COMPLETE SPECIFICATION

TITLE OF THE INVENTION

METHODS FOR SELECTIVE VISUAL DETECTION OF TNT

FIELD OF THE INVENTION

The present invention relates to methods for selective visual detection. The present invention further relates to selective visual detection of 2,4,6-trinitrotoluene (TNT).

BACKGROUND OF THE INVENTION

Ultralow detection of 2,4,6-trinitrotoluene (TNT) is important in terms of national security. Developing analytical methods, which are highly sensitive while being selective, is a challenging area of research. Chem. Rev. 2012, DOI: 10.1021/cr2001178, Chem. Soc. Rev. 2011, 40, 44, Anal. Chem. 2012, 84, 541 and J. Am. Chem. Soc. 2012, 134, 4834 describe the use of structural, functional and electronic features of nanomaterials to develop reliable analytical methods. Surface-enhanced spectroscopies of several kinds, particularly surface-enhanced Raman can be used for such applications, which may be further enhanced by spatially separating the analyte and the active plasmonic nanostructure with an insulator, termed as shell-isolated nanoparticle-enhanced Raman spectroscopy (SHINERS).

Creating uniform anisotropic structures with nanoscale attributes by simple solution chemistry and combining analyte-selective chemistry on such surfaces enables ultrasensitive and selective detection methodologies. Noble metal quantum clusters (QCs), a new family of atomically precise nanomolecules with intense luminescence, and their protein protected analogues are highly sensitive and selective toward specific analytes. Anchoring such QCs on mesoscale (100 nm to a few u.m) particles leading to surface-enhancement of their luminescence create a new platform for ultrasensitive detection, especially using optical microscopies. Gold mesoflowers (Au MFs) are anisotropic materials with unique five-fold symmetric stems with surface enhancing nanoscale features within. As MFs as a whole are a few micrometers in dimension, their distinct shapes are useful for unique identification by optical microscopy and changes in their properties are used for immediate and efficient detection of analytes.

Several literature and patent references may be found in this regard, for example, Ace. Chem. Res. 2008, 41, 1653, ACS Nano 2009, 3, 2859, J. Am. Chem. Soc. 2009, 131, 4616, Chem. Soc. Rev. 2008, 37, 955, Nano Lett. 2007, 7, 1591, Nature 2010, 464, 392, Anal. Chem. 2011, 83, 7061, J. Am. Chem. Soc. 2011, 133, 8424, J. Am. Chem. Soc. 2009, 131, 13806, J. Chem. Soc. Rev. 2012, 41, 1867, In Advanced Fluorescence Reporters in Chemistry and Biology II, Ed. Demchenko, A. P., Springer-Verlag Berlin, Heidelberg, 2010; Vol. 9, part 4, pp. 333- 353, and references cited therein, Nano Reviews, 2012, (DOI: 10.3402/nano.v3i0.14767), J. Am. Chem. Soc. 2010, 132, 16304, Angew. Chem. Int. Ed. 2012, 51, 2155, Angew. Chem. Int. Ed. 2009, 48, 2122, Chem. Phys. Lett. 2007, 449, 186, Anal. Chem. 2010, 82, 9194, Anal. Chem. 2011, 83, 9450, J. Nanoscale 2012, 4, 1968, Chem. Eur. J. 2010, 16, 10103, Nano Res. 2009, 2, 306, CN Application No. 102095711, 102183503 and 101597372, US Patent Application Publication No. 2011/0177606, PCT Publication No. WO 2011/154939, US Patent Application Publication No.2011/0015872, J. Am. Chem. Soc, 2011, 133, 8424, Nanoscale, 2012, 4, 4255, Talanla, 2011, Jan, 83, 1023, J. Am. Chem. Soc, 2009, 131, 7368, J. Am. Chem. Soc, 2009, 131, 13806, IEEE Sens. J., 2008, 8, 974 and Langmuir, 2010, 26, 456.

SUMMARY OF THE INVENTION

The present invention provides a method of selectively detecting 2, 4, 6-trinitrotoluene (TNT), wherein the method comprises,

a) anchoring fifteen atom silver clusters embedded in bovine serum albumin (Agl5 or Agl5@BSA) on silica-coated gold mesoflowers (Au@Si02 MFs) to obtain a combined structure (Au@Si02@Agl5 MFs), and

b) using Au@Si02@Agl5 MFs for detecting 2, 4, 6-trinitrotoluene (TNT).

