Ruthenium complex for use in nitrite detection

Abstract

In various embodiments the present invention is directed to a complex for use in detecting nitrite, a method for making the complex, and a method for detecting nitrite with the complex. The complex comprises a structure of Formula (I) ##STR00001## where R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7 and R.sub.8 are independently selected from hydrogen, a halogen atom, a C1-C4 straight or branched alkyl group, a C1-C4 straight or branched alkoxyl group, a phenyl group or a heterocyclic group, or any two of R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7 and R.sub.8 together form a phenyl group and the others are independently selected from hydrogen, a halogen atom, a C1-C4 straight or branched alkyl group, a C1-C4 straight or branched alkoxyl group, or a hydroxyl group, where said phenyl group is optionally substituted with a C1-C4 alkyl group or a halogen atom; and wherein n is an integer selected from 0, 1 or 2.

Claims

1. A complex comprising the following structure: ##STR00009## wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.7 and R.sub.8 are independently selected from hydrogen, a halogen atom, a C1-C4 straight or branched alkyl group, a C1-C4 straight or branched alkoxyl group, a phenyl group or a heterocyclic group.

2. A complex according to claim 1, wherein the complex comprises the following structure: ##STR00010## wherein R.sub.3 and R.sub.4 are independently selected from hydrogen, a halogen atom, or a C1-C4 straight or branched alkyl group.

3. A complex according to claim 1, wherein the complex comprises a structure of Formula (IV): ##STR00011##

4. A complex according to claim 1, wherein the complex comprises a structure of Formula (VII): ##STR00012##

5. A complex according to claim 1, wherein the complex comprises [Ru(npy)([9]aneS3)(CO)][ClO.sub.4], where npy is 2-(1-naphthyl)pyridine and [9]aneS3 is 1,4,7-trithiacyclononane.

6. A complex according to claim 1, wherein the complex is yellow in solution.

7. A dication comprising a structure of Formula (VIII): ##STR00013##

8. A method for making the complex of claim 1, comprising the steps of: mixing [Ru([9]aneS3)(CH.sub.3CN).sub.3](CF.sub.3SO.sub.3).sub.2, where [9]aneS3 is 1,4,7-trithiacyclononane, 2-(1-naphthyl)pyridine, and triethylamine (Et.sub.3N) in a first solvent of dimethylformamide (DMF) to form a first solution; warming the first solution in an inert atmosphere; removing the first solvent from the first solution to form a green residue; subjecting the green residue to chromatography to collect a yellow band of eluate; adding a saturated aqueous sodium perchlorate (NaClO.sub.4) solution to the collected yellow band of eluate to form yellow solids; charging the yellow solids with DMF into a pressurised container containing carbon monoxide (CO) gas to form an orange mixture; warming the orange mixture; removing solvent from the orange mixture to form yellow solids; and recrystallizing the yellow solids in a recrystallization solvent by slow diffusion of diethyl ether (Et.sub.2O) into the recrystallization solvent to form pale yellow crystals.

9. The method according to claim 8, wherein the [Ru([9]aneS3)(CH.sub.3CN).sub.3](CF.sub.3SO.sub.3).sub.2, 2-(1-naphthyl)pyridine, and Et.sub.3N are mixed in a molar ratio of approximately 10:15:17, respectively.

10. The method according to claim 8, wherein the first solution is warmed at around 60 C. for about 18 hours.

11. The method according to claim 8, wherein the inert atmosphere comprises argon.

12. The method according to claim 8, wherein the first solvent is removed after the first solution is allowed to cool to room temperature.

13. The method according to claim 8, wherein the green residue is subjected to basic alumina column chromatography using acetone ((CH.sub.3)CO) as eluent.

14. The method according to claim 13, wherein the eluent is removed under vacuum after the saturated aqueous NaClO.sub.4 solution is added to the collected yellow band of eluate.

15. The method according to claim 8, wherein the CO gas is pressurised at 3 bar.

16. The method according to claim 8, wherein the orange mixture is warmed at around 120 C. for about 18 hours.

17. The method according to claim 8, wherein the recrystallization solvent in which the pale yellow crystals are formed is an acetonitrile solution.

18. A method for detecting nitrite, comprising the steps of: adding an acid and a sample to a solution comprising the complex according to claim 1 to form a detection solution; mixing the detection solution at room temperature; and checking the detection solution for any change in colour.

