Visual detection of mercury ions

Composite materials comprising a mesoflower structure, methods of preparing the composite material, and methods of detecting heavy metal ion using the composite material are described herein. In some embodiments, a silica-coated gold mesoflower with a layer of silver quantum clusters may be capable of detecting Hg2+ ions in a sample at zeptomolar concentrations.

CLAIM OF PRIORITY

This application is a U.S. national stage filing under 35 U.S.C. §371 of International Application No. PCT/IB2012/001543, filed Aug. 10, 2012, which claims priority to Indian Application No. 1273/CHE/2012, filed Mar. 30, 2012, and entitled “Visual Detection of Mercury Ions,” respectively, the disclosure of which is incorporated by reference in its entirety.

BACKGROUND

Heavy metal ions have numerous adverse health effects due to their eco-toxic, carcinogenic and non-biodegradable nature. Detection of heavy metals using nanoparticle based sensors are typically based on monitoring changes in the properties of the nanoparticles, such as changes in surface plasmon resonance (SPR) absorptions, fluorescence, inter-nanoparticle interactions (ion-chelation-induced aggregation process), and others, upon binding of an analyte. These changes may be detected using absorption and fluorescence spectroscopy; however, they often require relatively larger sensor and analyte concentrations and may have anomalies due to ensemble averaging. Integration of chemosensors into a single particle sensory device would be an ideal prospect, as it increases the sensitivity of detection. Single particle sensors employed so far have disadvantages owing to difficulty in locating and distinguishing such particles with other impurities in the system as they may not have a unique shape. The limits of such detection systems have not extended beyond the femtomolar regime.

SUMMARY

This disclosure is not limited to the particular systems, devices and methods described, as these may vary. The terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope. While various compositions and methods are described in terms of “comprising” various components or steps (interpreted as meaning “including, but not limited to”), the compositions and methods can also “consist essentially of” or “consist of” the various components and steps, and such terminology should be interpreted as defining essentially closed-member groups.

Presently disclosed are metallic composite materials, methods of making the composite materials, and methods of detecting mercury ions at a very low concentration using the composite materials in a single particle-based detection approach. The methods described herein may be used for ultra-trace sensitivity detection of mercury ions.

Embodiments describe metallic composite materials comprising a mesoflower structure having a metal core and a plurality of luminescent nanoparticles affixed to the mesoflower structure, wherein the composite material has an observable luminescent emission when heavy metal ions are not bound to the composite material and the emission changes when heavy metal ions are bound to the composite material.

Embodiments describe methods of preparing a metallic composite material comprising: providing a mesoflower structure having a metal core; contacting the mesoflower structure with a mixture comprising a silane to produce a mixture comprising a silicon dioxide coated mesoflower structure; isolating the silicon dioxide coated mesoflower structure; providing a plurality of luminescent nanoparticles; and contacting the silicon dioxide coated mesoflower structure with the plurality of luminescent nanoparticles to produce a mixture comprising the composite material.

Embodiments describe methods of detecting heavy metal ions in a sample comprising: providing a metallic composite material; contacting the composite material with a sample containing, or suspected of containing, heavy metal ions; exposing the composite material to light comprising at least one wavelength capable of exciting the composite structure; and detecting the observable luminescent emission of the composite material, wherein a change in the observable luminescent emission of the composite material following contact with the sample indicates that heavy metal ions are bound to the composite material and that heavy metal ions are present in the sample.

Embodiments describe kits useful for detection of heavy metal ions, wherein the kit comprises a metallic composite material comprising a mesoflower structure having a metal core and a plurality of luminescent nanoparticles affixed to the mesoflower structure and at least one secondary reagent or reaction container.

DETAILED DESCRIPTION

Described herein are metallic composite materials, methods of making the composite materials, and methods of detecting mercury ions at a very low concentration using the composite materials in a single particle-based detection approach. In some embodiments, the composite material may be a composite material made of gold mesoflowers and silver quantum clusters.

In embodiments, a metallic composite material made by functionalizing gold mesoflowers with silver quantum clusters may be used in sensing applications in new and more sensitive formats. Detection of mercury ions may be achieved at a very low concentration (zeptomoles) using these methods. The methods described herein may provide a highly selective and sensitive way to recognize the presence of mercury ions in a mixture, well beyond the detection limit compared to conventional solution based approaches, by monitoring changes in the fluorescence of the composite material. In further embodiments, a turn-on sensor may be prepared by providing an additional functionality in the form of a fluorophore to the composite material. In embodiments, only a single particle may be required for the detection of an analyte.

