Organic small molecule fluorescent probe for detecting HYPO- and hyper-cholesterolemia

Abstract

The present disclosure relates to a non-enzymatic / metal-free small fluorescent probes for total cholesterol detection comprising a hydrophobic part; a spacer; and a hydrogen-bonding site. The present disclosure discloses a non-enzymatic / metal- free small fluorescent probe having a compound represented by the Formula (I) for total cholesterol detection. The present disclosure provides a method of detecting total cholesterol by contacting aqueous / ethanolic or serum samples with an aqueous solution of fluorescence probe compound to quantify the cholesterol presence in the solution using a fluorescence spectrometer / microplate reader. The present disclosure also provides a diagnostic probes for detection of total cholesterol (cholesterol and / or cholesteryl ester) having an aqueous solution of fluorescence probe compound represented by the Formula (I): Formula (I) wherein, R is selected from H, OH NH2, N(CH3)2, N(Ph)2, OH and B(OH)2.

Description

ORGANIC SMALL MOLECULE FLUORESCENT PROBE FOR DETECTING HYPO- AND HYPER CHOLESTEROLEMIAFIELD OF THE INVENTION

[0001] The present disclosure relates to the total cholesterol detection technique field. Particularly, the present disclosure provides a non-enzymatic / metal-free small fluorescent probe for total cholesterol detection. The present disclosure also provides a method for the detection of total cholesterol. The present disclosure also provides a detection probe for the total cholesterol.BACKGROUND OF THE INVENTION

[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any information provided herein is prior art or relevant to the presently claimed invention or that any publication specifically or implicitly referenced is prior art.

[0003] Cholesterol biomarkers are important for clinically diagnosing several serious health diseases correlated with their variations in human blood serum. It is a crucial sterol that maintains the integrity and fluidity of the cell membrane in all eukaryotic cells for the proper functioning of biological systems. The average cholesterol concentration in a healthy person's serum is 2.9-5.18 mM (110-200 mg dl-1). The presence of cholesterol concentration below average level may lead to hemorrhagic stroke and anemia, and above the average concentration may also lead to high blood pressure, atherosclerosis, cardiovascular diseases, plaque, and stroke. Thus, to maintain a healthy life and prevent serious disorders, checking the total cholesterol level in human serum is of utmost importance.

[0004] Unlike the currently used indirect methods, which deal with the H2O2 produced from the cholesterol oxidase enzyme-catalyzed oxidation of cholesterol and conversion of cholesterol, the cholesterol esterase enzyme is used to produce free cholesterol from cholesterol esters. These methods have the inherent drawback of non-reproducibility of 100% reaction of H2O2 release and its reaction with a detection probe to produce fluorescent or colorimetric species dependingon the practical conditions.

[0005] For example, Chou et al. (US11604148B2) reported a device or method which is used to determine the plasma high-density lipoprotein (HDL) cholesterol by using the colorimetric assay via the detection of H2O2. Bohidar, Rawat and coworkers reported the enzyme-free Laponite-Montmorillonite / Indium tin oxide (L- MMT / ITO) (IN201611029865A) films-based sensor was used for the electrochemical detection of cholesterol. Lu et al. (RSC Adv., 2020, 10, 39596) reported a carbon nitride quantum dot (CNQDs) nanocomposites for a rapid and non-enzymatic method for turn-off detection of cholesterol through the formation of hydrogen bonds between -NH2, -NH on the surface of CNQDs and cholesterol containing -OH and could detect as low as 10.93 pmol L-1. De et al. (Biosensors and Bioelectronics, 2015, 63, 212-217) reported the non-enzymatic P-cyclodextrin functionalized graphene -based method for electrochemical detection of cholesterol using methylene blue dye as redox indicator via host-guest interaction. Park et al. (Biosensors and Bioelectronics, 2010, 26, 1353-1358) reported a non- enzymatic macroporous Au electrode with a coral-like shape and a highly rough surface achieved through the use of Pt nanoparticles (macroporous Au- / nPts) based biosensor for highly sensitive and selective detection of cholesterol and not shown to detect cholesteryl ester.