19. A method according to claim 18, wherein the acid is hydrochloric acid and the complex comprises a structure of Formula (VII): ##STR00014##

20. The method according to claim 18, wherein 1 mL of hydrochloric acid at a concentration of 1 mol L.sup.1 and 0.2 mL of sample solution are added to 0.2 mL of a [Ru(npy)([9]aneS3)(CO)].sup.+ solution.

21. The method according to claim 18, wherein the detection solution changes from yellow to red if the sample contains nitrite.

22. The method according to claim 21, wherein a dynamic detection range for a change in colour of the detection solution is 1-840 mol L.sup.1 nitrite.

23. The method according to claim 21, wherein a change in colour of the detection solution is determined by the naked eye.

24. The method according to claim 21, wherein a change in colour of the detection solution is determined by a spectrophotometer measured in the range of 450-550 nm.

25. The method according to claim 24, wherein a change in colour of the detection solution is determined by a spectrophotometer measured at around 483 nm.

26. The method according to claim 21, wherein the colour of the detection solution is stabilised by adding ammonia solution to neutralise and/or alkalinize the detection solution.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) It will be convenient to further describe the present invention with respect to the accompanying drawings that illustrate possible arrangements of the invention. Other arrangements of the invention are possible, and consequently the particularity of the accompanying drawings is not to be understood as superseding the generality of the preceding description of the invention.

(2) FIG. 1 illustrates an insertion reaction of nitrosonium ion (NO.sup.+) into the RuC bond of cyclometalated ruthenium complexes (a) as previously reported; (b) according to an embodiment of the invention, showing the working principle of the RuNPY assay.

(3) FIG. 2 illustrates a perspective view of the RuPNY cation of FIG. 1b, with H atoms omitted for clarity

(4) FIG. 3 is a photograph of RuPNY solutions with different concentrations of acidified NO.sub.2 (0-840 mol L.sup.1), and pure water (labeled as H.sub.2O) included for color comparison.

(5) FIG. 4 is a chart illustrating time-dependent absorbance of RuNPY.sup.soln at 483 nm for 70 (closed circle) and 700 (open circle) mol L.sup.1 of NO.sub.2.sup. upon addition of HCl with concentrations of 1, 0.75 and 0.5 mol L.sup.1.

(6) FIG. 5 is a chart illustrating absorption spectra for the RuNPY assay in the presence of various standard NaNO.sub.2 solutions.

(7) FIG. 6 is a chart illustrating an analytical curve in the NO.sub.2.sup. concentration ranges of up to 840 (main curve) and 50 (inset curve) mol L.sup.1.

(8) FIG. 7 is a chart illustrating selectivity tests of the RuNPY assay: (a) the absorption spectra of the assay with 70 mol L.sup.1 of NO.sub.2.sup. or other species at higher concentrations (7000 or 700 mol L.sup.1); (b) relative absorbance response at 483 nm of the assay to 7 mol L.sup.1 of NO.sub.2.sup. in the presence and absence of 700 mol L.sup.1 of other species (exception: 70 mol L.sup.1 for Co.sup.2+, Ni.sup.2+, Cu.sup.2+, and uric acid).

DETAILED DESCRIPTION

(9) Regarding the working principle of NO.sub.2 detection, many colorimetric [19-25] and fluorometric assays [25-28] are based on trapping the nitrosonium ion (NO.sup.+) generated from acidified NO.sub.2 to form chromophores and luminophores for spectroscopic measurements. For example, NO.sup.+ is trapped by sulfanilamide for the formation of azo dye in the Griess assay, and is trapped by 2,3-diaminonaphthalene to give the fluorescent 2,3-naphthotriazole in the DAN assay. On the other hand, trapping of NO.sup.+ by inorganic species as a NO.sub.2 detection mechanism is sparse in the literature.

