WO2021124116A1

WO2021124116A1 - Composition and method for the detection of analytes

Info

Publication number: WO2021124116A1
Application number: PCT/IB2020/061982
Authority: WO
Inventors: Deniz YUKSEL YURT; Stephen B. Roscoe; Myungchan Kang; Minghua Dai; Federica SGOLASTRA
Original assignee: 3M Innovative Properties Company
Priority date: 2019-12-18
Filing date: 2020-12-15
Publication date: 2021-06-24

Abstract

A method of detecting an analyte. The method includes providing a composition, the composition comprising nanoparticles coupled to a first aptamer and nanorods coupled to a second aptamer, wherein the composition is substantially free of aggregates of the nanoparticles and nanorods; contacting a sample with the composition; adding a reagent to change the morphologies of the nanoparticles or nanorods; detecting a first color change of nanoparticles responsive to a first analyte in the sample; and detecting a second color change of nanorods responsive to a second analyte in the sample.

Description

COMPOSITION AND METHOD FOR THE DETECTION OF ANALYTES

BACKGROUND

The assurance of food and feed safety, including identification and monitoring of multiple small molecule contaminants, is of utmost importance in the food industry. Within this context, mycotoxins are globally widespread secondary metabolites which can contaminate crops either in fields or while in storage. They are highly toxic and have serious human and animal health impacts such as carcinogenic, teratogenic, and hepatotoxic effects. Hence, their presence is highly regulated and their detection in parts per billion (ppb) amounts is required in a variety of foods and animal feed.

The mycotoxin analysis involves sampling (milling and sub-sampling), preparation (extraction and purification), and analysis. Analysis could be either based on chromatographic techniques (thin layer chromatography (TLC), gas chromatography coupled with mass spectroscopy (GC-MS), or high- performance liquid chromatography (HPLC) or immunochemical methods such as enzyme linked immunosorbent assay (ELISA) or lateral flow assay (LFA).

There is a need for a better method to detect small molecules for many applications including drug discovery, metabolomics, food analysis, environmental monitoring and clinical diagnosis.

SUMMARY

Thus, in one aspect, the present disclosure provides a method of detecting an analyte, comprising: providing a composition, the composition comprising nanoparticles coupled to a first aptamer and nanorods coupled to a second aptamer, wherein the composition is substantially free of aggregates of the nanoparticles and nanorods; contacting a sample with the composition; adding a reagent to change the morphologies of the nanoparticles or nanorods; detecting a first color change of nanoparticles responsive to a first analyte in the sample; and detecting a second color change of nanorods responsive to a second analyte in the sample.

In another aspect, the present disclosure provides a composition, the composition comprising; nanoparticles coupled to a first aptamer; and nanorods coupled to a second aptamer; wherein the composition is substantially free of aggregates of the nanoparticles and nanorods.

Various aspects and advantages of exemplary embodiments of the present disclosure have been summarized. The above Summary is not intended to describe each illustrated embodiment or every implementation of the present disclosure. Further features and advantages are disclosed in the embodiments that follow. The Drawings and the Detailed Description that follow more particularly exemplify certain embodiments using the principles disclosed herein. DEFINITIONS

For the following defined terms, these definitions shall be applied for the entire Specification, including the claims, unless a different definition is provided in the claims or elsewhere in the Specification based upon a specific reference to a modification of a term used in the following definitions:

The terms “about” or “approximately” with reference to a numerical value or a shape means +/- five percent of the numerical value or property or characteristic, but also expressly includes any narrow range within the +/- five percent of the numerical value or property or characteristic as well as the exact numerical value. For example, a temperature of “about” 100°C refers to a temperature from 95°C to 105°C, but also expressly includes any narrower range of temperature or even a single temperature within that range, including, for example, a temperature of exactly 100°C. For example, a viscosity of “about” 1 Pa-sec refers to a viscosity from 0.95 to 1.05 Pa-sec, but also expressly includes a viscosity of exactly 1 Pa-sec. Similarly, a perimeter that is “substantially square” is intended to describe a geometric shape having four lateral edges in which each lateral edge has a length which is from 95% to 105% of the length of any other lateral edge, but which also includes a geometric shape in which each lateral edge has exactly the same length.

The term “substantially” with reference to a property or characteristic means that the property or characteristic is exhibited to a greater extent than the opposite of that property or characteristic is exhibited. For example, a substrate that is “substantially” transparent refers to a substrate that transmits more radiation (e.g. visible light) than it foils to transmit (e.g. absorbs and reflects). Thus, a substrate that transmits more than 50% of the visible light incident upon its surface is substantially transparent, but a substrate that transmits 50% or less of the visible light incident upon its surface is not substantially transparent.

The term “analyte” is defined as one or more substance potentially present in the sample. The analysis determines the presence, quantity, or concentration of the anal vie present in the sample.