[0006] Clinically used cholesterol detection probe has several drawbacks such asi) requirement of enzymes and special conditions for operation, ii) indirect method for cholesterol via H2O2 detection, iii) 200 nM detection limit (Thermofisher Inc Cat. Number A12216 and Sigma-Aldrich Cat. Number MAK043), iv) more ingredients (6-9) are required, hence expensive probes, v) transportation and storage require a freezer, vi) Many systems reported, such as nanocomposite, Metal-organic Framework (MOF), carbon dots, and metal oxides, which are hard to synthesize and less sensitive to cholesterol, vii) not shows a wide detection range, hence not useful for hypocholesterolemia, and viii) longer incubation time (30 to 60 minutes).

[0007] Thus, there is a need to develop a newer fluorescent probe for totalcholesterol detection, which can overcome the problems as discussed above in the prior arts.OBJECTS OF THE INVENTION

[0008] An object of the present disclosure is to provide a non-enzymatic / metal- free small fluorescent probe for total cholesterol detection.

[0009] Another object of the present disclosure is to provide a fluorescent probe for highly sensitive and selective detection of total cholesterol.

[0010] Another object of the present disclosure is to provide a fluorescent probe for both hypocholesterolemia and hypercholesterolemia.

[0011] Still, another object of the present disclosure is to provide a method for detecting total cholesterol with a shorter response time and is economical.

[0012] Yet another object of the present disclosure is to provide a non- enzymatic / metal-free detection probe and easy to operate.SUMMARY OF THE INVENTION

[0013] Aspects of the present disclosure relate to non-enzymatic / metal-free small fluorescence probes for total cholesterol detection comprising a hydrophobic part, a spacer, and a hydrogen-bonding site.

[0014] An aspect of the present disclosure provides a small fluorescence probe, represented by Formula (I) for total cholesterol detection comprising: a hydrophobic part wherein the hydrophobic part is derived from a polyaromatic system, a spacer wherein the spacer is an unsaturated hydrocarbon, and a hydrogen-bonding site denoted by R.Formula (I)

[0015] In an embodiment, R is selected from H, OH, NH2, N(CH3)2, N(Ph)2and B(OH)2. In a preferred embodiment, the probe is a non-enzymatic and metal-free probe. In another embodiment, the hydrophobic part is a polyaromatic system comprising pyrene or its derivatives. In a further embodiment, the spacer is an unsaturated hydrocarbon preferably an alkyne and wherein the carbon number is C2to C4.

[0016] Another aspect of the present disclosure provides a method for detection of total cholesterol comprising: preparation of a stock solution by dissolving the probe, in a polar solvent and wherein the concentration of stock solution is between 0.0005M and 0.005 M; dilution of a stock solution in a polar solvent with water to prepare a working aqueous solution wherein the concentration of working aqueous solution is 1-20 pM; preparation of a solution of biological sample in polar or nonpolar solvent titration of biological sample containing cholesterol and / or cholesteryl ester against the working aqueous solution until turnon detection response; and assessing the turn-on response and calculating the total cholesterol by comparing the intensity of the turn-on response in the biological analyte to standardized values, using a fluorescence detector.

[0017] Yet another aspect of the present disclosure provides a diagnostic probe for detecting total cholesterol (cholesterol and cholesteryl ester) comprising an aqueous solution of fluorescent probes.

[0018] Other aspects, advantages, and salient features of the invention will become apparent to those skilled in the art from the following detailed description, which, taken in conjunction with the exemplary embodiments of the invention.DESCRIPTION OF THE FIGURES OF THE INVENTION

[0019] The accompanying drawings are included to provide a further understanding of the present disclosure and are incorporated in and constitute a part of this specification. The drawings illustrate exemplary embodiments of the present disclosure and, together with the description, serve to explain the principles of the present disclosure.

[0020] Figure 1 illustrates rationally designed fluorescent probes for total cholesterol detection.