1. EXPERIMENTAL SECTION

(10) 1.1 Chemicals and Materials

(11) All reagents were used as received, and solvents were purified by standard methods. [Ru([9]aneS3)(dmso)Cl.sub.2] and [Ru([9]aneS3)(CH.sub.3CN).sub.3](CF.sub.3SO.sub.3).sub.2 were prepared according to literature procedure [29]. .sup.1H and .sup.13C{.sup.1H} NMR spectra were recorded on a Bruker 400 DRX FT-NMR spectrometer. Peak positions were calibrated with solvent residue peaks as internal standard. Electrospray mass spectrometry was performed on a PE-SCIEX API 3000 triple quadrupole mass spectrometer. Infrared spectrum was recorded as KBr plates on a Perkin-Elmer FTIR-1600 spectrophotometer. UV-visible spectra were recorded on a Shimadzu UV-1800 spectrophotometer. Elemental analyses were done on an Elementar Vario Micro Analyzer. The sensing solution was prepared by dissolving RuNPY in acetonitrile (2 mmol L.sup.1). This sensing solution was stable under ambient conditions for at least 1 month. Standard solutions of sodium nitrite were prepared by appropriate dilution of the stock solution (10 mmol L.sup.1). NaNO.sub.2, Na.sub.3PO.sub.4 and Cd(NO.sub.3).sub.2 were purchased from International Laboratory USA (South San Francisco, Calif.). NaCl, NaBr, NaHCO.sub.3, NaClO.sub.4, CuCl.sub.2 and Zn(NO.sub.3).sub.2 were purchased from Acros Organics (Geel, Belgium). KNO.sub.3, Na.sub.2SO.sub.3, Na.sub.2SO.sub.4, Co(OAc).sub.2, NiCl.sub.2, NaOH, HCl (>37%), NH.sub.4OH (ca. 25% assayed as NH.sub.3), urea and uric acid were purchased from Sigma-Aldrich (St. Louis, Mo.). ZnSO.sub.4 was purchased from BDH Chemicals (Poole, England). Na.sub.2CO.sub.3 and CaCl.sub.2) were purchased from Uni-chem (China). All chemicals used were of analytical grade and used as received. Acetonitrile of HPLC grade was purchased from Anaqua Chemical Supply (Houston, Tex.). Ultrapure water (Millipore, DirectQ system) with a resistivity of 18.2 M.Math.cm was used throughout the experiment.

(12) 1.2 X-Ray Crystallography

(13) Single crystals of RuNPY.CH.sub.3CN were obtained by slow diffusion of Et.sub.2O into an acetonitrile solution of RuNPY. A suitable crystal was selected and measured on an Oxford Diffraction Gemini S Ultra X-ray single crystal diffractometer. The crystal was kept at 173 K during data collection. Using Olex2 [30], the structure was solved with the ShelXS [31] structure solution program using Patterson Method and refined with the ShelXL [32] refinement package using Least Squares minimization.

(14) 1.3 Synthesis of RuNPY

(15) A mixture of [Ru([9]aneS3)(CH.sub.3CN).sub.3](CF.sub.3SO.sub.3).sub.2 (0.250 g, 0.355 mmol), 2-(1-naphthyl)pyridine (0.109 g, 0.533 mmol) and Et.sub.3N (0.061 g, 0.604 mmol) in 10 mL DMF were warmed at 60 C. for 18 h under an argon atmosphere. Upon cooling to room temperature, the solvent was removed to give a green residue. This crude product was eluted by column chromatography (basic alumina, (CH.sub.3).sub.2CO as eluent) and the yellow band was collected. A saturated aqueous NaClO.sub.4 solution (5 mL) was added and the (CH.sub.3).sub.2CO was removed under vacuum to give yellow solids. The solids, together with 10 mL of DMF, were charged into a sealed glass container pressurized with 3 bar of CO gas. The orange mixture was then warmed at 120 C. for 18 hr. Upon cooling to room temperature, the solvent was removed and the resultant yellow solids were recrystallized by slow diffusion of Et.sub.2O into an acetonitrile solution to give pale yellow crystals. Yield: 0.115 g, 60%. Anal. Calcd for C22H.sub.22NO.sub.5S.sub.3RuCl: C, 43.10; H, 3.62; N, 2.28. Found: C, 43.12; H, 3.65; N, 2.20. .sup.1H NMR (400 MHz, CD.sub.3CN): 2.31-2.38, 2.60-2.70, 2.76-2.91, 2.98-3.21 (m, 12H, [9]aneS3); 7.25 (t, 1H, J=6.5 Hz, npy), 7.44 (t, 1H, J=7.5 Hz, npy), 7.58 (t, 1H, J=7.7 Hz, npy), 7.68 (d, 1H, J=8.1 Hz, npy), 7.83, (d, 1H, J=8.1 Hz, npy), 7.93 (d, 1H, J=8.1 Hz, npy), 8.01 (t, 1H, J=7.9 Hz, npy), 8.50, (d, 1H, J=8.7 Hz, npy.), 8.55, (d, 1H, J=8.3 Hz, npy), 8.63, (d, 1H, J=5.5 Hz, npy). .sup.13C NMR (100 MHz, CD.sub.3CN): 31.1, 32.8, 34.4, 35.5, 36.1, 37.2 ([9]aneS3); 123.2, 123.4, 125.1, 125.5, 128.2, 130.2, 130.5, 137.6, 139.5, 155.0 (npy); 131.9, 133.4, 141.2, 167.4, 172.8 (5 quaternary carbons of npy), 195.9 (CO). IR (KBr, .sub.CO=1959. ESI-MS: m/z 514 [M.sup.+].

