further functionalized with a biological probe, wherein the biological probe is selected from the group consisting of a peptide-nucleic acid, an oligonucleotide, an aptamer, an antibody, an antibody fragment, a complementary DNA or RNA, a complementary oligonucleotide, a modified oligonucleotide, a locked nucleic acid, an enzyme, or a small molecule. In some embodiments, the capped nanoparticles in different nanoparticle ensembles are functionalized with different biological probes.

[0006] It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail below are contemplated as being part of the inventive subject matter disclosed herein and may be used to achieve the benefits and advantages described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Features of examples of the present disclosure will become apparent by reference to the following detailed description and drawings, in which like reference numerals correspond to similar, though perhaps not identical, components. For the sake of brevity, reference numerals or features having a previously described function may or may not be described in connection with other drawings in which they appear.

[0008] FIG. 1A depicts one embodiment of a plasmonic-resonance sensing device. FIG. 1B depicts one embodiment of an array of nanostructures in a sensor of the plasmonic- resonance sensing device.

[0009] FIG. 2A, 2B, 2C and 2D depict the optimization of deposition buffer composition for citrate-coated gold nanoparticle deposition onto amine substrate. FIG. 2A shows scanning electron microscope (SEM) images of colloidal gold deposition at 5 mM citrate phosphate buffer. FIG. 2B shows scanning electron microscope (SEM) images of colloidal gold deposition at 1 mM citrate phosphate buffer. FIG. 2C shows the Full width at half maximum (FWHM) for varying citrate phosphate buffer concentrations. FIG. 2D shows the average peak intensity for varying citrate phosphate buffer concentrations.

[0010] FIG. 3A and 3B depict non-limiting example schematics of micro-spotted nanosensors. FIG.3A illustrates a schematic of a manually micro-spotted substrate with 80 (5x16) low-density nanoparticle ensembles. FIG. 3B illustrates a schematic of planned micro-spotted nanosensor substrate layout.

[0011] FIG. 4A, 4B and 4C shows scanning electron microscope (SEM) images to monitor particle aggregation. FIG. 4A illustrates an acquired SEM image. FIG. 4B illustrates a binary image. FIG. 4C illustrates a masked image that depicts single particle versus aggregated particles.

[0012] FIG. 5 depicts the effect of varying particle optical density (OD) on particle aggregation.

[0013] FIG. 6 depicts the percentage of particles without a neighbor within 100nm as a function of optical density (OD).

[0014] FIG. 7A and 7B depicts the nucleic acid sensing using citrate-coated gold nanorods deposited onto amine functionalized surface. The citrate coated gold has a probe on it targeted to bind K. pneumoniae, and the nanoparticle is exposed to oligonucleotides from Klebsiella pneumoniae (target) and Escherichia coli (off-target control). FIG. 7A illustrates processed spectral data quantifying peak shift. FIG.7B illustrates centroid shift.

[0015] FIG. 8A and 8B depict the nucleic acid sensing using citrate-coated gold nanorods deposited onto amine functionalized surface using extracted genomic material from Klebsiella pneumoniae and E. faecium. FIG. 8A illustrates processed spectral data quantifying peak shift. FIG. 8B illustrates centroid shift.

[0016] FIG. 9 depicts the nucleic acid sensing using oligo-functionalized gold nanoparticles deposited onto amine functionalized surface using oligonucleotides from Klebsiella pneumoniae and Escherichia coli.

[0017] FIG. 10A and 10B depicts the nucleic acid sensing using cetyltrimethylammonium bromide (CTAB)-capped gold nanorods deposited onto thiol functionalized surface using oligonucleotides from Klebsiella pneumoniae and Escherichia.coli. FIG. 10A illustrates processed spectral data quantifying peak shift. FIG. 10B illustrates centroid shift.

[0018] FIG. 11 depicts the centroid shift of a nanoparticle ensemble as a function of time. The ensemble used was comprised of cetyltrimethylammonium bromide (CTAB)-capped gold nanorods with a probe targeted to bind K. pneumoniae deposited onto thiol functionalized surface. During the experiment, this nanoparticle ensemble is exposed to its target complement oligo. DETAILED DESCRIPTION

[0019] All patents, applications, published applications and other publications referred to herein are incorporated herein by reference to the referenced material and in their entireties. If a term or phrase is used herein in a way that is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the use herein prevails over the definition that is incorporated herein by reference.Plasmon-Resonance Sensing Devices

[0020] Disclosed herein is a plasmon-resonance sensing device. As shown in Figure 1A and 1B, the plasmon-resonance sensing device 100 comprises an array of sensors 101. Each sensor 101 comprises a plurality of nanoparticles 102 and is thus a nanoparticle ensemble. Preferably, the nanoparticles in the ensemble are homogeneously distributed. In some embodiments, the nanoparticles are metallic. For example, the nanoparticles may include one or more of the following: gold, platinum, aluminum, silver, copper, cesium oxide, metal coated iron oxide, and palladium. The nanoparticles may include nanospheres, nanorods, nanocubes, nanoshells, nanobipyramids, nanowires, nanoflakes or any other nano-sized particle shapes.

[0021] In some embodiments, the sensor (or nanoparticle ensemble) may have a diameter of about 0.01 mm to about 10 cm. In some embodiments, the sensor may have a diameter of about 0.01 mm to about 10 cm, about 0.01 mm to about 5 cm, about 0.5 mm to about 4 cm, about 0.05 mm to about 3 cm, about 0.5 mm to about 2 cm, about 0.5 mm to about 1 cm, or about 0.05 mm to about 5 mm, about 0.05 mm to about 2 mm, about 0.05 mm to about 1 mm, or about 0.05 mm to about 0.5 mm, or about 0.05 mm to about 0.3 mm, or about 0.05 mm to about 0.2 mm. In some embodiments, the sensor may have a diameter of about 0.1 mm. In some embodiments, the sensors may have an area of from about 1 μm2to about 5 mm2. In some embodiments, the sensors may have an area of from about 10 μm2to about 5 mm2about 50 μm2to about 5 mm2, about 100 μm2to about 5 mm2, about 200 μm2to about 5 mm2, about 400 μm2to about 5 mm2, about 500 μm2to about 5 mm2, about 1 mm2to about 2 mm2, about 2 mm2to about 5 mm2.