[0021] Figure 2 illustrates the color change in probe solution (1-4) by adding 5 equivalents of cholesterol and cholesteryl oleate.

[0022] Figure 3 illustrates probe 2 in the binary mixtures of DMSO and water: a) the picture of solution samples and b) emission spectra.

[0023] Figure 4 illustrates the picture of an aqueous solution of probe 2 in the presence of various analytes.

[0024] Figure 5 illustrates the change in a) emission spectra and b) intensity of probe 2 in the presence of various analytes.

[0025] Figure 6 illustrates the change in emission spectra of probe 1 with increasing concentrations of cholesterol and cholesteryl oleate in H2O.

[0026] Figure 7 illustrates the change in emission spectra and intensity of probe 2 in the presence of cholesterol.

[0027] Figure 8 illustrates the change in emission spectra and a plot of intensity at 460 nm vs cholesteryl oleate concentrations of probe 2.

[0028] Figure 9 illustrates the change in emission spectra and intensity of probe3 in the presence of cholesterol and cholesteryl oleate.

[0029] Figure 10 illustrates the emission spectra and intensity of probe 4 with the addition of increasing amounts of cholesterol and cholesteryl oleate.DETAILED DESCRIPTION OF THE INVENTION

[0030] The embodiments herein and the various features and advantageous details thereof are explained more comprehensively with reference to the nonlimiting embodiments that are detailed in the following description. Descriptions of well-known components and processing techniques are omitted so as to not unnecessarily obscure the embodiments herein. The examples used herein are intended merely to facilitate an understanding of the ways in which the embodiments herein may be practiced and to enable those of skill in the art further to practice the embodiments herein. Accordingly, the examples should not be construed as limiting the scope of the embodiments herein.

[0031] Unless otherwise specified, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of the ordinary skills in the art to which this invention belongs. By means of further guidance, term definitions may be included to better appreciate the present invention's teaching.

[0032] As used in the description herein, the meaning of “a,” “an,” and “the”includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.

[0033] As used herein, the terms “comprise”, “comprises”, “comprising”, “include”, “includes”, and “including” are meant to be non-limiting, i.e., other steps and other ingredients which do not affect the end of result can be added. The above terms encompass the terms “consisting of’ and “consisting essentially of’.

[0034] As used herein, the terms “blend”, and “mixture” are all intended to beused interchangeably.

[0035] The terms “weight percent”, “vol-%”, “percent by weight”, “% by weight”, and variations thereof, as used herein, refer to the concentration of a substance as the weight of that substance divided by the total weight of the composition and multiplied by 100. It is understood that, as used here, “percent”, “%”, and the like are intended to be synonymous with “weight percent”, “vol-%”, etc.

[0036] In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about”. Accordingly, in some embodiments, the numerical parameters set forth in the written description are approximations that can vary depending on the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable.

[0037] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

[0038] The term selectivity, as mentioned through the specification, denotes that the present invention can selectively detect cholesterols without interference from other biological molecules or analytes, such as amino acids, carbohydrates, vitamins, salts but is not limited to the same. The term biological analyte, as mentioned through the specification, may include various sources of cholesterol and cholesteryl ester, such as laboratory reagents, blood serum solutions, cells, orfood samples, but is not limited to the same.

[0039] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.

[0040] The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus, if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.

[0041] The present disclosure is on the premise of using a small organicmolecule -based fluorescent probe that is free from metal and a non- enzymatic approach that shows highly sensitive and turn-on detection of cholesterol and cholesteryl ester without interference from other biomolecules present in serum. The newly developed probe is designed to rapidly detect hyperand hypo- cholesterolemia.

[0042] The utilization of the small organic molecules-based fluorescent probes for highly sensitive and selective detection of total cholesterol via a self-assembly process led to the formation of nanovesicles, which showed a change from non- fluorescent to fluorescent color (Turn on). This method can be performed without converting cholesteryl ester into cholesterol, which is conventionally achieved using enzymes. The detection of cholesterol and cholesteryl ester can be observed with the naked eye by the changes in fluorescence color and does not require any instrument for the same. The difference in fluorescence intensity can be measured by a fluorescence spectrophotometer, microplate Reader, and other fluorescencebased instruments.