(16) 1.4 UV-Vis Absorption Measurements

(17) UV-Vis absorption spectra were recorded on a Shimadzu UV-1800 spectrophotometer with a quartz cuvette with optical path length of 1 cm. In a typical measurement, 1 mL of HCl (1 mol L.sup.1) was first mixed with 0.2 mL of samples or NO.sub.2.sup. standard solutions. Secondly, 0.2 mL of sensing solution (2 mmol L.sup.1 RuNPY) was added into the above mixture and vortexed for 1 min to produce color change. Finally, the resultant mixture was alkalinized by adding ammonium hydroxide (0.2 mL, ca. 25%) and the absorption spectra were collected by UV-Vis spectrophotometer.

(18) 1.5 Sample Preparation for Human Urine Analysis

(19) Human urine samples were obtained from three self-reported healthy male volunteers aged from 24-28. The analysis were performed within 2 hr after urine collection. After spiking known amount of NaNO.sub.2 into the urine samples, deproteinization was carried out according to literature reported method [33]. Briefly, 2.0 mL of urine spiked with NaNO.sub.2 was added into a mixture of 0.2 mL of 1 mol L.sup.1 NaOH and 1.2 mL of 0.15 mol L.sup.1 ZnSO.sub.4 at 0 C. and stirred for 15 min. The resultant mixture was centrifuged at 13000 rpm for 5 min and the supernatant was collected. Three urine aliquots (150 L) were spiked with 50 L of NO.sub.2.sup. standard solutions to give two concentrations (25 mol L.sup.1 and 50 mol L.sup.1). The NO.sub.2.sup. concentration in urine samples were then analyzed by standard addition method.

2. RESULTS AND DISCUSSION

(20) 2.1 Working Principle and Design of the RuNPY Assay

(21) The working principle of the RuNPY assay is based on a fast reaction between NO and cyclometalated Ru(II) complexes discovered previously [34-36]. Briefly, Ru(II) complexes bearing orthometalated 2-arylpyridine react with NO to give 2-(2-nitrosoaryl)pyridine-ligated Ru(II) complexes (FIG. 1(a)). Significantly, this reaction proceeds smoothly to completion within 1 min at room temperature, suggesting that the cyclometalated Ru(II) complexes are efficient NO trapping agents. In view of the fast kinetics, this reaction is exploited to develop a rapid and convenient spectrophotometric NO.sub.2 assay.

(22) FIG. 1(b) depicts the reactions involved in the RuNPY assay, which are (1) acidification of NO.sub.2.sup. to give NO.sup.+ (eq. 1); and (2) trapping of NO.sup.+ by RuNPY (eq. 2). Alkalinization of the reaction mixture can be done to stabilize the absorbance of the mixture if the solution is not measured immediately. RuNPY showcases excellent stability towards air and moisture compared with the previously reported [Ru(2-phenylpyridine)([9]aneS3)(solv)].sup.+ where solv=CH.sub.3CN, EtOH or DMSO (FIG. 1(a)) [34, 35]. More specifically, the sensing solution, a 2 mmol L.sup.1 acetonitrile solution of RuNPY (denoted as RuNPY.sup.soln), is stable for at least 1 month under ambient conditions. Furthermore, the more conjugated nature of npy makes both the RuNPY and the NO.sup.+ inserted complex [Ru(NO-npy)([9]aneS3)(CO)].sup.2+ absorb in the visible region, which not only enables visual detection of NO.sub.2.sup. but also avoids interference from shorter wavelength absorptions due to sample matrix. The crystal structure of RuNPY was determined by X-ray crystallography, and the perspective view of its cation is depicted in FIG. 2. Thermal ellipsoids are at the 30% probability level.