The present invention further provides a method of selectively detecting 2, 4, 6-trinitrotoluene (TNT), wherein the method comprises,

a) coating fluorescein isothiocyanate (FITC) on silica-coated gold mesoflowers (Au@Si02 MFs) to obtain Au@Si02-FITC MFs,

b) anchoring fifteen atom silver clusters embedded in bovine serum albumin (Agl5 or Agl5@BSA) on Au@Si02-FITC MFs to obtain a combined structure (Au@(Si02-FITC)@Agl5MFs),and

c) using Au@(Si02-FITC)@Ag15 MFs for detecting 2, 4, 6-trinitrotoluene (TNT).

The present invention further provides a method of selectively detecting 2, 4, 6-trinitrotoluene (TNT), wherein the method comprises,

a) anchoring fifteen atom silver clusters embedded in bovine serum albumin (Agl5 or Agl5@BSA) on bimetallic Ag-coated Au MFs (Au/Ag MFs) to obtain a combined structure (Au/Ag@Agl5 MFs), and

b) using Au/Ag@Ag]5 MFs for detecting 2, 4, 6-trinitrotoluene (TNT).

The present invention provides a simple and reliable method for detection of TNT from solution, for example, at sub-zeptomole level. Selectivity of the silver quantum clusters (Ag QCs) towards the analytes are exploited and the present invention can be extended to other QCs with brighter luminescence, protected with more specific ligands which may also enhance their chemical stability. The present invention has numerous applications in catalysis, bioimaging, and the like, wherein novel devices based on properties of both the constituents may be visualized, merged and tailored. The present invention can be used in terms of a single-particle, single-molecule detection technique which is more likely the ultimate in ultra-trace sensitivity.

BRIEF DESCRIPTION OF THE DRAWINGS:

Fig.l depicts (A-C) fluorescence images (excitation -490 nm) of Au@Si02@Agi5 MFs taken using a dark field fluorescence microscope. Optical images corresponding to the fluorescence images are shown in the inset. (D) Schematic of the sensor and TNT detection approach. Insets depict the cartoon representation of the sensing particle before exposure (red) and after exposure to TNT (green). (E) Effect of emission spectra of the bare cluster upon mixing with FITC dye and subsequent exposure to TNT of varying concentrations. Inset shows the photographs of solution containing a mixture of FITC dye and Ag15 cluster before and after TNT addition, taken under visible (1 and 2) and UV light (1' and 2'), respectively. Optical and fluorescence images of Au@(Si02-FITC) MFs before (F, G) and after (H, I) cluster functionalization as well as after exposure to 100 ppt and 10 ppb TNT (L-M) and (N-O), respectively. (J, K) and (P, Q) are large area optical and fluorescence images before and after 10 ppb TNT exposure, respectively.

Fig.2 depicts Raman spectra showing gradual evolution of TNT features with increasing concentration of TNT added to Au/Ag@Ag15 MFs. Insets 'a' and 'b' show the dark field and corresponding luminescence images of Au/Ag@Ag15 MF. Symmetric and asymmetric NO2 stretching bands in the SERS spectra of TNT before (red) and after Meisenheimer complex formation (green) are compared in inset 'c'. Inset 'd' shows the gradual appearance of Raman feature at 2960 cm"1.

Fig.3 depicts (A) large area SEM image and (B) EDAX spectrum of Au@SiO2 MFs, (C) a magnified TEM image of the tip of the MF showing the uniform silica coating on its surface (shown with an arrow) and (D-G) SEM and corresponding EDAX images of a single MF showing the presence of Au Ma, and Si Ka on the MF. Carbon is from the substrate used for measurement.

Fig.4 depicts SEM and EDAX images of the various cluster-loaded hybrid MFs (A-D) Au@Si02@Ag15 MF, (E-G) Au/Ag@Ag15 MF and (H-J) Au@Ag15MF. Coating of cluster on the MF surface is evident from the EDAX map of Ag L on the MFs.

Fig.5 depicts EDAX spectra of the various cluster-loaded MFs (A) Au@Si02@Ag15 MF, (B) Au/Ag@Ag15 MF and (C) Au@Ag15 MF. Carbon and aluminium are from the substrate used for the measurement.