The term “polynucleotide” as referred to herein means single-stranded or double-stranded nucleic acid polymers of at least 10 bases in length. In certain embodiments, the nucleotides comprising the polynucleotide can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. Said modifications include base modifications such as bromouridine, ribose modifications such as aiabinoside and 2', 3 '-dideoxyribose and intemucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate and phosphoroamidate . The term “polynucleotide” specifically includes single and double stranded forms of DNA.

The term “oligonucleotide” referred to herein includes naturally occurring, and modified nucleotides linked together by naturally occurring, and/or non-naturally occurring oligonucleotide linkages. Oligonucleotides are a polynucleotide subset comprising members that are generally single- stranded and have a length of 200 bases or fewer. In certain embodiments, oligonucleotides are lO to 60 bases in length. In certain embodiments, oligonucleotides are 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in length. Oligonucleotides may be single stranded or double stranded, e.g. for use in the construction of a gene mutant. Oligonucleotides of the invention may be sense or antisense oligonucleotides with reference to a protein-coding sequence.

The term “aptamers” refers to nucleic acids (typically DNA, RNA or oligonucleotides) that emerge from in vitro selections or other types of aptamer selection procedures well known in the art (e.g. bead-based selection with flow cytometry or high density aptamer arrays) when the nucleic acid is added to mixtures of molecules. Ligands that bind aptamers include but are not limited to small molecules, peptides, proteins, carbohydrates, hormones, sugar, metabolic byproducts, cofactors, drugs and toxins. Aptamers of the invention are preferably specific for a particular analyte. Aptamers can have diagnostic, target validation and therapeutic applications. The specificity of the binding is defined in terms of the dissociation constant Kd of the aptamer for its ligand. Aptamers can have high affinity with Kd range similar to antibody (pM to nM) and specificity similar/superior to antibody (Tuerk and Gold,

1990, Science, 249:505; Ellington and Szostak, 1990, Nature 346:818). An aptamer will typically be between 10 and 300 nucleotides in length. RNAs and DNAs aptamers can be generated from in vitro selection experiments such as SELEX (Systematic Evolution of Ligands by Exponential Enrichment). Examples of aptamer uses and technology are PhotoSELEX™ and Riboreporters™. Aptamers, their uses, and manufacture are described, for example, in U.S. Pat. Nos. 5,840,867, 6,001,648, 6225,058, 6,207,388 and U.S. patent publication 20020001810, the disclosures of all of which are incorporated by reference in their entireties.

The term “analyte” refers to a substance to be detected or assayed by die method of the invention. Typical analytes may include, but are not limited to proteins, peptides, nucleic acid segments, molecules, cells, microorganisms and fragments and products thereof, or any substance for which attachment sites, binding members or receptors (such as antibodies) can be developed. The analytes have at least one binding site, e.g., epitope, that can be targeted by an aptamer.

A “sample” as used herein refers to any quantity of a substance that can be used in a method of the invention. For example, the sample can be a biological sample or can be extracted from a biological sample derived from humans, animals, plants, fungi, yeast, bacteria, viruses, tissue cultures or viral cultures, or a combination of the above. They may contain or be extracted from solid tissues (e.g. bone marrow, lymph nodes, brain, skin), body fluids (e.g. serum, blood, urine, sputum, seminal or lymph fluids), skeletal tissues, or individual cells. Alternatively, the sample can comprise purified or partially purified nucleic acid molecules and, for example, buffers and/or reagents that are used to generate appropriate conditions for successfully performing a method of the invention.

The terms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a material containing “a compound” includes a mixture of two or more compounds. BRIEF DESCRIPTION OF THE DRAWINGS

The disclosure may be more completely understood in consideration of the following detailed description of various embodiments of the disclosure in connection with the accompanying figures, in which:

Fig. 1A is absorbance spectra for samples containing OTA aptamer-Au NR and AFB₁ aptamer-Au NP, and mixtures of OTA and AFB₁.

Fig. IB is normalized absorbance spectra for samples containing OTA aptamer-Au NR and AFB₁ aptamer-Au NP, and mixtures of OTA and AFB₁.

Fig 2 showes normalized absorbance versus wavelength graphs of grown Au NPs in the presence of different concentrations of OTA and AFB₁.

While the above-identified drawings, which may not be drawn to scale, set forth various embodiments of the present disclosure, other embodiments are also contemplated, as noted in the Detailed Description. In all cases, this disclosure describes the presently disclosed invention by way of representation of exemplary embodiments and not by express limitations. It should be understood that numerous other modifications and embodiments can be devised by those skilled in the art, which fall within the scope and spirit of this disclosure.

DETAILED DESCRIPTION

Before any embodiments of the present disclosure are explained in detail, it is understood that the invention is not limited in its application to the details of use, construction, and the arrangement of components set forth in the following description. The invention is capable of other embodiments and of being practiced or of being carried out in various ways that will become apparent to a person of ordinary skill in the art upon reading the present disclosure. Also, it is understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. It is understood that other embodiments may be utilized and structural or logical changes may be made without departing from the scope of the present disclosure.