In the following embodiments, a mesoflower may be unsymmetrical at the single particle level resembling natural objects, such as flowers, starfish, or sea urchins, and may comprise of a large number of stems with unusual pentagonal symmetry (the FIGURE). Mesoflowers may have a high degree of structural purity with star-shaped, nano-structured stems. In embodiments, mesoflowers may be obtained in high yield, essentially free of contaminating structures and its size may be tuned from nano- to meso-dimensions. In the following embodiments, a mesoflower structure may have a size of about 500 nm to about 10 μm. In some embodiments, a mesoflower structure may have a size of about 500 nm, about 1 μm, about 1.5 μm, about 2 μm, about 2.5 μm, about 3 μm, about 4 μm, about 5 μm, about 6 μm, about 8 μm, or about 10 μm. Specific examples of sizes include about 500 nm, about 1 μm, about 2 μm, about 3 μm, about 4 μm, about 4.5 μm, about 6 μm, about 10 μm, and ranges between any two of these sizes (for example, about 4 μm to about 10 μm).

In the following embodiments, a mesoflower structure may have a metal core. The metal core may be mono-metallic, bi-metallic, or tri-metallic. In some embodiments, the metal core may comprise gold. In these embodiments, the metal core may comprise a layer of silver, nickel, cobalt, platinum, or a combination thereof. In further embodiments, the metal core may comprise an additional layer of nickel, cobalt, or a combination thereof. For example, a bi-metallic metal core may be gold coated with silver, and a tri-metallic metal core may be gold coated with platinum coated with nickel.

In the following embodiments, luminescent nanoparticles may comprise quantum clusters. In some embodiments, the quantum clusters may comprise Au25, Au18, or Ag15clusters confined in bovine serum albumin or glutathione. In some embodiments, the quantum clusters may comprise Ag15clusters confined in bovine serum albumin. In these embodiments, a composite material comprising the luminescent nanoparticles may exhibit red luminescent emission when illuminated with light of a wavelength of about 490 nanometers and heavy metal ions are not bound to the composite material.

In an embodiment, a method of preparing a metallic composite material may comprise: providing a mesoflower structure having a metal core; contacting the mesoflower structure with a solution comprising tetraethyl orthosilicate or (3-aminopropyl)trimethoxysilane to produce a mixture comprising a silicon dioxide coated mesoflower structure; isolating the silicon dioxide coated mesoflower structure; providing a plurality of luminescent nanoparticles; and contacting the silicon dioxide coated mesoflower structure with the plurality of luminescent nanoparticles to produce a mixture comprising the composite material. In some embodiments, the method may further comprise centrifuging the mixture comprising the composite material to produce a residue comprising the composite material and adding water to the residue comprising the composite material to produce a mixture comprising purified composite material. In these embodiments, the method further may comprise repeating the centrifuging and adding water steps one to five times to produce a mixture consisting essentially of the composite material.

In some embodiments, isolating the silicon dioxide coated mesoflower structure may comprise centrifuging the mixture comprising the silicon dioxide coated mesoflower structure and decanting to produce a residue comprising the silicon dioxide coated mesoflower structure. In these embodiments, isolating the silicon dioxide coated mesoflower structure may further comprise adding water to the residue comprising the silicon dioxide coated mesoflower structure and repeating the centrifuging and decanting steps one to five times to produce a residue comprising a substantially pure silicon dioxide coated mesoflower structure.

In some embodiments, the solution comprising tetraethyl orthosilicate or (3-aminopropyl)trimethoxysilane further may comprise a fluorescent dye. In some embodiments, the fluorescent dye may be fluorescein isothiocyanate.

In an embodiment, a metallic composite material useful for the detection of heavy metal ions may comprise a mesoflower structure having a metal core and a plurality of luminescent nanoparticles affixed to the mesoflower structure. In embodiments, the composite material may have an observable luminescent emission when heavy metal atoms are not bound to the composite material, and the observable luminescent emission may change when heavy metal atoms are bound to the composite material. In embodiments, the change in the observable luminescent emission may be a shift to a different wavelength, a diminished intensity, an intensified intensity, or a combination thereof.