[0022] In some embodiments, the optical density (OD) of nanoparticles in each of the ensembles on the substrate is between about 0.001 and about 20. For example, the OD may be about 0.001, 0.005, 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20 or any integer that is between about 0.01 to 20. In some embodiments the OD of the nanoparticle ensembles is 0.001. In some embodiments the OD of the nanoparticle ensembles is 0.005. In some embodiments the OD of the nanoparticle ensembles is 0.01. In some embodiments the OD of the nanoparticle ensembles is 0.02. In some embodiments the OD of the nanoparticle ensembles is 0.05. In some embodiments the OD of the nanoparticle ensembles is 0.1. In someembodiments the OD of the nanoparticle ensembles is 0.5. In some embodiments the OD of the nanoparticle ensembles is 1. In some embodiments the OD of the nanoparticle ensembles is 5. In some embodiments the OD of the nanoparticle ensembles is 10. The OD (optical density) of the nanoparticles can be measured using spectrophotometry or dynamic light scattering and is correlated with the concentration of nanoparticles in the solution that is used to make nanoparticle ensembles. In some embodiments, the OD disclosed herein is the OD of nanoparticles that are spotted on the substrate. In some embodiments, the OD disclosed herein is the OD of nanoparticles in the solution that is used to make an array of spots.

[0023] In some embodiments, the nanoparticle ensembles are conjugated with a biological probe. The biological probe is configured to bind to an analyte. The binding of the analyte to the biological probe alters the electromagnetic surface properties of the nanostructure, thereby causing a change in localized surface plasmon resonance. In some embodiments, the biological probe comprises one or more of a protein, peptide strand, amino acid, RNA strand, DNA strand, oligonucleotide, and / or nucleotide. In some embodiments, the biological probe comprises one or more of a modified protein, modified peptide, modified amino acid, modified RNA strand, modified DNA strand, modified oligonucleotide, and / or modified nucleotide. In some embodiments, the biological probe comprises at least one of: a peptide-nucleic acid, an aptamer, an antibody, an antibody fragment, a complementary DNA, complementary oligonucleotide, 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, complementary oligonucleotide, and an enzyme.

[0024] In some embodiments, at least a first sensor 101a in the array of sensors comprises nanoparticles 102 conjugated with a first biological probe. In some embodiments, at least a second sensor 101b in the array of sensors comprises nanoparticles conjugated with a second biological probe. In some embodiments, at least a third sensor in the array of sensors comprises nanoparticles conjugated with a third biological probe. In some embodiments, at least a fourth sensor in the array of sensors comprises nanoparticles conjugated with a fourth biological probe. In some embodiments, at least a fifth sensor in the array of sensors comprises nanoparticles conjugated with a fifth biological probe. In some embodiments, a “n” number of sensors in an array of sensors comprises nanoparticles conjugated to an “n” number of biological probes,wherein “n” is any number from 1 to 2000 In some embodiments, 6 or 12 sensors may be presented in the array of sensors on a substrate 103.

[0025] The substrate 103 may be a dielectric or non-conductive substrate. In some embodiments, the substrate 103 is transparent to allow the sensors to be exposed to the incident light through the substrate 103. For example, the substrate 103 may be a glass substrate, a plastic substrate, or a polymeric substrate. The substrate and the sensor array on the substrate may be integrated with a microfluidic module to provide a means for introducing or exposing the sample to the sensors. Analyte Detection The sensing devices disclosed herein may be used for detecting an analyte in a sample. In some embodiments, the analyte comprises DNA, RNA and / or protein. In some embodiments, the method comprises exposing at least one sensor 101 in the plasmon-resonance sensing device 100 of any of the embodiments disclosed herein to a sample. The sample may or may not comprise the target analyte. The plasmon-resonance sensing device 100 can be utilized to detect the presence of an analyte (i.e., a target analyte). In some embodiments, the method comprises exposing at least two sensors in the plasmon-resonance sensing device 100 of any of the embodiments disclosed herein to a sample. In some embodiments, the method comprises exposing at least three sensors, at least four sensors, at least 5 sensors, or at least 6 sensors in the plasmon-resonance sensing device 100 of any of the embodiments disclosed herein to a sample. In some embodiments, the method comprises exposing an “n” number of sensors in the plasmon- resonance sensing device of any of the embodiments disclosed herein to a sample, wherein “n” is any number from 1 to 2000. In some embodiments, the array of sensors is exposed to the sample. The sample may comprise a bodily fluid, such as blood, plasma, mucus, serum, urine, or saliva, etc. Mucus can be collected via cervical swabs, vaginal swabs, or nasal swabs. When the at least one sensor 101 is exposed to the sample, the biological probe in each sensor would selectively bind to the analyte that the biological probe is configured to bind.

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

[0028] Optionally, at least one sensor may be incubated with additional biomolecules after exposure to the sample. In some embodiments, at least one sensor is exposed to a buffer or biomolecules. In some embodiments, this exposure occurs before, during, or after subsequent steps.

[0029] The method for detecting or sensing an analyte further comprises illuminating a light onto at least one sensor. In some embodiments, the method comprises illuminating a light at a series of wavelengths onto at least one sensor. In some embodiments, the light may be emitted from a light source in an apparatus for analyte detection. The light source may be configured to emit a series of wavelengths for illuminating the sensor. In some embodiments, the plasmonic sensing chip containing the sensors may be inserted into the apparatus for analyte detection. The apparatus is configured to emit a light at a series of wavelengths onto the sensors, and to collect an optical spectrum of the light transmitted through, absorbed by, or reflected from the sensors. For example, the apparatus can perform absorbance / transmittance measurements. In some embodiments the measurements are made at wavelengths ranging from 300-1100 nm.

[0030] The method further comprises collecting or measuring data from the sensor. In some embodiments, the method comprises collecting or measuring absorbance data from the sensor. In some embodiments, the method comprises collecting or measuring transmittance data from the sensor. In some embodiments, the method comprises collecting or measuring extinction data from the sensor. In some embodiments, the method comprises collecting or measuring scattered response from the sensor. In some embodiments, the method comprises collecting or measuring absorbance, transmittance, scattered response and / or extinction data of the sensor. In some embodiments, the method further comprises comparing collected or measured data with a baseline data of the sensor prior to the sample exposure. In some embodiments, the method further comprises comparing at least one of the collected or measured absorbance, transmittance, scattered response and / or extinction data with a baseline data of the sensor prior to the sample exposure. For example, the absorbance / transmittance measurements of functionalized sensors are made prior to exposure to the sample. The peak absorbance wavelength of the functionalized sensor (prior to bonding with a target analyte) is identified. The absorbance / transmittance of the sensors are made again after exposing to the sample, and a shift in peak absorbance can be observed if a target analyte is present in the sample and binds with the probe on the functionalized sensors. The shift represents the detection signal.