As used in this Specification, the recitation of numerical ranges by endpoints includes all numbers subsumed within that range (e.g. 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.8, 4, and 5, and the like).

Unless otherwise indicated, all numbers expressing quantities or ingredients, measurement of properties and so forth used in the Specification and embodiments are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the foregoing specification and attached listing of embodiments can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the teachings of the present disclosure. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claimed embodiments, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

The present application provides a composition and a method for the detection of the analytes based on tire use of nanoparticles and nanorods coupled to an aptamer. The sensitivity of detection could be improved using nanorods coupled to aptamer. Moreover, simultaneous use of nanoparticles and nanorods in solution has allowed detection of multiple targets in solution.

The composition of the present application can include nanoparticles coupled to a first aptamer and nanorods coupled to a second aptamer. The composition of the present specification is substantially fiee of aggregates of the nanoparticles and nanorods.

The term nanorods have been used herein to generally describe one morphology of nanoscale objects synthesized from metals with dimensions having aspect ratio greater than 1, it will be appreciated that particles of different suitable form having aspect ratio greater than 1 may also be used. Nanoparticles can include spherical, cylindrical nanoparticles or ellipsoid nanoparticles. The nanoparticles may have a major axis and a minor axis. The nanoparticles may have a curved surface. The form of the nanoparticles is clearly not limited to those of a particular lateral or longitudinal cross-section. It will be appreciated that nanoparticles of aspect ratio of 1 , or substantially 1, or generally spherical nanoparticles may also be used. The nanoparticles comprise localized or locally defined surface chemistries.

Suitable nanoparticles and nanorods may be made of a noble metals, such as gold, silver, copper, and platinum; semiconductors, such as CdSe, CdS, and CdS or CdSe coated with ZnS; and magnetic colloidal materials, such as those described in Josephson, Lee, et al.,Angewcmdte Chemie, international Edition (2001), 40(17), 3204-3206.

In some embodiments, the particles and nanorods are gold (Au) nanoparticles with an average size of 4 to 20 nm, and gold (Au) nanorods with an average width from 5 to 20 nanometers (nm) and length from 30 to 60 nrn. in some embodiments, gold nanoparticles and gold (Au) nanorods having an average diameter of from 4 to 20 nm are functionalized to the oligonucleotides, for example to aptamer.

Aptamers configured to bind to specific target analytes can be selected, for example, by synthesizing an initial heterogeneous population of oligonucleotides, and then selecting oligonucleotides within the population that bind tightly to a particular target analyte. Once an aptamer that binds to a particular target molecule has been identified, it can be replicated using a variety of techniques known in biological and other arts, for example, by cloning and polymerase chain reaction (PCR) amplification followed by transcription.

The synthesis of a heterogeneous population of oligonucleotides and the selection of aptamers within that population can be accomplished using a procedure known as the Systematic Evolution of Ligands by Exponential Enrichment or SELEX. The SELEX method is described in, for example, Gold et al., U.S. Pat. Nos. 5,270,163 and 5,567,588; Fitzwater et al., “A SELEX Primer,” Methods in Enzymology, 267:275-301 (1996); and in Ellington and Szostak, “In Vitro Selection of RNA Molecules that Bind Specific Ligands,” Nature, 346:818-22. For example, a heterogeneous DNA oligomer population can be synthesized to provide candidate oligomers for the in vitro selection of aptamers. The initial DNA oligomer population is a set of random sequences 15 to 100 nucleotides in length flanked by fixed 5' and 3' sequences 10 to 50 nucleotides in length. The fixed regions provide sites for PCR primer hybridization and, in one implementation, for initiation of transcription by an RNA polymerase to produce a population of RNA oligomers. The fixed regions also contain restriction sites for cloning selected aptamers. Many examples of fixed regions can be used in aptamer evolution. See, e.g., Conrad et al., “in Vitro Selection of Nucleic Acid Aptamers That Bind Proteins,” Methods in Enzymology, 267:336-83 (1996); Ciesiolka et al., “Affinity Selection-Amplification from Randomized Ribooligonucleotide Pools,” Methods in Enzymology, 267:315- 35 (1996); and Fitzwater et al., “A SELEX Primer,” Methods in Enzymology, 267:275-301 (1996). in some embodiments, a nanoparticle or nanorod can have a one, or a plurality' of aptamers, attached to il

Without being bound by theory, the method can include providing the composition of the present application and contacting a sample with the composition. Aptamer and target analyte interactions modulate the amount of aptamer strands adsorbed on the surface of aptamer-functionalized nanoparticles and nanorods. In the presence of target analyte, aptamer and target analyte interaction results in desorption of aptamer from the nanoparticles and nanorods surface. The first aptamer can desorb when the first aptamer coupled nanoparticles contact and bind with the first analyte. The second aptamer can desorb when the second aptamer coupled nanorods contact and bind with the second analyte. The higher the target analyte concentration is, the higher the amount of desorption occurs. In the absence of target analyte, aptamer can remain surface-bound on the nanoparticles and nanorods surface.