In some embodiments, the plurality of luminescent nanoparticles may be separated from the metal core by at least one inert layer. In these embodiments, the at least one inert layer may comprise a metal oxide. In some embodiments, the at least one inert layer may comprise aluminum oxide, titanium dioxide, zirconium oxide, silicon dioxide, or a combination thereof. In some embodiments, the at least one inert layer may comprise silicon dioxide.

In embodiments, the heavy metal ions may be selected from the group consisting of mercury, lead, nickel, arsenic, aluminum, cobalt, chromium, manganese, copper, cadmium, and combinations thereof. In some embodiments, the heavy metal ions may be mercury, copper, or a combination thereof. In some embodiments, the heavy metal ions may be mercury ions and they may be selectively bound by the composite material relative to other heavy metal ions. In some embodiments, the observable luminescent emission may have a diminished intensity when heavy metal atoms are bound to the composite material.

In further embodiments, the at least one inert layer may comprise a fluorescent dye. In some embodiments, the fluorescent dye may be fluorescein isothiocyanate. In these embodiments, the observable luminescent emission may have a shifted wavelength when heavy metal atoms are bound to the composite material.

As used herein, “sensitivity” is intended to define the ability to perceive a change in the observable luminescent emission due to heavy metal atoms binding to the composite material when the composite material is contacted with a sample containing an amount of heavy metal ions. In some embodiments, the change in the observable luminescent emission may have sensitivity to heavy metal ions at a concentration of about 0.1 zeptomolar to about 100 femtomolar. Specific examples of concentrations are about 0.1 zeptomolar, about 1 zeptomolar, about 10 zeptomolar, about 100 zeptomolar, about 1 attomolar, about 10 attomolar, about 100 attomolar, about 1 femtomolar, about 10 femtomolar, about 100 femtomolar, and any concentration or range of concentrations between those listed (for example, about 0.1 zeptomolar to about 10 attomolar). In some embodiments, the change in the observable luminescent emission may have sensitivity to heavy metal ions at a concentration of about 0.1 zeptomolar. In some embodiments, heavy metal ions may be within a test sample. In these embodiments, the sample may be a water sample or a biological sample (e.g., blood, urine, mucus, or tissue).

In an embodiment, a method of detecting heavy metal ions in a sample comprises: providing a metallic composite material described in the embodiments above; contacting the composite material with a sample containing, or suspected of containing, heavy metal ions; exposing the composite material to light comprising at least one wavelength capable of exciting the composite structure; and detecting the observable luminescent emission of the composite material. In embodiments, a change in the observable luminescent emission of the composite material following contact with the sample indicates that heavy metal ions are bound to the composite material and that heavy metal ions are present in the sample.

In some embodiments, providing a composite material may comprise making a mixture comprising the composite material. In these embodiments, contacting the composite material with a sample containing, or suspected of containing, heavy metal ions may comprise adding the sample containing, or suspected of containing, heavy metal ions to the mixture comprising the composite material. In other embodiments, providing a composite material may comprise making a drop-cast film of the composite material. In these embodiments, contacting the composite material with a sample containing, or suspected of containing, heavy metal ions may comprise adding the sample containing, or suspected of containing, heavy metal ions to the drop-cast film of the composite material.

In embodiments, the sample containing, or suspected of containing, heavy metal ions may be a water sample or a biological sample. In some embodiments, the heavy metal ions may be selected from the group consisting of chromium, zinc, silver, selenium, thorium, uranium, manganese, mercury, lead, nickel, arsenic, aluminum, cobalt, magnesium, copper, cadmium, and combinations thereof. In some embodiments, the heavy metal ions may be mercury, copper, or a combination thereof. Specific examples of heavy metal ions include, but are not limited to: Cr6+, Zn2+, Ag+, Se2+, Th4+, U6+, Mn2+, Co2+, Hg2+, Pb2+, Ni2+, Cd2+, Mg2+, and Cu2+. In some embodiments, the heavy metal ions may be mercury ions and the mercury ions may be selectively detected from other heavy metal ions.