Fig.6 (A) depicts luminescence profile of cluster solution (Ag15@BSA). Inset shows photographs of the cluster solution under visible light (a) and UV light (b). (B) MALDI MS of BSA (black trace) and cluster collected in linear positive ion mode using sinapic acid as the matrix (red trace).

Fig.7 depicts (A) schematic of the hyperspectral microscopic set-up used for collecting the images of the MFs showing a CCD camera/hyperspectralimager (8a), 100X oil objective (8b), cover slip (8c), glass slide (8d), dark field condenser (8e) and white light/mercury lamp light source (8f). Optical and fluorescence images of (B, E) Au@Ag15 MF, (C, F) Au@Si02@Ag15 MF and (D, G) Au/Ag@Ag15 MF obtained using a dark field fluorescence microscope, respectively. Corresponding spectra collected from the surface of the MF in each case is also shown in (H). The emitted light passes through a triple pass filter which cuts off the excitation light.

Fig.8 depicts (A, C) white light and fluorescence images of the as-prepared Au@Si02@Ag15 MFs. (B, D) Images collected from the same glass slide after 1 month in ambient conditions.

Fig.9 depicts (A) luminescence spectra collected after adding 100 ppm of various compounds into the Ag15 cluster solution. Inset shows the luminescence spectra collected from Au@Si02@Ag15 MF upon exposure to various concentrations of TNT. (B) Bar diagram shows the comparison of luminescence quenching phenomena at solution phase and single particle level upon exposure to various analytes (all at 100 ppm). (C) Emission spectra illustrating the effect of bare protein, BSA, with 0.1 mL of various concentrations of TNT solution indicated in the figure. (D) The luminescence spectra collected from Au@Si02-FITC MF. The emission is from the FITC molecules attached on Au@SiO2 MF. Inset shows the optical (a) and fluorescence images (b) of Au@(Si02-FITC) MF.

Fig.10 depicts UV-vis absorption spectra showing the effect of cluster solution with (A) various concentrations of TNT solution and (B) various nitro compounds and RDX.

Fig.11 depicts (A-C) variation of emission spectra of the Agis@BSA solution upon addition of equal amounts of various MFs. (D) Bar diagram showing the relative effect of luminescence intensity of the cluster solution with 0.2 mg of various MFs.

Fig.12 depicts (A) Raman spectra of solid TNT. (B) Raman spectra showing the effect of addition of 4-nitrotoluene (4-NT), 2,4-dinitrotoluene (2,4-DNT) and RDX to the luminescent Au/Ag@Ag15 MFs.

Referring to the drawings, the embodiments of the present invention are further described. The figures are not necessarily represented to scale, and in some instances the drawings have been exaggerated or simplified for illustrative purposes only. One of ordinary skill in the art may appreciate the many possible applications and variations of the present invention based on the following examples of possible embodiments of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention provides a method of selectively detecting 2, 4, 6-trinitrotoluene (TNT), wherein the method comprises,

a) anchoring fifteen atom silver clusters embedded in bovine serum albumin (Agl5 or Agl5@BSA)
on silica-coated gold mesoflowers (Au@Si02 MFs) to obtain a combined structure (Au@Si02@Agl5 MFs), and

b) using Au@SiO2@Agi5 MFs for detecting 2, 4, 6-trinitrotoluene (TNT).

The fifteen atom silver cluster embedded in BSA, hereinafter referred to as, Ag15 or Ag15@BSA, is generally a red luminescent water soluble quantum cluster (QC). It may be prepared according to the methods disclosed in J. Mater. Chem. 2011, 21, 11205. The essential characterization data may be found in Figure 6. Gold mesoflowers (Au MFs) may be prepared according to the methods disclosed in Nano Res. 2009, 2, 306. Au@SiO2 MFs are prepared by dispersing Au MFs in an alcoholic solvent, for example, isopropanol, and adding ammonia solution, and an orthosilicate, for example, tetraethyl orthosilicate (TEOS) under rapid stirring. The supernatant may be removed to arrest self nucleation of silica particles in the solution. The residue may be cleaned more than one time, for example, by centrifugation and redispersed in a mixture of water and an alcohol, for example water/isopropanol. The method provides a uniform coating of SiO2 of about 25 nm thickness on the MFs. The Au@Si02 MFs are generally of about 4 um tip-to-tip length as depicted in Figure 3.