In some embodiments, the method can further include adding a reagent to change the morphologies of the nanoparticles or nanorods. The reagent can include hydroxylamine (NHaOH) and/or hydrogen tetrachloroaurate(in) (HAuCl₄). Depending on the resulting aptamer coverage after the desorption of the aptamer, nanoparticles and nanorods can grow into morphologically varied nanostructures mediated by the reagent, which give rise to different colored solutions of grown nanoparticles and nanorods. In some embodiments, the change of the morphologies of the nanoparticles or nanorods can be that the size of the nanoparticles or nanorods grows. In some embodiments, the size of the nanoparticles or nanorods grows from 5 nm to 40 nm when the first aptamer coupled nanoparticles or second aptamer coupled nanorods contact the first or second analyte.

In some embodiments, nanoparticles with low aptamer coverage can grow into spherical nanoparticles after the first aptamer coupled nanoparticles contact the first analyte, which produce red- colored solutions, whereas nanoparticles with high aptamer coverage grow into branched nanoparticles, which produce blue-colored solutions. In some embodiments, nanorods with low aptamer coverage can grow in size after the second aptamer coupled nanorods contact the second analyte, which produce purple- colored solutions.

In some embodiments, the method can further include detecting a first color change of nanoparticles responsive to a first analyte in the sample and detecting a second color change of nanorods responsive to a second analyte in the sample. The degree the color changes in response to the analyte may be quantified by any suitable colorimetric quantification methods known to those of ordinary skill in the art, for example, UV-Vis spectra of the sample measured using a BioTek Synergy Neo2 plate reader (Winooski, VT). If higher precision is desired, various types of spectrophotometers may be used to plot a Beefs curve in the desired concentration range. The color of the sample may then be compared with the curve and the concentration of the analyte present in the sample determined. Suitable spectrophotometers include the Hewlett-Packard 8453 and the Bausch & Lomb Spec-20.

In some embodiments, the first or second analyte can be selected from the group consisting of large biomolecules, small biomolecules, organic molecules and inorganics. In one aspect, the analyte may be any biomolecule, for example large biomolecules, such as proteins (e.g. hormone, insulin), antibodies, growth factors, enzymes, virus, viral derived components, bacteria (e.g. anthrax), bacteria derived molecules and components (e.g. anthrax derived molecules), or cells. Biomolecules also may include small biomolecules, such as mycotoxins, amino acids (e.g. arginine), nucleotides (e.g. ATP, GTP), neurotransmitters (e.g. dopamine), cofactors (e.g. biotin), peptides, or amino-glycosides.

In another aspect, the analyte may be any organic molecule that causes the aptamer to fold. Preferable organic molecules include drugs, such as antibiotics and theophylline, or controlled substances, such as cocaine, dyes, oligosaccharides, polysaccharides, glucose, nitrogen fertilizers, pesticides, herbicides, hormones, feed additives such as ractopamine, pollutants such as dioxins, phenols, 2,4- dichlorophenoxyacetic acid, nerve gases, trinitrotoluene (TNT), or dinitrotoluene (DNT). In another aspect, the analyte may be any inorganics, for example, heavy metals. In some embodiments, the first analyte is aflatoxin B 1 and the second analyte is ochratoxin A.

The sensitivity of detection can be improved using nanorods compared to using nanoparticles. Moreover, simultaneous use of nanorods and nanoparticles allow detection of multiple targets in the sample .

The following working examples are intended to be illustrative of the present disclosure and not limiting.

EXAMPLES

The following abbreviations are used in this section: nm = nanometer, mM = millimolar, nM = nanomolar.

Materials

Phosphate buffered saline (PBS) pH 7.4, ochratoxin A (OTA), and aflatoxin Bi (AFB₁) were obtained from the Sigma-Aldrich Corporation, St. Louis, MO. Magnesium chloride (MgCl₂) was obtained from JT Baker, Phillipsbuig, NJ.

Hydrogen tetrachloroaurate (ΙΠ) (HAuCl₄) and hydroxylamine were obtained from Alfa Aesar, Haverhill,

MA Methanol was obtained from EMD Millipore, Billerica, MA.

Deionized (DI) water was purified using a MILLI-Q water purification system (EMD Millipore).

Gold nanoparticles (5 nm diameter, 71.8 nM in 2 mM aqueous citrate, product #AUCN5) and gold nanorods (45 nm by 17 nm size, 0.47 nM in MILLI-Q water, peak wavelength 660 nm, product #GRCN660) were obtained under the trade designation NanoXact from NanoComposix, San Diego, CA. Ochratoxin A aptamer (OTA-aptamer) [sequence: 5’-CGG GTG TGG GTG CCT TGA TCC AGG GAG TCT CTA ATC-3’]; and aflatoxin Biaptamer (AFB₁ -aptamer) [sequence: 5’-GTT GGG CAC GTG TTG TCT CTC TGT GTC TCG TGC CCT TCG CTA GGC CCA CA-3’] were obtained from Integrated DNA Technologies Incorporated, Coralville, IA.