(23) 2.2 Visual and Spectrophotometric NO.sub.2.sup. Detection

(24) The RuNPY method for visual detection of NO.sub.2.sup. was evaluated as follows: 1 mL of HCl (1 mol L.sup.1) was added to a mixture of 0.2 mL of standard NaNO.sub.2 solution and 0.2 mL of RuNPY, followed by a 1-min mixing at room temperature. The reaction mixture, which was originally pale yellow in color, becomes red in the presence of NO.sub.2.sup. with an onset NO.sub.2.sup. concentration between 21 and 42 mol L.sup.1 (FIG. 3). Because the guideline value and maximum contaminant level of NO.sub.2.sup. for drinking water set by WHO and U.S. EPA are 65 and 71 mol L.sup.1 respectively, the RuNPY method is sensitive enough for drinking water monitoring by the naked eye.

(25) The choices of the 1-min incubation time together with the concentration of HCl (1 mol L.sup.1) are based on kinetic and practical considerations. FIG. 4 shows the kinetic traces at 483 nm (the wavelength gives the largest absorbance response) upon adding HCl (0.5-1 mol L.sup.1) to mixtures of standard NaNO.sub.2 solution (70 and 700 mol L.sup.1) and RuNPY.sup.soln. The rate of color development was slower at lower HCl concentration, an expected consequence of slower NO.sup.+ generation according to eq.1 in FIG. 1(b). The HCl concentration of 1 mol L.sup.1 was found to allow completion of color development within 1 min for both high and low NaNO.sub.2 concentration, and was therefore chosen for the RuNPY protocol. Although the usage of even higher HCl concentration would definitely further shorten the time for color development, it is not favored in view of sample preparation and disposal, especially for on-site detections that the experimental settings lack laboratory capacity. It is worthwhile to mention that the absorbance is very stable if the acidic reaction mixture is alkalinized with ammonia solution. For example, we found that the addition of 0.2 mL of 25% NH.sub.4OH solution to the sample-RuNPY.sup.soln mixture 1 min after the acidification step could stabilize the absorbance for at least 12 hr. We therefore suggest the use of this stabilization technique for prolonged or delayed spectroscopic measurements.

(26) Absorption spectra for the RuNPY assay in the presence of various standard NaNO.sub.2 solutions are depicted in FIG. 5. Measurements were done with NH.sub.4OH stabilization. RuNPY.sup.soln alone shows an absorption peak at 389 nm with no apparent absorption beyond 450 nm. Upon addition of acidified NO.sub.2.sup. a new absorption band appears between 450 to 600 nm with absorption maximum at 483 nm, which is assigned as d(Ru).fwdarw.* (NO-npy) metal-to-ligand charge-transfer (MLCT) transition. Although any wavelength between 450 to 600 nm can be used for NO.sub.2.sup. quantification, absorbance at 483 nm shows the largest response towards NO.sub.2.sup..

(27) Correlation between the absorbance of the assay at 483 nm and the concentration of standard NaNO.sub.2 solutions is depicted in FIG. 6. The error bar represents the standard deviation of 10 measurements. Measurements were done with NH.sub.4OH stabilization. The response of the assay is highly linear, with a R.sup.2 value of 0.9996 for the NO.sub.2.sup. concentration ranging from 1 to 840 mol L.sup.1. The limit of detection (LOD), defined as 36 of the blank, is 0.39 mol L.sup.1. This LOD value is significantly lower than the guideline value (65 mol L.sup.1) and maximum contaminant level (71 mol L.sup.1) of NO.sub.2.sup. for drinking water set by WHO and U.S. EPA respectively, and is comparable with many other spectrophotometric NO.sub.2.sup. assays (Table 1).