Au@Si02@Ag15 MFs are prepared by anchoring Ag15@BSA on Au@SiO2 MFs. The process may be carried out by dispersing Au@SiO2 MFs in water and adding an alkoxy silane, for example, 3-aminopropyltrimethoxysilane (APTMS). The solution may be washed more than one time with water to remove excess APTMS and centrifuged. Agl5@BSA solution is added to the residue followed by incubation. The solution may be centrifuged again and the residue may be washed with water. The above process may be repeated to ensure the removal of unbound cluster from the solution. The sample may be spotted on a cover slip, dried and observed using a dark field microscope.

Ag15 @BSA has a high quantum yield of about 10 or above in water. Further, it is stable in a wide pH range and exhibits emission in the solid state. The Au@SiO2@Ag15 MFs is used for detecting 2, 4, 6-trinitrotoluene (TNT). Varying concentrations of TNT may be exposed to Au@SiO2@Ag15 MFs. Even a concentration less than one zeptomole of TNT per mesoflower can quench the luminescence of the mesoflowers within about 1 minute or less. Disappearance of the luminescence of Ag15 on the MF and simultaneous appearance of luminescence of another embedded fluorophore can be used for easy identification of the analyte.

Au@SiO2@Ag15 MFs are observable under an optical microscope. Schematic of the dark field fluorescence set-up used to collect Rayleigh images, luminescence spectra and images of such MFs is shown in Figures 7 and 8. Dark field microscopic image of such a particle generally shows well defined features of the MF and they appear like stars in a two dimensional projection (inset of Figure 1 A). The fluorescence image of the same sample (about 490 nm excitation, when emitted light is passed through a triple-pass filter and imaged) shows characteristic red emission due to the QCs anchored on its surface (Figure 1 A). The emission is not present before anchoring the QCs. Observation of the well-defined shapes of the mesoscale structures ensures that the desired particles alone are analyzed unlike other spherical single particle sensors, where there is a difficulty in locating and distinguishing them in light-based microscopies. An additional feature of the MFs is the thin inert layer of silica, which enhances the adsorption capacity of MFs towards analytes. Such Au core-silica shell structures can show phenomena such as enhanced fluorescence and Raman. Better stability of the QCs on the silica layer, reduction of luminescence quenching of QC on the MF surface, ease of functionalization of the adsorbed cluster, etc. are some of the added advantages of the present invention. Exposure of about 2.5 uL, about 1 ppt TNT to Au@SiO2@Ag15 MF decreases its luminescence intensity substantially while the optical image remains unaffected as depicted Figure IB. At 1 ppb of TNT, the luminescence feature disappears completely as depicted in Figure 1C. Spectral intensities collected from the surface of these MFs are depicted in Figure 9. Not wishing to be bound by any theory, the quenching of cluster luminescence may be due to the formation of a Meisenheimer complex by the chemical interaction between TNT and the free amino groups in BSA. The color of the solution turns dark red and the formation of the complex may be confirmed by the emergence of features at 340, 450 and 525 nm in the UV absorption spectra as depicted in Figure 10. Specificity of the Meisenheimer complexation makes the cluster selective to TNT and closely similar molecules such as 2, 4-dinitrotoluene (2, 4-DNT), cyclotrimethy-lenetrinitramine (RDX) and 4-nitrotoluene do not quench its luminescence as depicted in Figures 9 and 10. However, no change in absorption features is observable when the concentration of TNT is less than about 10 ppm in bulk solution phase measurements (Figure 10), which may indicate the limit of TNT detection by Agi5 cluster in solution. However, at the single particle level, a spectral change occurs even at about 1 ppt as depicted in the inset of Figure 9A. Figure 9B provides comparison of the luminescence spectra at identical concentrations (100 ppm) both in solution phase and at single particle level as bar diagram. Though the bare protein (BSA) exhibits an intrinsic blue fluorescence due to tryptophan with an excitation and emission maximum at 295 and 332 nm, respectively, it shows only a minimum quenching in presence of TNT (Figure 9C). Not wishing to be bound by any theory, this may be due to the proximity of the residue with the free amino groups in the same subdomain of the protein, which can undergo complexation with TNT. In the case of Agis, drastic quenching in luminescence of the cluster is observed. This may be attributed to the anionic a complex formed and subsequent sensitivity of the cluster to its immediate environment or as a result of the fluorescence resonance energy transfer between the cluster core and the protein.