Ultraviolet (UV)-visible spectra and absorbance values were recorded on a Synergy Neo2 (BioTek Instruments, Winooski, VT) plate reader or a SpectraMax M5 plate reader (Molecular Devices, LLC, San Jose, CA). Plate images were taken using a CanoScan 9000F scanner (Canon USA, Melville, NY).

Tissue culture treated 96-well microplates were obtained from the Coming Life Sciences, Tewksbury, MA.

Preparatory Example 1. Preparation and Optimization of OTA-Aptamer Adsorbed on Gold Nanoparticles (Au-NPs)

Wells in a 96-well microplate were individually filled by micropipette with 0.7 microliters of 5 nm diameter Au NPs (71.8 nM in 2 mM aqueous citrate), 20 microliters of MgCl₂ solution (1 mM solution in deionized water), 9.3 microliters of deionized water, and 150 microliters of an OTA-aptamer solution. The OTA-aptamer solution was prepared so that the final concentration of OTA-aptamer in a well was either 0 (blank), 15, 30, 60, 90, 120, or 240 nM. The microplate was incubated at room temperature overnight and then 20 microliters of PBS was added to each well. The microplate was allowed to incubate for 30 minutes at room temperature. Next, 5 microliters of hydroxylamine solution (130 mM in deionized water) and 6.55 microliters of HAuCl₄ solution (2.9 mM in deionized water) were added to each well and the contents were thoroughly mixed with a pipette. An additional 3 aliquots of HAuCl₄ solution (each aliquot 6.55 microliters of the 2.9 mM solution in deionized water) were sequentially added to each well with incubation periods of 5 minutes spaced between each addition. The colors of the sample wells changed from a bright red color to hues of purple with increasing amounts of aptamer. The microplate was imaged and the ultraviolet (UV)-visible spectra from each well were recorded. A graph of peak wavelength versus aptamer concentration was plotted and the optimum OTA- aptamer concentration was determined to be the concentration where the slope of this graph started to plateau. The corresponding peak shifts are reported in Table 1 (four replicates for each OTA-Aptamer concentration). Based on this data, a concentration of 90 nM was chosen for experiments performed with Au NPs and OTA-aptamer. Table 1. Peak Shifts for Grown Gold Nanoparticles (Au NPs) based on OTA-Aptamer Concentration

Preparatory Example 2. Preparation and Optimization of AFB₁-Aptamer Adsorbed on Gold Nanoparticles (Au NPs)

Wells in a 96-well microplate were individually filled by micropipette with 0.7 microliters of 5 nm diameter Au NPs (71.8 nM in 2 mM aqueous citrate), 20 microliters of MgCl₂ solution (1 mM solution in deionized water), 9.3 microliters of deionized water, and 150 microliters of an AFB₁-aptamer solution. The AFB₁-aptamer solution was prepared so that the final concentration of AFB₁-aptamer in a well was either 0 (blank), 15, 30, 60, 90, 120, or 240 nM. The microplate was incubated at room temperature overnight and then 20 microliters of PBS was added to each well. The microplate was allowed to incubate for 30 minutes at room temperature. Next, 5 microliters of hydroxylamine solution (130 mM in deionized water) and 6.55 microliters of HAuCl₄ solution (2.9 mM in deionized water) were added to each well and the contents were thoroughly mixed with a pipette. An additional 3 aliquots of HAuCl₄ solution (each aliquot 6.55 microliters of the 2.9 mM solution in deionized water) were sequentially added to each well with incubation periods of 5 minutes spaced between each addition. The colors of the sample wells changed from a bright red color to hues of purple with increasing amounts of aptamer. The microplate was imaged and the ultraviolet (UV)-visible spectra from each well were recorded. A graph of peak wavelength versus aptamer concentration was plotted and the optimum OTA- aptamer concentration was determined to be the concentration where the slope of this graph started to plateau. Based on this data, a concentration of 60 nM was chosen for experiments performed with Au NPs and AFB₁-aptamer.

