FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
AND
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10; rule 13)
TITLE OF THE INVENTION
A nanoparticle method of detecting the presence of bisulfite ion (HSO3") in a sample using a conjugate of calix[4]arene
APPLICANT
INDIAN INSTITUTE OF TECHNOLOGY, BOMBAY
DEPARTMENT OF CHEMISTRY, POWAI,
BOMBAY 400 076, MAHARASHTRA, INDIA.
PREAMBLE TO THE DESCRIPTION The following specification particularly describes the invention and the manner
in which it is to be performed

FIELD OF INVENTION
This invention relates to a nanoparticle method of detecting the presence of bisulfite ion (HSO3") in a sample using a conjugate of calix[4]arene
TECHNICAL FIELD
Anions, including oxo-based ones, exhibit important functions in biology. Of these, bisulfite (HSO3"), an oxo-anion of sulfur is certainly an important one. HSO3" has been used as anti oxidant in food and chemical industry. This ion is also being used in DNA sequencing. Studies show that HSO3" can reduce the neurotoxic damage induced by intrathecal local anesthetic. Therefore the detection of bisulfite ion is important. Till date, the official method for determination of sulfite in food involves Monier-Williams method which is very tedious and time consuming one. Development of nanoparticle (hereinafter referred as "NT" also) based detection in aqueous condition would enhance its application and may well enter into the arena of health care science. To our knowledge, there are no reports of NP detection of HSO3" using a conjugate of calix[4]arene (L).
An efficient sensing system will emerge only when the NPs are protected from agglomeration by the receptor molecular system and that too by a water soluble one. Such sensor systems can turn out to be an ideal one if the detection can bring visually observable colour changes. Therefore, this invention deals with the detection of bisulfite ion by Ag-nanoparticles (AgNPs) protected with water soluble lower rim diamine conjugate of calix[4]arene (hereinafter referenced as "L") also by colorimetry and absorption. Such coating utilizes the advantage of the macrocyclic effect in stabilizing the AgNPs. To our knowledge this is the first

report on the detection of bisulfite ion by water soluble AgNPs protected by calix[4]arene conjugates (L-AgNPs).
BRIEF DESCRIPTION OF THE INVNETION
This invention relates to a method of detecting the presence of bisulfite ions (HS03") in a sample which comprises adding to a solution of said sample, a solution of silver nano particles coated with water solution diamine tetra sulphonato conjugate of calix[4]arene, the presence of bisulfite ion being indicated by immediate colour change from orange yellow to orange red after addition.
DETAILED DESCRIPTION OF INVENTION.
Titrations with anions (visual colour change): Titrations of L-AgNPs with different anions were carried out by adding 2.5 ml of their sodium salts (3x10-2 M) to 2.5 ml of L-AgNP solution. Among the 19 anions, F-, CI-, Br-, I-, CH3COO-, N3- BF4- CIO4-, SCN- H2P04-, HPO4-, HS04- HS03-, S042- C032-, HC03- N02-, N03- and OH" studied, only HSO3" exhibited a colour change from orange yellow to orange red immediately after addition. Even the other related oxo-anions, such as, HSO4- and S04 2-, does not show any change in the colour of the solution
It has also been observed that among all the anions studied, I" and Br* decolorize the solution slowly and among these two, Br- does it much slower than I"
Control experiments carried out between simple L (without the support of Ag surface), and other anions, exhibited no change in the colour of L and hence establishes the positive role played by Ag surface in bringing the colour change .

When HS03" is added to L-AgNP, the pH of the medium changes from 9.2 to 5.8. When the pH of L-AgNP has been changed to 5.8 without the addition of HS03-, no such colour change was observed, suggesting that simple pH change of the NPs does not bring the colour change.
Titrations with anions (absorption studies): Absorption titration studies carried
out between L-AgNP and varying concentrations of HSO3-, showed decrease in
the absorbabnee of -398 nm band and increase in the ~454 nm band, which is
new. An isosbestic point has also been observed at 422 nm which clearly indicates
the complex formation between L-AgNP and HS03-. Only a nominal absorbance
increase was observed in case of -208 nm band. The observed -398 nm band
intensity reflects on the fact that HS03-, induces some changes in the AgNP
surface properties. Similar titrations carried out with H2P04- does not result in any
change of either of the band. This is in line with the fact that no colour change was
indeed observed.
The minimum detection limit of HS03- has been found to be (1.3 ± 0.17) * 10-4 M
by the absorption studies and 3 x 10-3 M by visual colour change.
L-A
Experimental details and examples are given below.
Synthesis of calix[4] arene conjugate L: The receptor L has been prepared in three steps starting from p-tertbuty\ calix[4]arene (LI) (Scheme 1). All the synthetic compounds including the final product have been checked for their purity, authenticity and were characterized by NMR, FTIR and ESI MS. Both the synthesis and characterization details are being given the experimental section.