[0043] The design of fluorescent probes for the detection of total cholesterolcontains a hydrophobic part for interaction with cholesterol, a spacer for increasing the conjugation / match the cholesterol length, and hydrogen (H) bonding donor / acceptors group, which provides the H-bonding sites to -OH group of cholesterol as shown in Figure 1.

[0044] An aspect of the present disclosure provides a non-enzymatic / metal-free small fluorescence probe for total cholesterol detection comprising a hydrophobic part, a spacer, and a hydrogen-bonding site.

[0045] In an embodiment, the hydrophobic part is derived from a polyaromatic system. The polyaromatic system is selected from a group consisting of pyrene and its derivatives. The hydrophobic part provides the hydrophobicity to the hydrophobic part of the cholesterol and cholesteryl ester structure.

[0046] In an embodiment, the spacer is selected from a group consisting of alkene, alkyne, phenylene, or a combination thereof. The spacer helps to increase the conjugation and maintain the length of probes to achieve better binding with cholesterol. Preferably, the spacer is C2-C4 alkyne. Most preferably, the spacer is ethyne.

[0047] In an embodiment, the hydrogen-bonding site is selected from a group consisting of H, OH, NH2, N(CH3)2, N(Ph)2and B(OH)2. Preferably, the hydrogen-bonding site is H, NH2, N(CH3)2, and N(Ph)2. The hydrogen-bonding site provides H-donor / acceptor systems to the probes to interact with the -OH group of cholesterol.

[0048] An aspect of the present disclosure provides a small fluorescence probe, represented by Formula (I) for total cholesterol detection comprising: a hydrophobic part wherein the hydrophobic part is derived from a polyaromatic system, a spacer wherein the spacer is an unsaturated hydrocarbon, and a hydrogen-bonding site denoted by R.Formula (I)

[0049] In an embodiment, R is selected from H, OH, NH2, N(CHs)2, N(Ph)2 and B(OH)2. In a preferred embodiment, the probe is a non-enzymatic and metal-free probe. In another embodiment, the hydrophobic part is a polyaromatic system comprising pyrene or its derivatives. In a further embodiment, the spacer is an unsaturated hydrocarbon preferably an alkyne and wherein the carbon number is C2 to C4.

[0050] In an embodiment, the fluorescence probe compounds are:

[0051] In an embodiment, the fluorescent probe is for nanomolar detection and highly selective for total cholesterol (cholesterol and cholesteryl ester).

[0052] In an embodiment, the probe with the addition of cholesterol and cholesteryl ester exhibits the turn-on response with enhancement in emission intensity in fast response time in the range of 1 to 10 seconds. Probe 2 undergoes aggregation with increasing fractions of water and becomes non-fluorescent, but the addition of cholesterol and cholesteryl ester exhibits the turn-on response withenhancement in emission intensity in a fast response time (2 seconds).

[0053] In an embodiment, the cholesterol and cholesteryl ester added to the solution of probe displays the change from non-fluorescent to cyan (490 nm) and blue (460 nm), respectively, and is detected as low as 10 nM (cholesterol) and 30 nM (cholesteryl oleate) in aqueous medium, preferably for probe.

[0054] In an embodiment, the probe aggregates in an aqueous solution and further exhibits a disaggregation process via the formation of self-assembly and form nanovesicles, on interaction with cholesterol or cholesteryl esters in biological sample. This would lead to enhancement in emission intensity of probe at 490 nm, preferably for probe 2.

[0055] In an embodiment, total cholesterol detection is performed without converting cholesteryl ester into cholesterol.

[0056] In an embodiment, probe is for both hypocholesterolemia and hypercholesterolemia.