[0031] In some embodiments, an array of sensors in the plasmon-resonance sensing device 100 of any of the present embodiments is exposed to the sample. In some embodiments, at least a first sensor 101a in the array of sensors 101 comprises nanoparticles conjugated with a first biological probe. In some embodiments, at least a second sensor 101b in the array of sensors 101 comprises nanoparticles conjugated with a second biological probe. In some embodiments, at least a third sensor in the array of sensors comprises nanoparticles conjugated with a third biological probe. In some embodiments, at least a fourth sensor in the array of sensors comprises nanoparticles conjugated with a fourth biological probe. In some embodiments, at least a fifth sensor in the array of sensors comprises nanoparticles conjugated with a fifth biological probe. In some embodiments, a “n” number of sensors in an array of sensors comprises nanoparticles conjugated to an “n” number of biological probes, wherein “n” is any number from 1 to 50,000. 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 a multiplex sensing capability. In this configuration, multiple analytes can be detected simultaneously.

[0032] In some embodiments, at least a first sensor 101a in the array of sensors comprises nanoparticles conjugated with a first biological probe and at least a second sensor 101b in the array of sensors comprises nanoparticles conjugated with a second biological probe. In some embodiments, a first set of sensors in the sensor array is functionalized with a first biological probe, and a second set of sensors in the sensor array is functionalized with a second biological probe. In some embodiments, the first biological probe and the second biological probe are different. In some embodiments, the first biological probe and the second biological probe are the same. In some embodiments, the first biological probe and the second biological probe independently comprise one or more of a protein, peptide strand, amino acid, RNA strand, DNA strand, oligonucleotide, or and / or nucleotide. In some embodiments, the first biological probe and the second biological probe independently comprise one or more of a modified protein, modified peptide, modified amino acid, modified RNA strand, modified DNA strand, modified oligonucleotide, and / or modified nucleotide. In some embodiments, the first biological probe and the second biological probe independently comprise at least one of: a peptide-nucleic acid, an aptamer, an antibody, an antibody fragment, a complimentary DNA, and / or an enzyme. In some embodiments, first biological probe and the second biological probe are independently selectedfrom the group consisting of a peptide-nucleic acid, an aptamer, an antibody, an antibody fragment, a complimentary DNA, and an enzyme.

[0033] The detection of analyte(s) is based on an optical phenomenon that occurs between a metal nanoparticle and a dielectric – localized surface plasmon resonance (LSPR). LSPR is observed when the wavelength of incident light is larger than the size of the conductive nanoparticles. The nanoparticles result in highly confined electric fields of LSPR modes, which serve as a sensitive transducer to changes in the local dielectric environment (binding event). The nanoparticles can be conjugated to / covalently functionalized with probes that can bind with target analytes. Upon binding with the target analyte(s), red shifts in the spectral peak can be observed. In some embodiments the amount of red shift may be observed as a function of target analyte concentration. In some embodiments, the sensors detect transmittance, reflectance, scattered response and / or absorbance at certain wavelength range.

[0034] In some embodiments, the sensors that have been exposed to the sample, thus having analyte(s) bound to selective biological probe on the sensors, can be further exposed to functionalized particles configured to bind to the sensors that have analyte(s) present and bound to the biological probe. The functionalize particles may be nanoparticles or microparticles. In some embodiments, the particles may be metal, polymer, glass, or any material with a high refractive index, for example, a refractive index of about 1.5 and higher. When the functionalized particles are bound to the sensors, it has the potential to improve both sensitivity and specificity of the sensors. Without being bound to the theory, the sensitivity improvements may be due to the fact that the functionalized particle increases the change in refractive index at the sensor surface in the presence of the analyte. The additional binding of the functionalized particles to the sensors may improve the sensor signal through a greater peak-shift in the optical measurement. Specificity improvements may be due to the fact that two selective binding events are required (i.e., first analyte must bind to the sensor, then the functionalized particle must bind to the sensor-bound analyte). In some embodiments, the functionalized particles are functionalized to bind to the analytes that have bound to the biological probes.

[0035] In some embodiments, a spectrum of the sensor comprising an array of functionalized nanoparticles ensembles may be obtained prior to exposure to a sample. This may provide baseline data for the determination and analysis of an analyte binding event. Nanoparticle Ensembles Fabrication

[0036] Disclosed herein is a method for making nanoparticle ensembles. The method includes providing a chemically functionalized substrate, providing a nanoparticle solution, depositing nanoparticle solution in an array of spots on the chemically functionalized substrate, and evaporating the nanoparticle solution under a high humidity condition to form an array of nanoparticles ensembles. The nanoparticle ensembles may be formed in an array on a substrate. In some embodiments, the nanoparticle ensembles may be configured as sensors. In some embodiments, the nanoparticle ensembles may be utilized in a sensing device, such as a plasmon- resonance sensing device. In some embodiments, the method can be used to make low-density nanoparticle ensembles. In some embodiments, a low-density nanoparticle ensemble includes an ensemble that contains a number of nanoparticles that is not enough to make a monolayer within said ensemble. In some embodiments, the low-density nanoparticle ensemble includes an ensemble that contains a number of nanoparticles such that the majority of nanoparticles are single and not within 10nm of another nanoparticle. In some embodiments, the low-density nanoparticle ensemble includes an ensemble that contains a number of nanoparticles such that the majority of nanoparticles are single and not within 50nm of another nanoparticle. In some embodiments, the low-density nanoparticle ensemble includes an ensemble that contains a number of nanoparticles such that the majority of nanoparticles are single and not within 100nm of another nanoparticle. Chemically functionalized substrate

[0037] Chemical functionalization of the surface or substrate is required prior to nanoparticle deposition for successful fabrication of low-density nanoparticle ensembles due to the required electrostatic or covalent interaction between nanoparticles and substrates. The chemically functionalized substrate may include thiol-functionalized, carboxy-functionalized, epoxy-functionalized, aldehyde-functionalized, NHS-functionalized, poly-L-lysine- functionalized, maleimide-functionalized, streptavidin-functionalized, neutravidin-functionalized, avidin-functionalized, or amine-functionalized substrate. In some embodiments, the chemically functionalized substrate may be purchased from a third party. In some embodiments, the chemically functionalized substrate is prepared in-house. In some embodiments the following steps may be used. In some embodiments, the substrate is a glass substrate. In some embodiments, the substrate is a plastic substrate. In some embodiments, the substrate is a polymeric substrate.