Another aspect of the present invention provides a method of selectively detecting 2, 4, 6-trinitrotoluene (TNT), wherein the method comprises,

a) coating fluorescein isothiocyanate (FITC) on silica-coated gold mesoflowers (Au@SiO2 MFs) to obtain Au@SiO2-FITC MFs,

b) anchoring fifteen atom silver clusters embedded in bovine serum albumin (Ag15 or Ag15@BSA) on Au@SiO2-FITC MFs to obtain a combined structure (Au@(SiO2-FITC)@Ag15 MFs), and

c) using Au@(SiO2-FITC)@Ag15 MFs for detecting 2, 4, 6-trinitrotoluene (TNT).

A TNT-insensitive fiuorophore, FITC, with the same excitation energy, but with different emission wavelength is coated on Au@SiO2 MFs to obtain Au@SiO2-FITC MFs. A schematic of the procedure and detection approach may be found in Figure ID. Au@SiO2-FITC MFs are prepared by dispersing Au MFs in an alcoholic solvent, for example, isopropanol, and adding ammonia solution, FITC and an orthosilicate, for example, tetraethyl orthosilicate (TEOS) under rapid stirring. The supernatant may be removed to arrest self nucleation of silica particles in the solution. The residue may be cleaned more than one time, for example, by centrifugation and redispersed in a mixture of water and an alcohol, for example water/isopropanol. The method provides a uniform coating of Si02 of about 25 nm thickness on the MFs. Au@SiO2-FITC MF particles show a bright green emission due to FITC as depicted in Figure 1F-G. The fluorescence spectrum of such a single particle is shown in Figure 9D. The emission observed around 540 nm indicates the adsorption of FITC on Au@SiO2 MF.

Au@(SiO2-FITC)@Ag15 MFs are prepared by anchoring Ag15@BSA on Au@SiO2-FITC MFs. The preparation is earned out by following the method described in the previous aspect of the present invention, or the method exemplified herein. Au@(SiO2-FITC)@Ag15 MFs show red emission as depicted in Figure 1H-I wherein the FITC emission is suppressed. Upon exposure to 10 ppb of TNT, green emission of the underlying FITC may be observed from the particle, as depicted in Figure 1N-0, as the red luminescence from the cluster is quenched completely. Even at further lower concentrations, for example, 100 ppt, an observable color change is evident as depicted in Figure 1L-M. Large area images of the MFs before (Figure 1J-K) and after TNT exposures (Figure 1P-Q) establish that the change occurs uniformly on all MFs. The observation of green luminescence is in agreement with the solution phase data wherein disappearance of cluster emission and emergence of FITC emission are observed upon TNT exposure as depicted in Figure IE, which also shows the solutions before and after TNT addition under visible and UV light, respectively.

Yet another aspect of the present invention provides a method of selectively detecting 2, 4, 6-trinitrotoluene (TNT), wherein the method comprises,

d) anchoring fifteen atom silver clusters embedded in bovine serum albumin (Ag15 or Ag15@BSA) on bimetallic Ag-coated Au MFs (Au/Ag MFs) to obtain a combined structure (Au/Ag@Ag15 MFs), and

e) using Au/Ag@Ag]5 MFs for detecting 2, 4, 6-trinitrotoluene (TNT).