[0057] In an embodiment, probe is highly selective and sensitive, with a more comprehensive detection range. Probe can be used for detecting / quantifying cholesterol in biological samples. In an embodiment, the range of detection of total cholesterol is between 40 mg / deci-litre to 400 mg / deci-litre. An aspect of the current invention is that the probe is selective in binding specifically to cholesterol and cholesteryl ester in biological samples wherein the biological sample may contain other analytes including carbohydrates, salts, proteins, vitamins and minerals but is not limited to the same.

[0058] Still another embodiment of the present disclosure provides a method of detecting total cholesterol comprising contacting aqueous / ethanolic or serum samples with an aqueous solution of fluorescent probe compound as discussed above to quantify the cholesterol presence in the solution using a fluorescence spectrometer / microplate reader.

[0059] In an embodiment of the present disclosure provides a method for the detection of total cholesterol in water with high sensitivity and selectivity based on a visual change in fluorescence color from non-fluorescent to cyan, as other biological (serum) analytes do not exhibit any significant change in fluorescent color.

[0060] The present disclosure includes a method of detecting total cholesterol using the small fluorescence probe, comprising: preparation of a stock solution by dissolving the probe, as claimed in claim 1, in a polar solvent and wherein the concentration of stock solution is between 0.0005M and 0.005 M; dilution of a stock solution in a polar solvent with water to prepare a working aqueous solution wherein the concentration of working aqueous solution is 1-20 pM; preparation of a solution of biological sample in polar or nonpolar solvent titration of working aqueous solution against biological sample containing cholesterol and cholesteryl ester until detection of tum-on response; and assessing the turn-on response and calculating the total cholesterol by comparing the intensity of the turn-on response in the biological analyte to standardized values, using a fluorescence detector.

[0061] In an embodiment, the modification the biological samples, with the addition of different concentrations of ethanol, one can estimate the HDL and LDL values which is essential parameter for blood lipid profile estimation.

[0062] In an embodiment, the polar solvent is either water, dimethyl sulphoxide (DMSO) or a combination of the same.

[0063] In an embodiment, the current method does not rely on reactivity with cholesterol. Instead, it operates through a physical mechanism involving strong binding interactions driven by supramolecular self-assembly, as evidenced by the higher association constants — 40,000 M-1for cholesterol and 33,300 M-1for cholesteryl oleate ester.

[0064] Yet another embodiment of the present disclosure provides a diagnostic probe comprising an aqueous solution of fluorescent probe as discussed above. The designed fluorescence probe provides fast, simple, and real-time detection of cholesterol. The present probe is estimated to be highly economical because it is free of enzymes. The quantitative detection of cholesterol can be performed using either a fluorescence spectrophotometer or a microplate reader.

[0065] In an embodiment, wherein the method is capable of detecting total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), HDL:LDL ratio and triglycerides in a biological analyte. The designed diagnostic probe based on fluorescent probes follows a novel process that detects the cholesterol in serum and water samples. The process demonstrates that adding cholesterol to the designed probe displays the increase in emission intensity with a change in fluorescent color from non-fluorescent to cyan in solution form. The probe has unique characteristics, such as detecting both hypo- and hypercholesterolemia as well as LDL / HDL ratio with a wide detection range (10 nM-6 mM for cholesterol; 30 nM-0.1 mM cholesteryl oleate ester). The probe follows the direct approach for detecting cholesterol in serum samples with high sensitivity and selectivity, unlike the prior art enzymatic methods based on an indirect approach that quantifies H2O2 rather than cholesterol. The present innovation deals with a final diagnostic probe, an aqueous solution made using organic probe molecules that will be treated with aqueous / ethanolic or serum samples to quantify the cholesterol presence in those solutions using a fluorescence spectrometer / microplate reader. It can also be used in cholesterol estimation on tissue samples removed from the human body and will not be practiced on a living body. Further, this probe will be used to quantify and estimate cholesterol amounts from the different samples collected from hospitals.