In some embodiments, the change in the observable luminescent emission of the composite material following contact with the sample may have a magnitude proportional to the amount of heavy metal in the sample. In embodiments, the sample containing, or suspected of containing, heavy metal ions contains heavy metal ions at a femtomolar, attomolar, or zeptomolar concentration, and the change in the observable luminescent emission of the composite material following contact with the sample may be observable. In embodiments, the sample containing, or suspected of containing, heavy metal ions contains heavy metal ions at a concentration of at least about 0.1 zeptomolar, and the change in the observable luminescent emission of the composite material following contact with the sample may be observable.

In an embodiment, a kit useful for detection of heavy metal ions comprises: a metallic composite material comprising a mesoflower structure having a metal core and a plurality of luminescent nanoparticles affixed to the mesoflower structure, wherein the composite material has an observable luminescent emission when heavy metal ions are not bound to the composite material and there is a change in the observable luminescent emission when heavy metal ions are bound to the composite material; and at least one secondary reagent or reaction container.

EXAMPLES

Example 1: Synthesis of Au/Oligoaniline Nanoparticles

Citric acid (25 mg) of was dissolved in 35 mL of distilled water and maintained at 80° C. To this solution was added 1 mL of 25 mmol/L HAuCl4. After the color changed from pale yellow to pink, 100 μL distilled aniline was added immediately followed by 500 μL of 25 mmol/L HAuCl4and heating was continued for 5 more min. The mixture cooled to room temperature. The mixture was kept at room temperature for 5 h and then centrifuged at 4000 rpm. The light pink supernatant, containing Au/oligoaniline nanoparticles, was collected and used for further reactions. Au/oligoaniline nanoparticles are raspberry-like aggregates of small nanoparticles (˜3 nm diameter), forming 75-100 nm structures, which are embedded in an oligoaniline matrix.

Example 2: Synthesis of Silica Coated Gold Mesoflowers

To a 20 mL CTAB solution (100 mM) in a beaker, 335 μL HAuC14(25 mM), 125 μL AgNO3(10 mM) and 135 μL ascorbic acid (100 mM) were added sequentially. To this solution, 2 mL of the Au/oligoaniline nanoparticles from Example 1 were added, and the mixture was maintained at 80° C. for 1 hour. It was then cooled to room temperature and the mixture was centrifuged at 3500 rpm for 4 minutes. The residue containing gold mesoflowers was washed with water three times in order to remove excess CTAB and other unwanted materials. The gold mesoflowers were dispersed in isopropanol (2 mg in 10 mL). Tetraethyl orthosilicate TEOS (120 μL) and ammonia (1.5 mL) solutions were added under rapid stirring. The mixture was centrifuged and the supernatant was removed to arrest self nucleation of silica particles. The residue was cleaned a couple of times by centrifugation and redispersed in water/isopropanol. This yielded a uniform coating of silica (approximately 25 nm) on the gold mesoflowers.

Example 3: Synthesis of Ag15Quantum Clusters Confined in Bovine Serum Albumin

A silver nitrate solution (5 mL at 10 mM) was added to bovine serum albumin powder in distilled water (250 mg in 5 mL) with vigorous stirring at room temperature. About 0.3 mL NaOH (1 M) was added, followed by drop-wise addition of a 10 mM NaBH4solution until the solution turned from colorless to reddish brown (about 120 μL); indicating the formation of Ag15quantum clusters confined in bovine serum albumin.

Example 4: Synthesis of Silica Coated Gold Mesoflowers Loaded with Ag15Quantum Clusters Confined in Bovine Serum Albumin

Gold mesoflowers from Example 1 were dispersed in distilled water, and (3-aminopropyl)trimethoxysilane (APTMS) was added. The mixture was kept for 15 minutes and centrifuged. To the residue, a mixture containing Ag15quantum clusters confined in bovine serum albumin, from Example 2, was added and incubated for 30 minutes. The mixture was again centrifuged and the residue was washed with water, this process was repeated three times to ensure the removal of unbound quantum cluster from the mixture. Attachment of silver clusters to the surface of the gold mesoflowers was verified using energy-dispersive X-ray spectroscopy (EDAX).