[0038] In some embodiments, the method of preparing a chemically functionalized substrate comprises cleaning the substrate by sonication, adding a functionalization solution to thecleaned substrate, washing the substrate to remove any excess functionalization solution that is not bound to the substrate and drying the functionalized substrate. In some embodiments, the substrate is cleaned before being functionalized. In some embodiments, the substrate is placed in a fluid that is agitated by ultrasound. In some embodiments, the substrate is cleaned using a sonicator. In some embodiments, the fluid for cleaning the substrate comprises one or more detergents. In some embodiments, the concentration of detergent in the fluid is about 1%, about 2 %, about 3 %, about 4 %, about 5 %, about 6 %, about 8 %, about 10 %, or any % that is between about 2 % and about 10 %. In some embodiments, the concentration of detergent in the fluid is about 2 %. In some embodiments, the detergent solution comprises anionic surfactants. In some embodiments, the detergent solution comprises nonionic surfactants.

[0039] In some embodiments, the substrate may further be cleaned by a solution containing one or more alcohol. In some embodiments, the alcohol includes methanol. In some embodiments, the alcohol includes ethanol. In some embodiments, the substrate may further be cleaned by a solution containing one or more acids. In some embodiments, the acid includes hydrochloric acid and sulfuric acid. In some embodiments, the substrate may further be cleaned by a solution containing one or more peroxides. In some embodiments, the peroxide includes hydrogen peroxide. In some embodiments, the substrate may further be cleaned by plasma etching. In some embodiments, the cleaning fluid may comprise a combination of alcohol(s), peroxide(s) and acid(s). In some embodiments, the substrate is cleaned by a mixture of methanol and hydrochloric acid. The substrate is further rinsed / washed by ultra-pure water, such as HPLC grade water. The substrate can then be dried in room temperature or in an elevated temperature.

[0040] In some embodiments, the substrate is allowed to incubate for about 10 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 10 hours, about 20 hours, about 24 hours, about 30 hours or any amount of time that is between 1 hour and 30 hours. In some embodiments, the temperature at which the substrate is allowed to dry is about 0°C , about 10°C , about 23°C, about 27°C, about 30°C, about 37°C, about 40°C, about 50°C, about 60°C, about 70°C, about 80°C, about 90°C, about 100°C, or any temperature that is between about 23°C and about 100°C.

[0041] In some embodiments, the pre-cleaned substrates are chemically functionalized using the method outlined herein. In some embodiments, the substrates are thiol functionalized. In some embodiments, the substrates are amine functionalized.

[0042] Thiol-functionalized substrates can be prepared by exposing the substrates to (3-Mercaptopropyl)trimethoxysilane (MPTMS) or 3-mercaptopropyl triethoxysilane (mptes) (MPTES). Amine-functionalized substrates can be prepared by exposing the substrates to one or more organosilanes. For example, a mixture of 3-aminopropyltriethoxysilane (APTES) and 3- aminopropyltrimethoxysilane (APTMS) can be used in functionalization of the substrate. The functionalized substrate may further be washed in anhydrous ethanol and sonicated. In one embodiment, the substrate is exposed to APTES for 15 minutes at room temperature. Nanoparticles Deposition

[0043] Colloidal nanoparticles may be electrostatically deposited onto or covalently attached to the functionalized substrate. Nanoparticles are homogenously deposited on the functionalized surface. Nanoparticles may be modified for deposition or covalent bonding to the substrate. A nanoparticle solution comprising modified nanoparticles may be purchased or prepared in-house.

[0044] For electrostatic deposition of nanoparticles, the nanoparticles may be capped or coated with a substance that allows for electrostatic interaction between the nanoparticles and the functionalized substrate surface. In some embodiments, the nanoparticles are coated with a substance that allows for electrostatic interaction between the nanoparticles and the functionalized substrate surface. In some embodiments, the nanoparticles are capped. For example, the cap may comprise citrate, silica, poly-ethylene glycol, polyvinylpyrrolidone, or polyacrylic acid. In some embodiments, the citrate-capped nanoparticles may be suspended in a buffer, such as a citrate phosphate buffer. In some embodiments, the nanoparticles are functionalized with a biorecognition molecule. In some embodiments, the nanoparticles are functionalized with an oligonucleotide or a DNA sequence. In some embodiments, the nanoparticles are functionalized with amino acids. In some embodiments, the biorecognition molecule is selected from the group consisting of a peptide- nucleic acid, an oligonucleotide, an aptamer, an antibody, an antibody fragment, a complimentary DNA or RNA, a complementary oligonucleotide, a modified oligonucleotide, a locked nucleic acid, a small molecule or an enzyme. In some embodiments, the nanoparticles may be functionalized by an oligonucleotide that has a sequence complementary to a biorecognition molecules. In some embodiments, the modified nanoparticles for electrostatic deposition may interact with amine functionalized substrate. The modified nanoparticles may be suspended in citrate phosphate buffer or water.

[0045] In some embodiments, the nanoparticles are capped with a cap selected from a group consisting of citrate, cetyltrimethylammonium bromide (CTAB), Polyethylene glycol (PEG), Polyvinylpyrrolidone (PVP), Polyvinyl alcohol (PVA), Bovine serum albumin (BSA), and Ethylene diamine tetra acetic acid (EDTA). In some embodiments, the capped nanoparticles in different nanoparticle ensembles are functionalized with different biological probes. In some embodiments, the capped nanoparticles in different nanoparticle ensembles are functionalized with same biological probes.

[0046] For covalent bonding or physisorption to a functionalized substrate surface, the nanoparticles may be capped with cetyltrimethylammonium bromide (CTAB). In some embodiments, the CTAB-capped nanoparticles may be suspended in water. In some embodiments, the CTAB-capped nanoparticles may be suspended in solutions of CTAB in water. In some embodiments, the CTAB-capped nanoparticles may be suspended in citrate or phosphate buffer. In some embodiments, the CTAB-capped nanoparticles can be immobilized onto a thiol- functionalized substrate surface.

[0047] In some embodiments, the nanoparticles may be sonicated prior to spotting on to the substrate. In some embodiments, the nanoparticles are sonicated for about 2 seconds, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 60 minutes or any amount of time that is between 2 and 60 minutes.

[0048] The method further comprises deposition of nanoparticles onto the substrate. In some embodiments, the nanoparticles are manually micro-spotted on the substrate. In some embodiments, the nanoparticles are micro-spotted using an automatic pipetting system. In some embodiments, the nanoparticles are spotted using high-throughput precision dispensing technology.

[0049] In some embodiments, the dispense volume of nanoparticles that are spotted on the substrate is about 0.01μl, about 0.02μl, about 0.04μl, about 0.05μl, about 0.1μl, about 0.2μl, about 0.5μl, about 1μl, about 1.5μl, about 2μl, about 2.5μl, about 3μl, about 3.5μl, about 4μl, about 4.5μl, about 5μl, about 10μl or any integer that is between about 0.01μl to 10μl.