[0066] The foregoing description of the specific embodiments will so fully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and / or adapt for various applications such specific embodiments without departing from the generic concept and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein.EXAMPLES

[0067] The present invention is further explained in the form of the following 10 examples. However, it is to be understood that the following examples are merely illustrative and are not to be taken as limitations upon the scope of the invention.Example 1: Preparation of probe 2 solution

[0068] In this example, the present invention provides a probe for detecting and estimating total cholesterol among the various biomolecules. The 3 mg of probe 2 was dissolved in 10 mL DMSO solvent as a stock solution. After that 30 pL of probe solution from the stock solution was added to 2970 pL of water for each sample.Example 2: Detection of total cholesterol using fluorescent probes

[0069] The present invention describes the development of a novel fluorescencebased protocol for detecting total cholesterol-health disease biomarkers. For example, the solution of fluorescent probe 2 (10 pM) on interaction with cholesterol and cholesteryl ester undergoes a change in its fluorescence color from non-fluorescent to cyan and blue, respectively, as shown in Figure 2.

[0070] The emission spectra of probe 2 (10 pM) in DMSO solvents exhibitedtwo emission maxima at 430 and 540 nm, which correspond to the monomer and excimer of pyrene upon excitation at 380 nm and the color of solution appears as white under 365 nm light. The addition of water fraction from 0 to 99.9% shows the decrease in intensity at both maxima as well as the disappearance of emission maxima due to the formation of aggregates as shown in Figure 3.

[0071] The emission spectra of probe 2 in water displays the non-fluorescent upon excitation at 380 nm. The addition of other analytes like Na+, K+, Mg2+, Ca2+, glucose, galactose, fructose, sucrose, lactose, mannose, ascorbic acid, urea glutathione, cysteine, alanine, histidine, tryptophan, and glutamic acid did not show any visible change in fluorescence color of the solution of probe 2 (Figure 4). Interestingly, the addition of cholesterol to the probe 2 solution showed an enhancement of fluorescence intensity at 490 nm with a high quantum yield (Figure 5). This was associated with a change in the fluorescence color of the solution from non-fluorescent to cyan. The fluorescence color change with cholesterol remains achievable even in the presence of other biomolecules.

[0072] This change in fluorescence color on the interaction of probe 2 with cholesteryl ester is associated with turn-on detection and an increase in fluorescence intensity at 460 nm with a change in the emission color from non- fluorescent to blue.

[0073] The solution of probe 1 (10 pM) in H2O with the addition of an increasing amount of cholesterol and cholesteryl oleate exhibits 9-fold and 120- fold enhancement in emission intensity at 422 nm, respectively. While simultaneous change at 514 nm exhibited fluorescent color change from green to blue. Probe 1 is more sensitive toward cholesteryl oleate as compared to cholesterol (Figure 6).

[0074] The aggregates of probe 2 (10 pM) on gradual addition of cholesterol 20 exhibited 250-fold enhancement in fluorescence intensity at 490 nm with fluorescent color change from non-fluorescent to cyan. The plot of fluorescence intensities at 490 nm vs cholesterol concentration shows a linear increase influorescent intensity in the range from 0 to 100 pM and can detect as low as 10 nM cholesterol as shown in Figure 7. Probe 2 (10 gM) in water upon the addition of an increasing amount of cholesteryl oleate (cholesteryl ester) exhibited a 126- fold increase in emission intensity at 460 nm with a change in solution color from non-fluorescent to blue. The plot of intensity at 460 nm vs cholesteryl oleate concentrations reveals a linear increase in emission intensity in the range of 0 to 20 pM and can detect as low as 30 nM cholesteryl oleate (Figure 8). Probe 2 is highly sensitive to both cholesterol and cholesteryl ester. In an embodiment, the turn-on response and enhancement in emission intensity is due to the physical association of the probe with the cholesterol and cholesteryl ester and wherein the association constant is upto 40,000 M-1for cholesterol and 33,300 M-1for cholesteryl ester.

