Claims:The scope of the invention is defined by the following claims:
Claims:
1. A chalcone based biosensor probe to detect hepatocytes comprising:
a) The color-tunable emission property was truly harmonized when these compounds were subjected to biosensing of hepatocytes. The hepatocytes were visualized in the fluorescent image under UV irradiation of 361-389 nm and 430-490 nm. Interestingly 400x image showed endosomes with brilliant blue when irradiated with 361-389 nm for all the probes but also stained it to green when excited with 430-490 nm.
b) The excitation-dependent fluorescence (EDF) spectra were recorded by varying the excitation wavelength from 310 nm to 450 nm with a 10 nm interval. With a gradual increase in the excitation wave-length, the emission wavelength of all the compounds also increased due to Red-edge effect.
c) The violation of Kasha’s rule was observed due to the dependence of emission on excitation wavelength. A representative compound, Formula I in Figure 2 shows the EDF in hexane.
d) The fluorophores are typically more tightly hydrogen bonded to the solvent, and thus displayed a red-shifted emission.

2. As per claim 1, molecules where one part of it acts as an electron acceptor and another as electron donor generally exhibit TICT states. Upon photoexcitation, intramolecular charge-transfer occurs between the donor and the acceptor part of the molecule. Importantly, this charge-transfer is dependent on the ability of the molecule to twist, mostly from an all-planar geometry to 90 degrees twisted geometry.

3. According to claim 1, the emission spectrum of all the compounds exhibited a dual peak in acetone, due to twisted intramolecular charge transfer complex formation, especially when excited with 340-360 nm. The photophysical properties of such TICT complexes depend on their environment.

4. According to claim 1, all the compounds exhibited solvatochromic fluorescence when excited with 310-450 nm radiation. An interesting behavior observed is the solvatochromic twist at 320-330 nm excitations in case of ethanol and acetone solution.

5. According to claim 1, the compound at 0.1x10-10 M concentration most probably impermeable to the living cell, entered into the cell through endocytosis as evident from the marginalized endosomes. , Description:Field of Invention
The present invention relates to identification and visualization of murine hepatocyte cells as fluorescent image in brilliant blue and green when subjected to UV irradiation of 361-389 nm and 430-490 nm respectively.
Background of the Invention
Fluorescent molecules have always been an important segment for biochemical and cell biological processes as described by Haugland et al., The Handbook: A Guide to Fluorescent Probes and La-beling Technologies, 10th ed., Molecular Probes, Eugene, OR (2005). Major applications of fluorescent materials include fluorescence bio-imaging and labeling. The hunt for small molecule-based environment-sensitive fluorophores having color-tunable fluorescence with large Stokes shifts is an emerging field of interest for probing inter-biomolecular interactions, structures/chemical entities and dynamics, comprising living cells. Prior art with indolizine, 1,2-dihydropyrrolo[3, 4-b]indolizin-3-one derivatives, core-based probes stained A375 cells only at a particular wavelength as described by Kim et al., ) J. Am. Chem. Soc., 130:12206 (2008).and Liu et al., Chem. Eur. J. 18: 1599 – 1603 (2012). Multi-step synthetic procedure limits the scope.

Usually, rhodamine and functionalized rhodamine molecules were designed to resolve the crisis of bulk fluorescence. Majority of such fluorophores are solvatochromic in nature but have a short and strict excitation-emission window. Moreover, such probe becomes unsuitable if any of the background materials also fluorescence at that excitation wavelength. Again, the quenching fluorescence by electron donors like guanine, tryptophan of fluorophores still resides. Biosensing ability of any fluorophore was never achieved while varying wavelength so that blue or green fluorescence would be accessible while using a single probe. The problems can be solved by proper designing of fluorescent probes such that red edge effect could be incorporated in them but in a cheap and environment-friendly route. Demchenko et al., J. Lumin. 17: 19–42 (2002) and Guha et al., Biochemistry. 35: 13426–13433 (1996) showed that excitation Dependent Fluorescence (EDF) is an uncommon fluorescence exhibited by some polar molecules where the fluorescence bands can shift towards longer wavelengths with an increase in the excitation wavelength, commonly known as Red-edge effect.