[0050] In some embodiments, the spot size of the nanoparticle ensembles is about 0.01 mm to about 10 cm. In some embodiments, the sensor may have a diameter of about 0.01 mm to about 10 cm, about 0.05 mm to about 5 cm about 0.5 mm to about 4 cm, about 0.05 mm to about3 cm, about 0.5 mm to about 2 cm, about 0.5 mm to about 1 cm, or about 0.05 mm to about 5 mm, about 0.05 mm to about 2 mm, about 0.05 mm to about 1 mm, or about 0.05 mm to about 0.5 mm, or about 0.05 mm to about 0.3 mm, or about 0.05 mm to about 0.2 mm. In some embodiments, the spot size may have a minimum diameter of about 0.1 mm.

[0051] In some embodiments, a sensing device comprising an array of nanoparticle ensembles on a substrate is disclosed. In some embodiments, each of the nanoparticle ensembles comprises a homogenous distribution of nanoparticles wherein the nanoparticle ensembles have a nanoparticle optical density ranging from 0.02 to 20.

[0052] In some embodiments, the optical density (OD) of nanoparticle ensembles on the substrate is between about 0.01 and about 20. For example, the OD may be about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20 or any number that is between about 0.01 to 20. In some embodiments the OD of the nanoparticle ensembles is 0.01. In some embodiments the OD of the nanoparticle ensembles is 0.02. In some embodiments the OD of the nanoparticle ensembles is 0.05. In some embodiments the OD of the nanoparticle ensembles is 0.1. In some embodiments the OD of the nanoparticle ensembles is 0.5. In some embodiments the OD of the nanoparticle ensembles is 1. In some embodiments the OD of the nanoparticle ensembles is 5. In some embodiments the OD of the nanoparticle ensembles is 10.

[0053] In some embodiments, the detection sensitivity may be improved by minimizing particle aggregation (defined as a particle physically touching another particle) of nanoparticles on the substrate surface. Particle aggregations may mute the spectral shift expected from a biomolecule binding event when compared to a binding event occurring on a single particle. This is largely due to interparticle couplings that hypsochromically shift the resonance of the aggregated particles compared to a single particle on the surface. Assuming no limitations in optical detection capability, continuing to step down in nanoparticle density will increase detection sensitivity. More specifically, decreased particle aggregation means that each nanoparticle will be substantially free of interactions with neighbors. As a result, the biomolecular target will be bound to a smaller number of sensors resulting in a larger ensemble colorimetric shift.

[0054] In some embodiments, the dispense volume of the nanoparticle solutions may be adjusted depending on the contact angle of the substrate surface for spotting. The OD of the nanoparticle ensembles may be adjusted in view of the changes in surface tension and / or other conditions. In some conditions, and dependent on the contact angle, it may be advantageous to increase the OD of particles, which can be achieved through particle centrifugation and redispersion into a lower volume of buffer. In other conditions, and dependent on the contact angle, it may be advantageous to decrease the OD of the particles, which can be achieved through dilution in additional buffer with controlled salt concentration and / or pH.

[0055] Following the nanoparticle spotting / deposition, the substrate is placed in a high humidity environment / chamber for the solution to evaporate. The relative humidity of such high humidity environment may be about 60% to 100%, about 65% to 100%, about 70% to 100%, about 75% to 100%, about 80% to 100%, about 85% to 100%, about 90% to 100%, or about 95% to 100%. In some embodiments, the environment is cooled to be about 18°C to 24°C. In some embodiments, the incubation time is about 10 mins to about 1 hour, about 1 to about 5 hours, about 1.5 to about 4 hours, about 2 to 3 hours, or about 2 hours. In some embodiments, the nanoparticle functionalized substrates are dried in a low light condition. In some embodiments, the nanoparticle functionalized substrates may be subsequently submerged in high-purity water after the drying.

[0056] In some embodiments, the deposition of nanoparticles onto the substrate under the conditions disclosed herein reduces the aggregation of nanoparticles. In some embodiments, the deposition of nanoparticles onto the substrate under the conditions disclosed herein results in homogenous deposition of nanoparticles. In some embodiments, the deposition of nanoparticles onto the substrate under the conditions disclosed herein results in evenly spaced nanoparticles. In some embodiments, the deposition of nanoparticles onto the substrate under the conditions disclosed herein reduces nanoparticle-nanoparticle interaction. In some embodiments, deposition / functionalization steps described herein allows for more uniformly distributed nanoparticles in the nanoparticle ensembles (sensors), especially when the nanoparticle ensemble contains a concentration of the nanoparticles that is less than needed for forming a monolayer in the spot / ensemble on the substrate surface. In some embodiments, the nanoparticles in the nanoparticle ensemble are homogeneously distributed.

[0057] The method may further comprise a surface blocking step following the nanoparticle deposition to avoid nonspecific surface binding. In some embodiments, NHS-conjugated molecules are used for surface blocking following the nanoparticle deposition on an amine-functionalized substrate. In some embodiments, NHS-conjugated molecule is NHS-PEG. In some embodiments, blocking molecule may be NHS-Acetate, bovine serum albumin, succinic anhydride in 1-methyl-2-pyrrolidon or acetic anhydride in dichloroethane. Blocking can also be performed with negatively charged polymers, such as Dextran Sulfate or DNA such as Salmon Sperm DNA. Additional blocking buffers such as BlockAid, Thermo Superblock (single glycoprotein), Pierce Blocking Buffer, Denhardt's Solution (1% Ficoll (type 400), 1% polyvinylpyrrolidone, and 1% bovine serum albumin)

[0058] In some embodiments, thiol reactive molecules may be used for surface blocking following nanoparticle deposition on a thiol-functionalized substrate. Examples of the thiol reactive molecules include mmPEG12 (methyl-PEG-maleimide reagent), iodoacetic acid, and ethylene maleimide.

[0059] The method may further comprise functionalization of the deposited nanoparticles with biological probes. In some embodiments, the biological probe is selected from the group consisting of a peptide-nucleic acid (PNA), an oligonucleotide, an aptamer, an antibody, an antibody fragment, a complementary DNA, a complementary oligonucleotide, and an enzyme. In some embodiments, the biological probe is a PNA. In some embodiments, gold nanoparticle ensembles can be functionalized with PNA. PNA sequences can be designed to bind sequences specific to target organisms. In some embodiments, the PNA probes include a thiol-linker, which enables direct covalent binding to gold nanoparticle ensembles. In some embodiments, the biological probe is a complementary oligonucleotide. In some embodiments, gold nanoparticle ensembles can be functionalized with this oligonucleotide. Oligonucleotide sequences can be designed to bind sequences specific to target organisms. In some embodiments, the oligonucleotide probes include a thiol-linker, which enables direct covalent binding to gold nanoparticle ensembles. Definitions

[0060] All technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs unless clearly indicated otherwise.