Example 5: Detection of Heavy Metal Ions Using Silica Coated Gold Mesoflowers Loaded with Ag15Quantum Clusters Confined in Bovine Serum Albumin

A batch of mesoflowers from Example 3 were drop casted on to a glass slide, and one drop of each metal ion solution (Hg2+, Pb2+, Ni2+, Cd2+, and Cu2+) was added separately, dried, and monitored under a fluorescence microscope (CytoViva). Fluorescence images (490 nm excitation) of metal ion-treated gold mesoflowers were compared with untreated gold mesoflowers. Quenching of cluster luminescence was more pronounced in the case of Cu2+and Hg2+ions than for the other ions at 100 ppm each. A feeble luminescence pattern was observed in case of Cu2+ions (10 ppm), but Hg2+ions (10 ppm) resulted in complete quenching of cluster luminescence.

In order to check the lower detection limit, experiments using 10 ppb, 500 ppt and 10 ppt of Hg2+ions were conducted. While a concentration of 10 ppb of Hg2+ions showed complete quenching of cluster luminescence, a substantial quenching was also observed for 500 ppt Hg2+ions. A slight decrease in fluorescence intensity was also noted for 10 ppt Hg2+ions.

In the 500 ppt example, 2.5 μL of a 500 ppt mercury solution was used to wet the gold mesoflowers. A large gold mesoflower of 4 μm length has a volume of about 34 femtoliters and that volume of 500 ppt solution contains about 50 Hg2+ions (or 0.083*10−21moles). Thus, about 0.1 zeptomoles of Hg2+ions are being detected. The success of this sensing methodology lies in the nanostructures which enhance the sensitivity and the specificity of the protein protected quantum clusters to the analyte.

Example 6: Synthesis of Fluorescent Silica Coated Gold Mesoflowers

To a 20 mL CTAB solution (100 mM) in a beaker, 335 μL HAuC14(25 mM), 125 μL AgNO3(10 mM) and 135 μL ascorbic acid (100 mM) were added sequentially. To this solution, 2 mL of the Au/oligoaniline nanoparticles from Example 1 were added, and the mixture was maintained at 80° C. for 1 hour. It was then cooled to room temperature and the mixture was centrifuged at 3500 rpm for 4 minutes. The residue containing gold mesoflowers was washed with water three times in order to remove excess CTAB and other unwanted materials. The gold mesoflowers were dispersed in isopropanol (2 mg in 10 mL). Tetraethyl orthosilicate TEOS (120 μL), ammonia (1.5 mL), and fluorescein isothiocyanate (1 mL at 25 mM) solutions were added under rapid stirring. The mixture was centrifuged and the supernatant was removed to arrest self nucleation of silica particles. The residue was cleaned a couple of times by centrifugation and redispersed in water/isopropanol. This yielded a uniform coating of fluorescent silica (approximately 25 nm) on the gold mesoflowers which showed green luminescence under 490 nm excitation. Similar to Example 3, these mesoflowers were loaded with Ag15quantum clusters confined in bovine serum albumin. These loaded mesoflowers showed red luminescence under 490 nm excitation.

Example 7: Detection of Heavy Metal Ions Using Fluorescent Silica Coated Sold Mesoflowers Loaded with Ag15Quantum Clusters Confined in Bovine Serum Albumin

Similar to Example 4, fluorescent silica coated gold mesoflowers loaded with Ag15quantum clusters confined in bovine serum albumin were drop cast and expose to heavy metal ions. In presence of Hg2+ions (1 ppb) the red luminescence of the cluster was completely quenched and the gold mesoflowers appeared green under 490 nm excitation due to the underlying fluorescein incorporated silica layer. Whereas, for the Gold mesoflowers treated with 500 ppt of Hg2+ions, the luminescence from the gold mesoflowers changed from red to yellowish orange. The yellow color observed may be due to the additive effect of the unquenched red luminescent cluster on the gold mesoflower surface and the underlying green luminescent fluorescein functionalised silica shell. This easily observable color change (red to green) in presence of Hg2+ions in solution enables ppt level detection of such ions.

In the present disclosure, reference is made to the accompanying FIGURE, which forms a part hereof. The illustrative embodiments described in the detailed description, the FIGURE, and claims are not meant to be limiting. Other embodiments may be used, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the FIGURE, may be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein.

While various compositions, methods, and devices are described in terms of “comprising” various components or steps (interpreted as meaning “including, but not limited to”), the compositions, methods, and devices can also “consist essentially of” or “consist of” the various components and steps, and such terminology should be interpreted as defining essentially closed-member groups.