In the prior work WO2009002565A1, a 'systems biology' approach to predict hepatotoxicity and other responses of biological hepatocytes which can result from acquaintance to the test substances. Prior work WO2015/001286Al, describes that the sensor was able to deliver a specific sensitive device which can monitor many biomolecules in real-time and US20050019799A1 presents the invention related to an optical biosensor encompassing a porous matrix. Biosensing ability of any fluorophore was never achieved while varying wavelength so that blue or green fluorescence would be accessible while using a single probe. There is an urgent need of proper designing of fluorescent biosensors in a cheap and environment-friendly route that can solve aforementioned problems. The present invention addresses the problems observed in prior arts and discloses herein small organic fluorophore molecule displaying ‘excitation dependent fluorescence’ which can act in various wavelength to detect hepatocytes devoid of the above-mentioned drawbacks and explores the detailed fluorescence property, their solvatochromism (in a polar solvent and nonpolar solvent), and biosensing ability of some chalcones while using cheap, green, easy synthetic method. Here chalcone derivatives were chosen because of its numerous applications in biological and material science.

Summary of the Invention
The aforementioned drawbacks in the prior art, the current invention purposes to detect hepatocyte cells with a biosensor.
The specific objective of the invention is to design a chalcone based biosensor molecule which can detect, identify and distinguish the murine hepatocyte cells with different fluorescence colors. A further specific objective of the invention is to eliminate multistep synthesis and to incorporate excitation dependent emission property in the molecule itself. The chalcones were designed with Donor (D) and Acceptor (A) groups as internal charge transfer (ICT) takes place in chalcones with Donor-Acceptor (D-A) groups, it can be tuned to exhibit fluorescence. In such D–A-molecules, a large fluctuation in charge distribution can be tempted in the excited state upon photons absorption leading to a huge increase of the dipole moment. As a result, a marked solvatochromic effect and a huge Stokes shift can be observed because of strong interaction with the neighboring medium, Xu et al., Spectrochim. Acta Part A Mol. Biomol. Spectrosc. 62: 987–990 (2005).

The present invention reports the detailed fluorescence property, their solvatochromism (in a polar solvent and nonpolar solvent), and cell imaging capability of some chalcones while using cheap & green synthetic method. Chalcone derivatives has numerous applications in biological and material science, so suitably substituted chalcones were synthesized using Claisen Schmidt method and the compounds thus synthesized as shown in Figure 1 with formula I and formula II, were characterized using 1H NMR, UV, Fluorescence. Formula I is (E)-3-(4-hydroxy-3-nitrophenyl)-1-(4-methoxyphenyl)prop-2-en-1-one, and Formula II is (E)-1-(4-chlorophenyl)-3-(4-hydroxy-3-nitrophenyl)prop-2-en-1-one. Computation studies and then systematically the solvatochromic studies were also carried out.