(28) TABLE-US-00001 TABLE 1 Performance Comparison of the RuNPY assay with other NO.sub.2.sup. detection assays reported recently Time required for Dynamic Detection color range limit development Temperature for Assays (mol L.sup.1) (mol L.sup.1) (min) detection ( C.) References Modified gold 5.2-100 .sup.a 10 95 [12] nanorods RB-PDA 2-10 .sup.a 10 RT.sup.b [37] Gold nanorods 1-15 0.5 10 55 [38] TMB 0.5-30 0.1 1 RT.sup.b [39] Ag@Au 1-20 0.1 165 RT.sup.b [40] nanoparticles RuNPY 1-840 0.39 1 RT.sup.b This work .sup.aNot mentioned in the literature report. .sup.bRT = room temperature.

(29) 2.3 Selectivity Tests

(30) The detection of NO.sub.2.sup. by the RuNPY assay is highly selective. FIG. 7(a) depicts the absorption spectra of the assay in the presence of NO.sub.2 (70 mol L.sup.1) or other species at higher concentrations including 7000 mol L.sup.1 of F.sup., Cl.sup., Br.sup., HCO.sub.3.sup., NO.sub.3.sup., ClO.sub.4, Cl.sub.3.sup.2, SO.sub.3.sup.2, SO.sub.4.sup.2, PO.sub.4.sup.3, Ca.sup.2+, Zn.sup.2+, Cd.sup.2+ or urea, which clearly show that only NO.sub.2.sup. can induce a spectroscopic change to the assay, even the concentrations of other species are 100-fold of that of NO.sub.2. Similar observation also holds for 700 mol L.sup.1 of Co.sup.2+, Ni.sup.2+, Cu.sup.2+ or uric acid (remark: the former three ions have intrinsic absorptions in the visible region, whereas uric acid has a low solubility in water). Moreover, interference tests using the aforementioned species demonstrate that the performance of the RuNPY assay towards NO.sub.2.sup. is not downgraded by the coexistence of other species. For example, the difference in the absorbance response to 7 mol L.sup.1 of NO.sub.2.sup. between assays with and without the coexistence of 700 mol L.sup.1 of other species (70 mol L.sup.1 for Co.sup.2+, Ni.sup.2+, Cu.sup.2+ and uric acid) are within 10% (FIG. 7(b)).

(31) 2.4 Tap Water and Human Urine Testing

(32) Determination of NO.sub.2.sup. in tap water and human urine samples were attempted to demonstrate the practicability of the RuNPY assay. For urine samples, standard addition was employed to eliminate matrix effects. Three tap water and three human urine samples, which originally had no detectable NO.sub.2.sup., were spiked with known amounts of NO.sub.2.sup. to give two concentrations (25 and 50 mol L.sup.1). Recovery % ranging from 94-105 were obtained (Table 2), suggesting that the RuNPY assay is suitable for tap water and human urine testing.

(33) TABLE-US-00002 TABLE 2 Recovery of NO.sub.2.sup. from Three Tap Water and Three Human Urine Samples NO.sub.2.sup. added NO.sub.2.sup. recovered Sample (mol L.sup.1) (mol L.sup.1a) Recovery (%) Tap Water 0 .sup.b 25 23.5 0.3; 24.1 1.3; 94 1.3; 96 5.4; 26.3 1.5 105 5.7 50 48.9 3.2; 49.2 3.0; 98 6.5; 98 6.1; 52.2 1.2 104 2.3 Human 0 .sup.b Urine 25 23.9 0.1; 24.7 0.4; 96 0.4; 99 1.6; 25.9 0.1 104 0.4 50 46.9 2.0; 47.9 1.6; 94 4.3; 96 3.4; 52.1 2.7 104 5.2 .sup.aMean Standard Derivation (n = 3). .sup.bNot detected.

3. CONCLUSIONS

(34) The rapidity, simplicity and selectivity of the newly developed RuNPY assay for NO.sub.2 detection have been verified. Its limit of detection is well below the guideline values for drinking water recommended by WHO and U.S. EPA. Practical applications for tap water and human urine testing were successfully demonstrated. Overall, this method holds great potentials for on-site environmental and biological investigations.

(35) It will be appreciated by persons skilled in the art that the present invention may also include further additional modifications made to the system which does not affect the overall functioning of the system.

REFERENCES

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