Preparatory Example 3. Preparation and Optimization of OTA-Aptamer Adsorbed on Gold Nanorods (Au NRs)

Wells in a 96-well microplate were individually filled by micropipette with 85 microliters of Au NRs (0.47 nM in Milli-Q water), 20 microliters of MgCl₂ solution (1 mM solution in deionized water), 15 microliters of deionized water, and 60 microliters of an OTA-aptamer solution. The OTA-aptamer solution was prepared so that the final concentration of OTA-aptamer in a well was either 0 (blank), 5, 10, 15, 20, 30, or 60 nM. The microplate was incubated at room temperature overnight and then 20 microliters of PBS was added to each well. The microplate was allowed to incubate for 30 minutes at room temperature. Next, 5 microliters of hydroxylamine solution (130 mM in deionized water) and 6.55 microliters of HAuCl₄ solution (2.9 mM in deionized water) were added to each well and the contents were thoroughly mixed with a pipette. An additional 3 aliquots of HAuCl₄ solution (each aliquot 6.55 microliters of the 2.9 mM solution in deionized water) were sequentially added to each well with incubation periods of 5 minutes spaced between each addition. The microplate was imaged and the ultraviolet (UV)-visible spectra from each well were recorded. A graph of peak wavelength versus aptamer concentration was plotted and the optimum OTA-aptamer concentration was determined to be the concentration where the slope of this graph started to plateau. Based on this data, an OTA-aptamer concentration of 20 nM was chosen for experiments performed with Au NRs and OTA-aptamer.

Preparatory Example 4. Detection of OTA using Gold Nanoparticles (Au NPs) and OTA-Aptamer

OTA was solubilized in methanol at 1 mg/mL (10⁶ ppb) and serially diluted in PBS buffer, pH 7.4.

Wells in a 96-well microplate were individually filled by micropipette with 9 microliters of OTA- aptamer solution (2 micromolar in deionized water), 0.7 microliters of 5 nm diameter Au NPs (71.8 nM in 2 mM aqueous citrate), 20 microliters of MgCl₂ solution (1 mM solution in deionized water), and 150.3 microliters of deionized water. The microplate was incubated at room temperature overnight. Individual analyte samples were prepared containing OTA in varying concentrations. The concentrations of OTA in the analyte samples were either 0, 10, 40, 400, or 4000 ppb. Each analyte sample (20 microliters) was added to a separate well. The microplate was incubated for 30 minutes at room temperature. Next, 5 microliters of hydroxylamine solution (130 mM in deionized water) and 6.55 microliters of HAuCl₄ solution (2.9 mM in deionized water) were sequentially added to each well and the contents were mixed using a pipette. An additional 3 aliquots of HAuCl₄ solution (each aliquot 6.55 microliters of the 2.9 mM solution in deionized water) were sequentially added to each well with incubation periods of 5 minutes spaced between each addition. Samples were mixed thoroughly by pipetting. The plate was then imaged and the ultraviolet (UV)-visible spectra from each well were recorded. Grown Au NPs varied from a deep violet color at low concentrations of OTA to a red color with increasing concentrations of OTA. A graph of peak shift versus OTA concentration was plotted and showed that OTA could be detected at 40 ppb. The results for observed Peak Shift versus OTA concentration are reported in Table 2 (3 replicates). Table 2.

Preparatory Example 5. Detection of AFB₁ using Gold Nanoparticles (Au NPs) and AFB₁-Aptamer AFB₁ was solubilized in methanol at 1 mg/mL (10⁶ ppb) and serially diluted further in PBS buffer, pH 7.4.

Wells in a 96-well microplate were individually filled by micropipette with 6 microliters of AFB₁-aptamer solution (2 micromolar in deionized water), 0.7 microliters of 5 nm diameter Au NPs (71.8 nM in 2 mM aqueous citrate), 20 microliters of MgCl₂ solution ( 1 mM solution in deionized water), and 153.3 microliters of deionized water. The microplate was incubated at room temperature overnight. Individual analyte samples were prepared containing AFB₁ in varying concentrations. The concentrations of AFB₁ in the analyte samples were either 0, 5, 50, 500, or 5000 ppb. Each analyte sample (20 microliters) was added to a separate well. The microplate was incubated for 30 minutes at room temperature. Next, 5 microliters of hydroxylamine solution (130 mM in deionized water) and 6.55 microliters of HAuCl₄ solution (2.9 mM in deionized water) were sequentially added to each well and the contents were mixed using a pipette . An additional 3 aliquots of HAuCl₄ solution (each aliquot 6.55 microliters of the 2.9 mM solution in deionized water) were sequentially added to each well with incubation periods of 5 minutes spaced between each addition. Samples were mixed thoroughly by pipetting. The plate was then imaged and the ultraviolet (UV)-visible spectra from each well were recorded. Grown Au NPs varied from a deep violet color to a red color with increasing concentrations of AFB₁. A graph of peak shift versus AFB₁ concentration was plotted and showed that AFB₁ could be detected at 5 ppb. The results for observed peak shift versus AFB₁ concentration are reported in Table 3 (1 replicate).

Table 3.

Preparatory Example 6. Detection of OTA using Gold Nanorods (Au NRs) and OTA-Aptamer

OTA was solubilized in methanol at 1 mg/mL (10⁶ ppb) and serially diluted further in PBS buffer, pH 7.4.