[0061] As used herein, the singular forms “a”, “and”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a sequence” may include a plurality of such sequences, and so forth.

[0062] The terms “comprising,” “including,” “containing,” and various forms of these terms are synonymous with each other and are meant to be equally broad. Moreover, unless explicitly stated to the contrary, examples comprising, including, or having an element or a plurality of elements having a particular property may include additional elements, whether or not the additional elements have that property.

[0063] The term “nanoparticle,” as used herein, refers to any particle that is between about molecular size, to about microscopic size. nanoparticles comprise nanomaterials, which can be any material in which a single unit is sized at about 1 nm to about 200 nm. Nanoparticles include nanorods, nanosquares, nanocubes, gradient multilayer nanofilm (GML nanofilm), icosahedral twins, nanocages, magnetic nanochains, nanocomposite, nanofabrics, nanofiber, nanoflower, nanofoam, nanohole, nanomesh, nanopillar, nanopin film, nanoplatelet, nanoribbon, nanoring, nanobipyramids, irregular nanoparticles, nanosheet, nanoshell, nanotip, nanoflake, nanoplate, nanowire, and nanostructured film. It will be understood that a nanoparticle can have various geometric shapes and properties based on the components of that nanoparticle.

[0064] The term “analyte” refers to a substance or chemical constituent that is of interest. For examples, analyte may include biological or chemical substance that may be detected by a sensing device and may be of interest for diagnosing a disease or a condition.

[0065] The term “density” refers to the surface particle density of the nanoparticles deposited on the surface of the substrate. The term “density” may be defined with respect to the interparticle distance between the nanoparticles. It may also be defined with respect to surface coverage – the percentage of the substrate surface that is covered by nanoparticles. Deposition of nanoparticle solutions of higher optical density (OD) result in higher density nanoparticle assemblies.

[0066] The term “homogeneous distribution” refers to a surface distribution of nanoparticles on substrate wherein the ensemble are not aggregated, and are instead evenly spaced apart. This can be determined based on a spectrometric (absorbance, reflectance, extinction) measurement through the substrate.

[0067] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[0068] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0069] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.EXAMPLES

[0070] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting. Example 1: Preparation of Chemically Functionalized Substrates

[0071] Glass microscope slides were used as substrates for preparing the sensor / nanoparticle ensemble arrays. The glass slides were cleaned by sonication in a heateddetergent solution (2% RBS 35) prior to functionalization, the washed with water. The slideswere also sonicated in a mixture of methanol and HCl, then washed with water and let dried. Amino-functionalized substrate were prepared by immersing the glass slides in a solution of 10% (v / v) ATPES ((3-Aminopropyl)triethoxysilane) solution or 10% (v / v) MPTMS ((3- mercaptopropyl) trimethoxysilane)) in anhydrous ethanol at room temperature. The slides were removed from the solution, sonicated in anhydrous ethanol, then placed in the oven at 120°C for 3 hours. After the 3-hour incubation, the slides were removed from the oven allowed to come to room temperature. Example 2: Electrostatic Deposition of Citrate Coated Gold Nanoparticles onto Amine Functionalized Surface

[0072] Citrate coated gold nanoparticles were electrostatically deposited onto amine functionalized surface. Stock solution of citrate-capped gold nanorods were vortexed and sonicated before it was diluted to desired Optical Density (OD) with 0.1mM citrate phosphate buffer. Citrate phosphate buffer could minimize aggregation of the gold nanorods on the surface due its highly negative charge that effectively forms a stabilizing and displaceable barrier around the nanoparticle. Gold nanoroads were suspended in citrate phosphate buffer at a concentration of 0.01 mM to 5mM. The pH of the buffer was below the pKa of the amine on the surface, and the salt concentration was optimized to disperse the nanorods across the surface. As shown in Figures 2A and 2B, as the salt concentration was lowered, the rod density on the surface went down. The effect of the salt concentration on dispersion levels off at 0.1 mM as shown in Figures 2C and 2D. These results indicate that as the salt concentration was lowered below 0.1 mM, the peak intensity and the full width at half max (FWHM), metrics for plasmonic signal quality and / or sensitivity flattened out, indicating that maximum dispersity was achieved with this buffer.

[0073] Following the dilution of nanoparticles in 0.1mM citrate phosphate buffer, desired volume of the nanoparticle solution was pipetted onto 3D amine-functionalized slide to form an array of nanoparticle ensembles as shown in Figures 3A and 3B. The desired volume depends on contact angle of the surface for spotting. For example, 1μL dispense resulted in ~2mm diameter spots while 2.5μL dispense resulted in ~3.4mm spots. The OD may be adjusted based on any changes to surface tension or condition. The dispensing can be carried out manually (via micropipette) and using a high-throughput precision dispensing technology (e.g., Scienion Ag, Berlin, Germany).

[0074] The slides were then placed into high humidity environment and were incubated for 2 hours in a low-light environment. For maintaining high humidity, a petri dish was filled with 15mL of water, a slide was added on top of stand in petri dish that kept it off of wetted surface and the petri dish was then covered and sealed with parafilm to form a humidity chamber. The slides were removed from humidity chamber and were submerged in MilliQ water. The slides were subsequently removed from the MilliQ water, and stored dry or in water bath, face up, at room temperature.

[0075] Next, surface blocking was performed on the functionalized substrate surface with citrate coated gold nanoparticles. First, 100mg / mL NHS-PEG suspended in 10mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) was prepared and vortexed for 30 seconds. The dried slide surface was then coated in NHS-PEG solution using a manual or automated micro- pipettor, ensuring that entire volume spreads evenly across slide surface. The slides were incubated at room temperature for 1 hour followed by washing with MilliQ water and dried.

[0076] Example 3: Deposition of Oligo-Functionalized Gold Nanoparticles onto Amine Functionalized Surface

[0077] The ability to deposit nanoparticles that have been functionalized with a biorecognition molecule (i.e., oligo) prior to deposition onto the substrate was demonstrated. Gold nanorods functionalized with a specified oligonucleotide were spotted onto amine functionalized slides using the protocols specified in Example 2. Example 4: Covalent Linkage of CTAB-Capped Gold Nanoparticles onto Thiol Functionalized Surface

[0078] A thiol-functionalized slide was pretreated by submersing in a solution of dithiothreitol (DTT) in phosphate buffered saline (PBS) then washed with MilliQ water and airdried.Excess CTAB from CTAB-capped gold nanorods was removed by adding CTAB-capped gold nanorods to HPLC water and centrifuged at 1900 RCF for 5 min. Some supernatant was then removed without disturbing the pellet, and the pellet was resuspend using a pipette tip, and MilliQ water was added. This suspension can be used directly for spotting or as a starting point for further OD dilutions.