Owing to their highly anisotropic nature, MFs can act as highly sensitive SERS probes. Au/Ag MFs may be prepared according to the methods provided in Langmuir 2010, 26, 8901. It allows ultrasensitive Raman detection compared to bare MFs. Au/Ag@Ag15 MFs are prepared by anchoring Ag15@BSA on Au/Ag MFs. The preparation is carried out by following the method described in the previous aspects of the present invention, or the method exemplified herein. Ag QCs when coated directly on such MFs results in unique metal-enhanced luminescence as depicted in Figures 7 and 11. Upon exposure to TNT, luminescence from the QC on the MF is lost and Raman features due to TNT at 1209, 1361, 1535, 1619 and 2960 cm"1 are detectable from the particle at 633 nm excitation. The gradual evolution of the Raman features of TNT with increase in TNT concentration is shown in Figure 2. Appearance of specific TNT features as depicted in Figure 12 ensures that the observed changes are only due to the analyte. Such changes are not observable with other analytes as depicted in Figure 12. The Meisenheimer complex formation may be confirmed by comparing the SERS spectra of TNT before and after complexation as depicted in inset 'c' of Figure 2. After complex formation, the symmetric and asymmetric stretching bands of NO2 group observed at 1372 and 1545 cm"1, respectively in TNT are shifted to lower wave numbers, 1361 and 1535 cm"1, respectively. Not wishing to be bound by any theory, this marked decrease in the vibrational frequencies can be attributed to electron derealization in the ring and reduction in symmetry, post-complexation. The emergence of peak at 2960 cm"1 as depicted in the inset 'd' of Figure 2 may also be attributed to the Meisenheimer complex formed due to NH2 symmetric stretch. Since the same region also corresponds to the C-H stretching and CH2 asymmetric stretching vibration of TNT, it may make an exact assignment difficult. However, by combining the high sensitivity and selectivity offered by SERS along with the instant sensing protocol, the present invention enhances the accuracy and reliability of the detection technique.

Since both the measurements are conducted at the single particle level, number of molecules truly responsible for such changes is very small. Taking the dimension of a single MF as about 4 um, the volume of the analyte solution required to completely wet the MF surface can be taken as 34 femtolitres. This volume of 100 ppt TNT solution uses amounts to 0.015 zeptomoles or about 9 molecules of TNT. Therefore, detection far below zeptomole level is possible with the present invention. As two methods of analyses are combined at the single particle level, false alarms are unlikely. The primary advantage of the present method is that only a single particle is required for the detection of an analyte allowing miniaturization of devices. The amount of gold required to make one MF of about 4 urn edge length is about 0.288 ng and it can be recovered and reused.

Scanning electron microscopic (SEM) images and energy dispersive analysis of X-ray (EDAX) images were obtained using a FEI QUANTA-200 SEM. For the SEM and EDAX measurements, samples were spotted on a carbon tape and dried in ambient condition. Luminescence measurements were carried out on a Jobin Yvon NanoLog instrument. The bandpass for excitation and emission was set as 5 nm. Raman measurements were done with a WiTec GmbH, Alpha-SNOM CRM 200 confocal Raman microscope having a 633 nm laser as the excitation source. This excitation source (instead of 532 nm) was used to suppress the cluster emission. For Raman studies, the material was carefully transferred onto a cover glass and dried in ambience. Then the MF-coated glass plates were mounted on the sample stage of the confocal Raman microscope. A supernotch filter placed in the path of the signal effectively cuts off the excitation radiation. The signal was then dispersed using a 600 grooves/mm grating, and the dispersed light was collected by a Peltier cooled charge coupled device (CCD). Matrix assisted laser desorption ionization mass spectrometric (MALDI MS) studies were conducted using a Voyager DE PRO Biospectrometry Workstation (Applied Biosystems) matrix assisted laser desorption ionization time-of-flight mass spectrometer (MALDI TOF MS). UV-vis spectra were recorded using Perkin Elmer Lambda 25 UV-vis spectrometer. Spectra were typically measured in the range of 200-1100 nm.

Dark-field imaging of the gold MFs was done using an Olympus BX-51 microscope and 100 W quartz halogen light source on a CytoViva microscope set-up. Here, a broadband white light was shown on the particles from an oblique angle via a dark field condenser. The scattered/emitted light from the particle was collected by a 100x oil immersion objective and imaged by a true-color charge-coupled device (CCD) camera or by a spectrophotometer. Each spectrum shown (in Figure 7, for example) was collected from a single pixel and was approximately 64 nm in size. Spectral analysis was performed with hyperspectral image analysis software. For fluorescence imaging, a mercury lamp light source was used. Light after passing through specific excitation (bandpass) filter fell on the sample. The emitted light was passed through a 460-500 nm band pass filter and fluorescence (if any) emitted by the particle was imaged or the spectrum was collected with a spectrometer.

It may be appreciated by those skilled in the art having the benefit of this disclosure that this invention provides a methodology for detection of TNT. It should be understood that the drawings and detailed description herein are to be regarded in an illustrative rather than a restrictive manner.