Wells in a 96-well microplate were individually filled by micropipette with 20 microliters of OTA-aptamer solution (0.2 micromolar in deionized water), 85 microliters of Au NRs (0.47 nM in MILLI-Q water), 20 microliters of MgCl₂ solution (1 mM solution in deionized water), and 55 microliters of deionized water. The microplate was incubated at room temperature overnight. Individual analyte samples were prepared containing OTA in varying concentrations. The concentrations of OTA in the analyte samples were either 0, 10, 40, 400, or 4000 ppb. Each analyte sample (20 microliters) was added to a separate well. The microplate was incubated for 30 minutes at room temperature. Next, 5 microliters of hydroxylamine solution (130 mM in deionized water) and 6.55 microliters of HAuCl₄ solution (2.9 mM in deionized water) were sequentially added to each well and the contents were mixed using a pipette. An additional 3 aliquots of HAuCl₄ solution (each aliquot 6.55 microliters of the 2.9 mM solution in deionized water) were sequentially added to each well with incubation periods of 5 minutes spaced between each addition. Samples were mixed thoroughly by pipetting. The plate was then imaged and the ultraviolet (UV)-visible spectra from each well were recorded. The spectra of grown Au NRs in the presence of different concentrations of OTA showed a blue shift as OTA concentration increased. A graph of peak shift versus OTA concentration was plotted and showed that OTA could be detected at 4 ppb. Due to larger shifts observed in the spectra upon growth of Au NRs, the sensitivity of OTA detection improved 10-fold compared to OTA detection with Au NPs (see Preparatory Example 4). The results for observed Peak Shift versus OTA concentration are reported in Table 4 (2 replicates).

Table 4.

Example 1. Successful Multi-analyte Detection of OTA and AFB₁ using Gold Nanoparticles (Au NPs) and Gold Nanorods (Au NRs)

OTA and AFB₁ were separately solubilized in methanol at 1 mg/mL (10⁶ ppb) and serially diluted further using PBS buffer, pH 7.4.

A stock solution of AFB₁-aptamer adsorbed on Au NPs (AFB₁-aptamer-Au NP) was prepared by mixing AFB₁-aptamer (2 micromolar in deionized water), with Au NPs (71.8 nM in 2 mM aqueous citrate), and MgCl₂ solution (1 mM in deionized water) in a 6:0.7: 10 ratio by volumes. A stock solution of OTA-aptamer adsorbed on Au NRs (OTA-aptamer-Au NR) was prepared by mixing OTA-aptamer (0.2 micromolar in deionized water), with Au NRs (0.47 nM in MILLI-Q water) and MgCl₂ solution (1 mM in deionized water) in a 20:85: 10 ratio by volumes. Both stock solutions were incubated at room temperature overnight. OTA-aptamer-Au NR stock solution (115 microliters), AFB₁-aptamer-Au NP stock solution (16.7 microliters), and deionized water (48.3 microliters) were added to each well in a 96- well microplate and thoroughly mixed with a pipette. Individual analyte samples were prepared containing mixtures of OTA and AFB₁ in varying concentrations. The concentrations of OTA in the analyte samples were either 4, 40, 400, or 4000 ppb. The concentrations of AFB₁ in the analyte samples were either 5, 50, 500, or 5000 ppb. Each analyte sample (20 microliters) was added to a separate well. The microplate was incubated for 30 minutes at room temperature. Next, 2.5 microliters of hydroxylamine solution (130 mM in deionized water) and 6.55 microliters of HAuCl₄ solution (2.9 mM in deionized water) were sequentially added to each well. An additional 3 aliquots of HAuCl₄ solution (each aliquot 6.55 microliters of the 2.9 mM solution in deionized water) were sequentially added to each well with incubation periods of 5 minutes spaced between each addition. Samples were mixed thoroughly by pipetting. The plate was then imaged and the ultraviolet (UV)-visible spectra from each well were recorded and analyzed. The presence of OTA in a sample was determined based on a shoulder peak (only present in Au NR spectrum) which shifted between 650-750 nm and the presence of a second peak between 950-990 nm (Fig. IB). The limit of detection of OTA was 40 ppb in this experiment. The presence of AFB₁ in a sample was determined from the decrease in the absorbance of the main absorbance peak between 550-650 nm (Fig. 1A). The limit of detection of AFB₁ was 50 ppb in this experiment.

Comparative Example A. Failed Multi-analyte Detection of OTA and AFB₁ using only Gold Nanoparticles (Au NPs)

OTA and AFB₁ were separately solubilized in methanol at 1 mg/mL (10⁶ ppb) and serially diluted further in PBS buffer, pH 7.4.

A stock solution of AFB₁-aptamer adsorbed on Au NPs (AFB₁-aptamer-Au NP) was prepared by mixing AFB₁-aptamer (2 micromolar in deionized water), with Au NPs (71.8 nM in 2 mM aqueous citrate), and MgCl₂ solution (1 mM in deionized water) in a 6:0.7: 10 ratio by volumes. A stock solution of OTA-aptamer adsorbed on Au NPs (OTA-aptamer-Au NP) was prepared by mixing OTA-aptamer (0.2 micromolar in deionized water) Au NPs (71.8 nM in 2 mM aqueous citrate) and MgCl₂ solution (1 mM in deionized water) in a 9:0.7: 10 ratio by volumes. Both stock solutions were incubated at room temperature overnight.