Synthesis of L-AgNP: To a two ml of 3 X 10-2 M AgN03 solution (60 ml of water), 2 ml of 3 x 10-2 M aqueous solution of L was added as stabilizer-cum-receptor with stirring for 20 min. This was then treated with 3 X 10-1 M of NaBH4 solution by continuously stirring for 1 h to result in silver nano particles as orange-yellow colored solution.4 The characteristic surface plasmon resonance (SPR) band of Ag(0) observed at ~ 398 nm confirms the formation of L-AgNPs. Based on the absorption spectra, L-AgNPs were found to be stable for more than a week.
Example for preparing the water soluble conjugate with diamine at the lower rim and tetra sulphonate at the upper rim is given below.
Synthesis of the intermediate L2: Compound L2 was synthesized as reported in the literature, with some modification. A mixture of p-tert-butyi calix[4]arene Li (l.Og, 1.55mmol), K2C03 (0.6g, 7.25mmol) and bromoacetonitrile (0.8g, 8.31mmol) in 50 ml acetonitrile was refluxed under nitrogen atmosphere for 7 h. The reaction mixture was allowed to cool down to room temperature and was filtered through celite to obtain clear ight brown solution. The filtrate was concentrated to give brown solid, which was recrystalized from chloroform/methanol to give white crystalline solid. Yield 80%. C48H58N204 (726.44): C 79.30, H 8.04, N 3.85; found: C 79.49, H 8.30, N 3.92%. FTIR: (KBr,

cm-1): 1482 (vCN), 3515 (vOH). 'H NMR: (CDC13, δ ppm): 7.119 (s, 4H, Ar-H), 6.727 (s, 4H, Ar-H), 4.808 (s, 4H, OCH2)L221 (d, J= 13.55 Hz, 4H, Ar-CH2-Ar), 3.447 (d, J= 13.55 Hz, 4H, Ar-CH2-Ar), 1.324 (s, 18H, C(CH3)3), 0.880, (s, 18H,C(CH3)3).
(b) Synthesis of diamine intermediate (L3): To a vigorously stirred solution of L2 (0.58 g, 0.81 mmol) in 32 ml diethylether, LiAlH4 (0.25 g, 7.13 mmol) was added and the reaction mixture was refluxed for 5 h. Then the reaction flask was immersed into an icewater bath and excess LiAlH4 was destroyed by the addition of wet benzene into the reaction mixture. The clear organic layer was decanted and the inorganic salts were rinsed with benzene. The combined organic layers were evaporated to dryness to yield diamine, as a light yellow solid. Yield (470 mg) 85%. C48H66N204 (734.50): C 78.43, H 9.05, N 3.81; found: C 78.29, H 8.37, N 3.97%. FTIR (KBr, cm-1) 1484 (vCN), 3368 vOH/NH ). 'H NMR: (CDC13, 6 ppm): 7.04 (s, 4H, Ar-H), 6.97 (s, 4H, Ar-H), 4.32 (d, J= 2.82 Hz, 4H, Ar-CH2. Ar), 4.07 (t, J= 4.76, 4.76 Hz, 4H, OCH2), 3.37 (d, J= 12.82 Hz, H, Ar-CH2-Ar), 3.29 (t, J- 4.76, 4.76, 4H5 NCH2), 1.24 (s, 18H, C(CH3)3), 1.10 (s, 8H, C(CH3)3). ,3C-NMR (400 MHz, CDC13): 31.28, 37.78 (C(CH3)), 33.98, 34.30 C(CH3)), 32.32 (Ar-CH2-Ar), 42.74 (-CH2-N-), 78.7 (-0-CH2-), 125.58, 126.01, 127.75, 133.31, 142.21, 147.66, 149.27, 150.42 (Aromatic carbons), m/z (ES-MS) 735.26 ([M+H]+, 100%).
(c) Synthesis calix[4]arene conjugate (L): Cone. H2S04 (5.0 ml) was added to L3 (0.2 g, 0.272 mmol) and heated at 50° C for 1 day. A black colored solution was formed which upon treatment with diethyl ether resulted in brown precipitate. This precipitate was filtered in N2 atmosphere and quickly transferred to NMR tube. A part of the same was dissolved in water and to it 15N NaOH solution was added

(pH ~ 8). The solution was evaporated to dryness using reduced pressure. The white colored product was isolated and kept for aif drying. ES-MS: m/z = 831.3 ([M+Hf, 100%).
Synthesis of L-AgNP from this calix[4]arene conjugate and Ag nano particles to produce LAgNP has been disclosed herein before.
Obvious modifications and alterations known to Persons skilled in the art are within the scope of the appended claims.

We claim:
1. A method of detecting the presence of bisulfite ion (HS03-) in a sample comprising adding to a solution of said sample a solution of silver nano particles coated with water soluble diamine tetra sulphenato conjugate of calix[4] arene, in the presence of bisulfite ion being indicated by immediate colour change from orange yellow to orange red after addition.
2. The method as claimed in claim 1 wherein said conjugate has diamine moites at its lower rim and sulphonate moites at its upper rim.
3. The method as claimed in claims 1 & 2 wherein silver nanoparticles coated with said conjugate are prepared by adding an aqueous solution of said conjugate to silver nitrate solution under stirring and then treating the same with a solution of a NaBH4 under stirring to produce an orange yellow coloured solution of silver nano particles coated with said conjugate.
4. A method of detecting the presence of bisulfite ions in a sample substantially as herein described and exemplified.