[0075] The solution of probe 3 (10 pM) in the presence of cholesterol and cholesteryl oleate exhibits 8-fold and 50-fold enhancement in emission intensity respectively with a blue-shift in emission maxima from 530 nm to 490 nm (cholesterol) and 470 nm (cholesteryl oleate) in the aqueous medium. Probe 3 is highly sensitive toward cholesteryl oleate as compared to cholesterol (Figure 9).

[0076] The increasing amounts of cholesterol in the solution of probe 4 (10 pM) exhibit a 7-fold enhancement in emission intensity with a blue-shift in emission maxima from 500 nm to 470 nm but the addition of cholesteryl oleate does not show a significant change in emission intensity. Probe 4 is very sensitive toward cholesterol as compared to cholesteryl oleate (Figure 10). Emission intensity and response time of probes 1-4 are given in Table 1.Table 1: Emission intensity and response time of probes 1-4.

[0077] The foregoing examples are merely illustrative and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the scope of the invention.ADVANTAGES OF THE PRESENT INVENTION

[0078] The present invention designed a fluorescent probe- for the detection of total cholesterol (cholesterol and cholesteryl oleate (cholesteryl ester)The advantages of the present invention are:

[0079] It is a non-enzymatic / metal-free detection probes and easy to operate.

[0080] Direct detection of total cholesterol is possible.

[0081] 10 nM detection limit with high selectivity is achieved.

[0082] Less ingredients used in the process thus it is economical method.

[0083] Freezer not required, hence easy to transport.

[0084] The simple organic fluorescent probe, which is easy to synthesize and characterize.

[0085] A wide detection range enables both hypocholesterolemia and hypercholesterolemia.

[0086] Shorter incubation time (1-10 seconds) is required.

[0087] It measures total cholesterol (cholesterol and cholesteryl ester)

Claims

We Claim:

1. A small fluorescence probe, represented by Formula (I) for total cholesterol detection comprising: a hydrophobic part wherein the hydrophobic part is derived from a polyaromatic system, a spacer wherein the spacer is an unsaturated hydrocarbon, and a hydrogen-bonding site denoted by R2. The small fluorescence probe as claimed in claim 1, wherein the hydrophobic part is a polyaromatic system comprising pyrene or its derivatives.

3. The small fluorescence probe as claimed in claim 1, wherein the spacer is selected from a group consisting of alkene, alkyne, phenylene and a combination thereof.

4. The small fluorescence probe as claimed in claim 1, wherein R is selected from a group consisting of H, OH, NH2, N(CH3)2, N(Ph)2and B(OH)2.

5. The small fluorescence probe as claimed in claim 1, wherein the probe is a non-enzymatic and metal-free probe.

6. The small fluorescence probe as claimed in claim 1, wherein the fluorescent probe is capable of detecting total cholesterol including cholesterol and cholesteryl esters in biological sampleswithout converting cholesteryl ester to cholesterol.

7. The fluorescence probe as claimed in claim 6, wherein the probe is selective in binding specifically to cholesterol and cholesteryl ester in biological samples.

8. The small fluorescence probe as claimed in claim 6, wherein the range of detection of total cholesterol is between 40 mg / deci-litre to 400 mg / deci-litre.

9. The fluorescence probe as claimed in claim 1, wherein the probe with addition of cholesterol and cholesteryl ester exhibits a turnon response in the form of enhancement in emission intensity.

10. The fluorescent probe as claimed in claim 9, wherein the time for the turn-on is in the range of Isec to 10 sec.

11. A method of detecting total cholesterol comprising: preparation of a solution of biological sample in polar or non-polar solvent; titration of the biological sample against working aqueous solution of the fluorescence probe of claim 1 ; and assessing tum-on response and calculating the total cholesterol using a fluorescence detector.

12. A method of detecting high-density lipoprotein (HDL), low- density lipoprotein (LDL), HDL: LDL ratio and triglycerides, said method comprising preparation of a solution of biological sample in polar or non-polar solvent; titration of the biological sample against working aqueous solution of the fluorescence probe of claim 1 ; and detecting HDL, LDL, HDL: LDL ratio and triglycerides in the sample.

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