The color-tunable emission property was truly harmonized when these compounds were subjected to hepatocytes sensing. The hepatocytes were visualized in the fluorescent image under UV irradiation of 361-389 nm and 430-490 nm. Interestingly 400x image showed endosomes with brilliant blue when irradiated with 361-389 nm and compound with Formula I & II, also stained it to green when excited with 430-490 nm (Figure 3, Figure 4; Compound with Formula I, Compound with Formula II). The compound at 0.1x10-10 M concentration most probably impermeable to the living cell, entered into the cell through endocytosis as evident from the marginalized endosomes as seen in the micrograph (Figure 3, Figure 4).
The facility to control the emission frequency connects these compounds as an imperative and functional member in material applications. As the ‘input’ and ‘output’ frequencies/shift can be somewhat ‘chosen’, they are employed in tailor-made high contrast fluorophores transducing agent in bio-& chemical-sensing, biological imaging, NLO materials etc.
Brief Description of Drawings
The invention will be explained in detail with reference to the representative pictures shown in the figures wherein:
Figure 1 Reaction scheme for the synthesis of 1-(4-substituted)-3-(4-hydroxy-3-nitrophenyl) prop-2-en-1- one
Figure 2 Exhibition of excitation dependent fluorescence
Figure 3 Fluorescence micrograph of Compound with formula I and formula II
Detailed Description of the Invention
The compounds were synthesized using Figure 1. The method of synthesis and characterization data for compound with Formula I & II are can be found by in the prior work of Saha et al., Chem. Phys. Lett. 653: 184-189 (2016). The absorption spectra were recorded in hexane, chloroform, acetone and ethanol at room temperature with a concentration of 0.5×10-8 M. The absorbance maxima (nm) of Formula I in Hexane was 313, 376; in Chloroform 321, 393; in Acetone 330, 393; in Ethanol 272, 325 and for Formula II in Hexane 310, 375; in Chloroform 316, 386; in Acetone 328; 270, in Ethanol 324. Two characteristic spectral band due to n-π* and π-π* was obtained followed by a bathochromic shift in the absorbance maxima, when the solvent polarity was changed from hexane-chloroform-acetone. On further increase in solvent polarity to ethanol, both the compounds displayed a modest blue shift in absorption maximum.
The emission spectra were recorded in a different solvent with increasing polarity for e.g., hexane, chloroform, acetone, and ethanol. The excitation wavelength was varied from 310 nm to 450 nm with a 10 nm interval to detect any change in emission spectra. Excitation-dependent fluorescence (EDF) spectra were recorded by varying the excitation wavelength from 310 nm to 450 nm with a 10 nm interval. With a gradual increase in the excitation wave-length, the emission wavelength of all the compounds also increased, this effect is known as a Red-edge effect. A representative compound, Formula I in Figure 2 shows the EDF in hexane. Violation of Kasha’s rule was observed due to the dependence of emission on excitation wavelength. This unique property can be explained by the knowledge that fluorescence is a haphazard event, where fluorophore emits at different times & the decay rate is nothing but the average of a group of fluorophores. It has been mentioned earlier that, the energy levels of these compounds are close enough, which probably increases the possibility of transition of an electron in the higher energy levels. This densely spaced electronic state makes it easier for the transiting electrons to “always” get an appropriate energy level available even with a shift in excitation frequency. Consequently, fluorescence behavior and variable light emitting property are also imparted in this series of compounds.
The continuous spectral shift showed no change in spectral shape and emission occurred during solvent relaxation represented an average of the partially relaxed emission. The shift occurs due to emission from the unrelaxed state. Gradually more of the molecules got relaxed to show longer wavelength emission. At transitional times, emission from both the species would be observed and a blue or red shift was observed. The redshift observed at around 370 nm excitation these fluorophores have a solvent configuration which is like the relaxed state. These fluorophores have more hydrogen bonded interaction to the solvent, and this is the reason behind the red-shifted emission. The densely spaced electronic state assumed from theoretical studies seems to make it easier for the transiting electrons to “always” get an appropriate energy level available even with a shift in excitation frequency.
Molecules in which one portion acts as an electron acceptor and another as electron donor generally exhibit TICT states. Upon photoexcitation, intramolecular charge-transfer occurs between the donor and the acceptor part of the molecule. Importantly, this charge-transfer is dependent on the ability of the molecule to twist, mostly from an all-planar geometry to 90 degrees twisted geometry was explained by Al-Hassan et al., J. Fluoresc. 23: 1197–1206 (2013). The emission spectrum of all the compounds exhibited a dual peak in acetone, due to twisted intramolecular charge transfer complex formation, especially when excited with 340-360 nm.
The photophysical properties of such Twisted Intramolecular Charge Transfer (TICT) complexes depend on their environment. In the present molecular system, the electron withdrawing and electron donor moieties are attached through a linker of alternating single and double bonds. These two units are able to rotate relative to each other. The extent of charge transfer was tuned through the incorporation of –OH, –NO2, and –Cl, –OCH3 groups. in the excited-state, after photon absorption, this twisting motion changes the emission characteristics of fluorescence. De-excitation from the TICT state occurs at a lower energy level and lead to a red-shifted second emission peak. Jozefowicz et al., J. Fluoresc. 21: 239–245 (2011) showed that as polar solvents promote the shift of the molecule from its local excited (LE) state into a TICT state by rotating around a single bond, two-peak emission in the present cases have been observed with acetone as the solvent as explained by Haidekker et al., Bioorg. Chem. 33: 415–425 (2005).

All the compounds exhibited solvatochromic fluorescence when excited with 310-450 nm radiation. An interesting behavior observed is the solvatochromic twist at 320-330 nm excitations in case of ethanol and acetone solution was observed. Application of these compounds as fluorescent probes this, murine hepatocytes were isolated by reported method [28]. For Information. The color-tunable emission property was truly harmonized when these compounds were subjected to cell staining. The hepatocytes were visualized in the fluorescent image under UV irradiation of 361-389 nm and 430-490 nm. Interestingly 400x image showed endosomes with brilliant blue when irradiated with 361-389 nm for all the probes but also stained it to green when excited with 430-490 nm (Figure 3 & Figure 4; Formula I, Formula II). The compound at 0.1x10-10 M concentration most probably impermeable to the living cell, entered into the cell through endocytosis as evident from the marginalized endosomes as seen in the micrograph (Figure 3, Figure 4). These compounds could be used for both fixing and staining the cells nonspecifically.
The facility to control the emission frequency connects these compounds as an imperative and functional member in material applications. As the ‘input’ and ‘output’ frequencies/shift can be somewhat ‘chosen’, they are employed in tailor-made high contrast fluorophores transducing agent in bio-& chemical-sensing, biological imaging, NLO materials etc.
5 claims & 3 Figures