[0079] 1μL of the nanoparticle solution was pipetted onto thiol functionalized slides to form an array of nanoparticle ensembles, the resulted spot diameter was about 2 mm. The OD may be adjusted based on any changes to surface tension or condition. The slides were then placed into high humidity environment and were incubated for 2 hours in a low-light environment as described in Example 2. The slides were removed from humidity chamber and were submerged in MilliQ water briefly and removed, stored dry or in water bath, face up, and at room temperature. Example 5: Surface Blocking

[0080] Surface blocking was performed on the functionalized substrate surface with CTAB-Capped Gold Nanoparticles prior to further functionalization. The functionalized slide surface was coated in a 12.5mM PEG12 solution (Methyl-PEG-Maleimide Reagent suspended in 100% DMSO and diluted with PBS) using a manual or automated micro-pipettor, ensuring that entire volume spreads evenly across slide surface. The slides were incubated at room temperature for approximately 2.5 hours followed by washing with MilliQ water and dried. In addition to PEG12, other thiol reactive molecules, such as idoacetic acid or ethylene maleimide can also be used for surface blocking. Example 6: Nanoparticle Density and Aggregation Analysis

[0081] In this example, the effect of nanoparticle surface density on biomolecule sensing was evaluated. Nanosensor sensitivity is enhanced when single nanoparticles are not aggregated or not interacting with their neighbors. Quantitative analyses of SEM images were used to evaluate particle aggregation and particle interactions with neighbors. Analysis of SEM images was completed using ImageJ, a National Institutes of Health and the Laboratory for Optical and Computational Instrumentation developed freeware designed for analysis of a wide range of image processing techniques.

[0082] For this analysis, images were converted to binary using ImageJ’s AutoThresholding function. Threshold values for the binary cutoff were determined based on SEM light intensity levels and were tuned for each image batch. Once the binary was acquired, theAnalyze Particles function was used to determine the total number of particles and surface area covered by those particles including determination between a single particle in space and a group of aggregated particles. Image conversion and masking steps are shown in Figures 4A-4C. The effect of varying particle optical density (OD) on particle aggregation is shown in Figure 5. All OD conditions were spotted in 0.1mM citrate phosphate buffer onto an amine functionalized surface. It was observed that particle aggregation increased in magnitude with high particle OD. At a higher surface coverage, we found that a larger portion of the substrate was covered by single particles, which could be advantageous for biosensing.

[0083] The interaction of nanoparticles with their neighbors may be evaluated by using the Analyze Particle function (ImageJ, NIH) to establish the boundary coordinates of each particle. Following export of these coordinates, the percentage of particles that did not have another particle within 100nm of its location was quantified. The 100nm threshold was determined by completing a decay length analysis in Finite-Difference Time-Domain (FDTD) simulations for the 124x40nm gold nanorods that were immobilized for the SEM images analyzed. For pixel resolution of the SEM images, 100nm between particles was equivalent to 16 pixels separating the boundary of each particle. Figure 6 illustrates the percentage of particles without a neighbor within 100nm as a function of OD. This analysis was completed for the same images that were used for Figure 5. Example 7: Nanosensor Functionalization with Biorecognition Molecule (Peptide Nucleic Acid)

[0084] Nanoparticle ensembles were functionalized with peptide nucleic acids (PNAs) purchased from PNA BIO Inc. (Newbury Park, CA). All PNA probes also included a thiol-linker (-SH) to enable direct covalent binding to gold nanoparticle ensembles. PNA stock was thawed and a 1uM PNA solution was prepared via dilution in HPLC water with 0.1% v / v Tween20. The PNA solution was then spotted (manually or via automated precision dispensing technology) onto the nanoparticle ensembles and incubated at room temperature for 1 hour, then aspirated off the substrate. The surface was washed with Saline-Sodium Citrate (SSC) buffer to remove residual PNA from the sensing surface. Transmission spectra were collected before and after the PNA functionalization to characterize successful PNA conjugation. Transmittance through the samples was measured using Nanopath’s custom readout instrumentation. For seamless integration with the readout instrumentation, the final geometries were fabricated as to fully cover the light source spot size, minimizing signal loss. Detailed specifications of the readout instrument and user interface are described in PCT / US2022 / 075993, which is incorporated by reference herein.Example 8: Nucleic Acid Sensing using Citrate-Coated Gold Nanorods Deposited onto Amine Surface

[0085] Nucleic acids detection utilizing citrate-coated gold nanorods deposited onto an amine surface was performed on samples containing either genomic material or complement oligonucleotides. Genomic extractions were prepared from bacterial culture using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) quantified using a NanoDrop UV-Vis Spectrometer (Thermo Scientific, Waltham, MA) and / or The Qubit® dsDNA HS Assay and diluted to the desired concentration. Oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA) and diluted in SSC buffer to the desired concentration. Following preparation, the samples were spotted onto sensing substrates at a range of concentrations and transmission spectra were collected to determine if hybridization to PNA-functionalized sensor occurred. The sensing substrate was functionalized with a PNA designed to bind Klebsiella pneumoniae.

[0086] Oligonucleotides ranged in length from 15 base pairs (exact complement) to 200 base pairs were exposed to the functionalized sensing substrate. Two unique 200 base pair sequences were evaluated: 1) an oligo with the genomic sequence flanking the target region, and 2) an oligo synthetic sequence flanking the target region. Concentrations of oligonucleotides ranged from 0.01 uM to 0.1 uM and were suspended in SCC buffer. As an off-target control, E. coli oligonucleotides (0.01 uM to 0.1 uM suspended in SCC buffer) were exposed to the functionalized sensing substrate. Similarly, a 15 base pair oligo and two 200 base pair oligos with genomic and synthetic flanking sequences, respectively were also designed. When exposed to on- target K. pneumoniae oligonucleotides, a significant shift in the peak absorbance wavelength of the sensor in all three oligonucleotides conditions was observed as illustrated in Figures 7A and 7B. No significant peak shift was observed when the sensor was exposed to off-target E. coli oligonucleotides as illustrated in Figures 7A and 7B.