OTA-aptamer-Au NP stock solution (19.7 microliters), AFB₁-aptamer-Au NP stock solution (16.7 microliters), and deionized water (143.6 microliters) were added to each well in a 96-well microplate and thoroughly mixed with a pipette. Individual analyte samples were prepared containing mixtures of OTA and AFB₁ in varying concentrations. The concentrations of OTA in the analyte samples were either 4, 40, 400, or 4000 ppb. The concentrations of AFB₁ in the analyte samples were either 5, 50, 500, or 5000 ppb. Each analyte sample (20 microliters) was added to a separate well. The microplate was incubated for 30 minutes at room temperature. Next, 2.5 microliters of hydroxy lamine solution (130 mM in deionized water) and 6.55 microliters of HAuCl₄ solution (2.9 mM in deionized water) were sequentially added to each well. An additional 3 aliquots of HAuCl₄ solution (each aliquot 6.55 microliters of the 2.9 mM solution in deionized water) were sequentially added to each well with incubation periods of 5 minutes spaced between each addition. Samples were mixed thoroughly by pipetting. The plate was then imaged and the ultraviolet (UV)-visible spectra from each well were recorded and analyzed. Normalized absorbance versus wavelength graphs of grown Au NPs in the presence of different concentrations of OTA and AFB₁ demonstrated a clear blue shift in the presence of all OTA and AFB₁ concentrations tested (Fig. 2). However, the assay could not distinguish whether OTA and/or AFB₁ was present in the samples tested as the observed peak shifts could be due to either analyte. All references and publications cited herein are expressly incorporated herein by reference in their entirety into this disclosure. Illustrative embodiments of this invention are discussed and reference has been made to possible variations within the scope of this invention. For example, features depicted in connection with one illustrative embodiment may be used in connection with other embodiments of the invention. These and other variations and modifications in the invention will be apparent to those skilled in the art without departing from the scope of the invention, and it should be understood that this invention is not limited to the illustrative embodiments set forth herein. Accordingly, the invention is to be limited only by the claims provided below and equivalents thereof.

Claims

What is claimed is:

1. A method of detecting an analyte, comprising: providing a composition, the composition comprising nanoparticles coupled to a first aptamer and nanorods coupled to a second aptamer, wherein the composition is substantially free of aggregates of the nanoparticles and nanorods; contacting a sample with the composition; adding a reagent to change the morphologies of the nanoparticles or nanorods; detecting a first color change of nanoparticles responsive to a first analyte in the sample; and detecting a second color change of nanorods responsive to a second analyte in the sample.

2. The method of claim 1, wherein the nanoparticles and nanorods are made of a noble metal.

3. The method of any of claims 1 to 2, wherein the nanoparticles and nanorods are made of gold or silver.

4. The method of any of claims 1 to 3, wherein the nanoparticles and nanorods are made of gold.

5. The method of any of claims 1 to 4, wherein the first aptamer desorbs when the first aptamer coupled nanoparticles contact the first analyte.

6. The method of any of claims 1 to 5, wherein the second aptamer desorbs when the second aptamer coupled nanorods contact the second analyte.

7. The method of any of claims 1 to 6, wherein the reagent comprises hydroxylamine (NH2OH) and/or hydrogen tetrachloroaurate(III) (HAuCl₄).

8. The method of any of claims 1 to 7, wherein the change of the morphologies of the nanoparticles or nanorods comprises that size of the nanoparticles or nanorods grows.

9. The method of claim 8, wherein the diameter of the nanoparticles or nanorods grows from 5 nm to 40 nm when the first aptamer coupled nanoparticles or second aptamer coupled nanorods contact the first or second analyte.

10. The method of any of claims 1 to 9, wherein detecting an analyte comprises detecting the change in size of the nanoparticles or nanorods.

11. The method of any of claims 1 to 10, wherein detecting an analyte comprises detecting the change in shape of the nanoparticles or nanorods

12. The method of any of claims 1 to 11, wherein the nanoparticles grow into spherical nanoparticles when the first aptamer coupled nanoparticles contact the first analyte.

13. The method of any of claims 1 to 12, wherein the first or second analyte is selected from the group consisting of large biomolecules, small biomolecules, organic molecules and inorganics.

14. The method of any of claims 1 to 13, wherein the first analyte is aflatoxin B 1.

15. The method of any of claims 1 to 14, wherein the second analyte is ochratoxin A.

16. A composition, the composition comprising; nanoparticles coupled to a first aptamer; and nanorods coupled to a second aptamer; wherein the composition is substantially free of aggregates of the nanoparticles and nanorods.