EXAMPLE 1

SYNTHESIS OF Au@SiO2 MESOFLOWERS:

To a solution of 2 mg of Au MFs dispersed in 10 mL isopropanol, 1.5 mL ammonia solution, and 120 u.L tetraethyl orthosilicate (TEOS) was added under rapid stirring for 1 h. The supernatant was removed to arrest self nucleation of silica particles in the solution. Residue was cleaned a couple of times by centrifugation and redispersed in water/isopropanol. This procedure yielded a uniform coating of SiO2 of approximately 25 nm thickness on the MFs.

For preparing fluorescein isothiocyanate (FITC) functionalized mesoflowers (Au@(SiO2-FITC MFs), FITC was added to the reaction medium prior to addition of TEOS in the above procedure.

EXAMPLE 2

SYNTHESIS OF Ag15@BSA LOADED Au MFs, Au/Ag MFs AND Au@SiO2 MFs:

To 2 mg of the MFs dispersed in 1 mL distilled water, 0.5 mL 3-aminopropyltrimethoxysilane (APTMS) was added and left for 15 minutes. The solution was washed repeatedly with water to remove excess APTMS and centrifuged. To the residue 1 mL Ag15@BSA solution was added and incubated for 30 minutes. The solution was again centrifuged and residue was washed with water, this process was repeated to ensure the removal of unbound cluster from the solution. The sample was spotted on a cover slip, dried and observed using a dark field microscope.

EXAMPLE 3

DETECTION OF TNT:

A stock solution containing 2 mg of each composite MF dispersed in 2 mL water/isopropanol was prepared. For each analysis, 10 u.L of this dispersion was drop-casted and dried on a glass slide. 2.5 uL of TNT solution of various concentrations were added on the prepared glass slide and dried rapidly after 1 minute exposure. The number of analyte molecules/ions in contact with the MF is calculated from the molarity of the solution used. Molar concentrations of 100 ppt TNT (MW 227.1) is 4.403*10"10M. The sample was covered using a cover slip and imaged using a dark field fluorescence microscope.

We Claim:

1. The said invention is a methodology for selective identification of sub-zeptomole level (visual detection) of 2, 4, 6-trinitrotoluene (TNT).

2. The method according to claim 1, uses a fifteen atom anchored silver clusters, embedded in bovine serum albumin (BSA) on silica-coated Au MF resulting in the formation of Au@SiO2@Ag15 MFs.

3. The fifteen atom anchored silver clusters according to claim 2, wherein, the 15 atom silver cluster protected with BSA are referred to as Agl5 is a red luminescent water soluble QC.

4. The fifteen atom anchored silver clusters according to claim 2, exhibits high quantum yield (10.7 %) in water, it is stable in a wide pH range and exhibits emission in the solid state. The embedded clusters are termed as Au@SiO2@Agis MFs.

5. The method according to claim 1, uses a dye wherein, the said dye is fluorescein isothiocyanate (FITC)

6. The method according to claim 1, wherein the FITC associates with Au@SiO2@Agi5 MFs resulting in the formation of Au@SiO2-FITC MF.

7. The method according to claim 1, wherein Au@Si02-FITC MF shows a bright green emission due to the presence of FITC.

8. According to any of claims 6 and 7, wherein the Au@(Si02-FITC)@Ag15 MFs after further functionalization with Ag QCs, shows red emission wherein the green emission of FITC is suppressed.

9. The method according to claim 1, wherein varying concentrations of TNT is exposed to Au@(SiO2-FITC)@Ag15 MFs.

10. The method according to claim 1, wherein exposure of TNT to Au@(SiO2-FITC)@Ag15 MFs decreases its luminescence intensity significantly while the optical image remains unaffected. At 1 ppb (parts per billion) of TNT, the luminescence feature disappears completely.

11. The method according to claim 1, wherein green emission of the under lying FITC was observed from the particle as the red luminescence from the cluster was quenched completely

12. The method according to claim 9, wherein the quenching of cluster luminescence is due to the formation of a Meisenheimer complex by the chemical interaction between TNT and the free amino groups in BSA. Colour of the solution turns dark red and the formation of the complex was confirmed by the emergence of features at 340, 450 and 525 nm in the UV absorption spectra.

13. The method according to claim 12, wherein specificity of the Meisenheimer complexation makes the cluster selective to TNT and closely similar molecules do not quench its luminescence.

14. The method according to claim 1, disappearance of the luminescence of Agl5 on the MF and simultaneous appearance of luminescence of another embedded fluorophore can be used for easy identification of the analyte.