[0087] Three concentrations of extracted K. pneumoniae genomic material, ranging from 103-105 copies / μL were exposed to the functionalized sensing substrate. As an off-target control, three concentrations of Enterococcus faecium genomic material, ranging from 103-105 copies / μL were exposed to the functionalized sensing substrate. A significant shift in the peak absorbance wavelength of the sensor was observed when exposed to K. pneumoniae as illustrated in Figures 8A and 8B. No significant peak shift was observed when the sensor was exposed to off-target E. faecium genomic material as shown in Figures 8A and 8B. This data supports that high- throughput method developed for homogenous deposition of citrate-coated gold nanoparticles can be successfully utilized for robust molecular plasmonic sensing. Example 9: Nucleic Acid Sensing using Oligo Functionalized Gold Nanoparticles Deposited onto Amine Functionalized Surface

[0088] Nucleic acids detection utilizing oligo functionalized gold nanoparticles deposited onto an amine surface was performed on samples containing complement oligonucleotides. Gold nanorods functionalized with an oligonucleotide specific to Klebsiella pneumoniae were purchased from Nanopartz Inc. (Loveland, CO). The functionalized gold nanorods were spotted onto amine-functionalized slides using the protocol specified in Example 2.

[0089] Three different nanoparticle ensembles densities ranging from OD1 to OD6 were tested. Direct complement oligonucleotide specific to K. pneumoniae was exposed to the sensing substrate. As an off-target control, oligonucleotide specific to E. coli to the sensing substrate was exposed to the sensing substrate. A significant shift in the peak absorbance wavelength of the sensor when exposed to on-target K. pneumoniae oligo complement relative to the peak shift was observed when the sensor was exposed to the off-target E. coli oligo was observed as illustrated in Figure 9. This data supports that a high-throughput method developed for homogenous deposition of oligo functionalized colloidal gold nanoparticles onto the substrate can be successfully utilized for robust molecular plasmonic sensing. Example 10: Nucleic Acid Sensing using CTAB-Capped Gold Nanorods Deposited onto Thiol Surface

[0090] Nucleic acids detection utilizing CTAB-coated gold nanorods deposited onto a thiol surface was tested performed on samples containing a complement oligonucleotide. The sensing substrate was functionalized with a PNA designed to bind Klebsiella pneumoniae.0.5 uM of direct complement oligonucleotide specific to K. pneumoniae was exposed to the sensing substrate. As an off-target control, 0.5 uM of oligonucleotide specific to E. coli was exposed to the sensing substrate. A significant shift in the peak absorbance wavelength of the sensor was observed when exposed to on-target K. pneumoniae oligo complement as illustrated in Figures 10A and 10B. No significant peak shift was observed when the sensor was exposed to the off-target E. coli oligo as illustrated in Figures 10A and 10B. It was also evident that the majority of target bindingto sensor occurred within the first approximately 10 min of incubation as shown in Figure 11. This demonstrates the ability to enable rapid nucleic acid profiling using such low-density nanoparticle ensembles as disclosed herein. This data supports that a high-throughput method developed for homogenous deposition of CTAB-capped gold nanoparticles can be successfully utilized for rapid and robust molecular plasmonic sensing.

[0091] The scope of the present disclosure is not intended to be limited by the specific disclosures of examples in this section or elsewhere in this specification and may be defined by claims as presented in this section or elsewhere in this specification or as presented in the future. The language of the claims is to be interpreted broadly based on the language employed in the claims and not limited to the examples described in the present specification or during the prosecution of the application, which examples are to be construed as non-exclusive.

Claims

WHAT IS CLAIMED IS:

1. A method of making nanoparticle ensembles comprising: providing a chemically functionalized substrate; providing a nanoparticle solution; homogenously depositing the nanoparticle solution in an array of spots on the chemically functionalized substrate; and incubating the nanoparticle solution under a high humidity condition to form an array of nanoparticle ensembles.

2. The method of claim 1, wherein the nanoparticle is selected from the group comprising gold, platinum, palladium, aluminum, silver, metal coated iron oxide, copper, and cesium oxide.

3. The method of claim 1, wherein prior to deposition, the nanoparticles are conjugated to a biological probe, wherein the biological probe is selected from the group consisting of a peptide-nucleic acid, an oligonucleotide, an aptamer, an antibody, an antibody fragment, a complimentary DNA or RNA, a complementary oligonucleotide, a modified oligonucleotide, a locked nucleic acid, an enzyme, and a small molecule.

4. The method of claim 1, wherein the nanoparticle solution is deposited on the chemically functionalized substrate at an optical density of 0.02 to 20.

5. The method of claim 1, wherein the nanoparticles are capped.

6. The method of claim 5, wherein the nanoparticles are capped with a cap selected from a group consisting of citrate, cetyltrimethylammonium bromide (CTAB), Polyethylene glycol (PEG), Polyvinylpyrrolidone (PVP), Polyvinyl alcohol (PVA), Bovine serum albumin (BSA), Ethylene diamine tetra acetic acid (EDTA), and a combination thereof.

7. The method of claim 5, wherein the capped nanoparticles are further functionalized with a biological probe, wherein the biological probe is selected from the group consisting of a peptide-nucleic acid, an oligonucleotide, an aptamer, an antibody, an antibody fragment, a complementary DNA or RNA, a complementary oligonucleotide, a modified oligonucleotide, a locked nucleic acid, an enzyme, or a small molecule.

8. The method of claim 1, wherein the spot size of the nanoparticle solution deposited on the chemically functionalized substrate is 0.01 mm to 50 mm in diameter.

9. A sensing device comprising an array of nanoparticle ensembles on a substrate, wherein each of the nanoparticle ensembles comprises a homogenous distribution of nanoparticles, and the nanoparticle ensembles have a nanoparticle optical density ranging from 0.02 to 20.

10. The sensing device of claim 9, wherein the nanoparticles are capped.

11. The sensing device of claim 10, wherein the nanoparticles are capped with a cap selected from a group consisting of citrate, cetyltrimethylammonium bromide (CTAB), Polyethylene glycol (PEG), Polyvinylpyrrolidone (PVP), Polyvinyl alcohol (PVA), Bovine serum albumin (BSA), Ethylene diamine tetra acetic acid (EDTA), and a combination thereof.

12. The sensing device of claim 10, wherein the capped nanoparticles are further functionalized with a biological probe, wherein the biological probe is selected from the group consisting of a peptide-nucleic acid, an oligonucleotide, an aptamer, an antibody, an antibody fragment, a complementary DNA or RNA, a complementary oligonucleotide, a modified oligonucleotide, a locked nucleic acid, an enzyme, or a small molecule.

13. The sensing device of claim 12, wherein the capped nanoparticles in different nanoparticle ensembles are functionalized with different biological probes.

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