Please use this identifier to cite or link to this item: http://dspace.iitrpr.ac.in:8080/xmlui/handle/123456789/1713
Title: Design and synthesis of fluorescent probes for recognition of enzymes/biomolecules as disease biomarkers
Authors: Sidhu, J.S.
Issue Date: 19-Jan-2021
Abstract: Fluorescent spectroscopy is an important tool for the monitoring of biochemical activities and cellular imaging. These biochemical activities are the result of enzymatic catalysis reactions for normal and abnormal cells. Among the various classes of enzymes, oxidoreductase is a diverse class of enzymes that regulate the redox homeostasis of the cellular system via various reaction mechanisms. Therefore, to recognize/monitor the enzymes/biomolecules by measuring the fluorescence emission, provides new insights to understand the biochemical reactions and disease diagnosis. In this context, fluorogenic scaffold were modified with an enzyme-specific substrate unit that increases its selectivity for a particular enzyme and thus recorded their activity by probing fluorescence response. We have synthesized Carbon Dots (CDs) and Naphthalimide based fluorescent probes for the detection of enzymes/biomolecules. CDs have fascinated significant devotion for the detection of biological analytes and cellular imaging owing to its biocompatibility, cell wall crossing ability and aqueous solubility. CDs are the zerodimensional structured nano-material (particle size <10 nm) and possess low toxicity to living tissues. In addition to their high biocompatibility, the CDs also offer high photochemical stability, impressive water solubility, and outstanding fluorescence behavior, which make them excellent fluorescent tools for cellular imaging. Thus, we have utilized them for recognition of enzymes and biomolecules. 1,8-naphthalimide based organic fluorophores were also developed and utilized for sensing studies. Moreover, these organic fluorophores were combined with CDs for the generation of the FRET mechanism. Thus, synthesized nano assemblies produced ratiometric changes in fluorescence emission in response to different analytes. FRET system reduces the fluctuation in detection limit and effect of external factor by considering the fluorescence intensity ratio of two interconnected donor and acceptor units that minimize the error arises from background signals. Moreover, other mechanisms such as PET and ICT based fluorescent probes were also developed. Along with that the colorimetric sensor was also synthesized using azo dye and the response of this dye towards cysteine was easily detected by naked eyes. The colorimetric response to cysteine was also extracted into red green and blue components for real-time applications. Herein in this dissertation, we have targeted to recognize Thioredoxin reductase, Tyrosinase, cysteine and xanthine oxidase enzyme using fluorescent and colorimetric sensors. The endogenous images of these enzymes/biomolecules were also recorded in different cell lines. The outline of the thesis is summarized into the following chapters. The content of each chapter is briefly discussed as: Chapter 1: Introduction This chapter includes the introduction of fluorescent sensors and different reaction mechanisms linked with fluorescence response. The chemistry of different enzymes and recently reported fluorophores were briefly discussed in this part. Designing of the fluorescent sensor for enzymes and their reaction mechanism is also described. Different strategies to improve the selectivity of probes for particular enzymes and their structureactivity relationship is the spotlight of this chapter. The conclusion and future outlook of the literature review are also briefly discussed here. Chapter 2: Utilization of Carbon Dots as a Turn on/Ratiometric Fluorescent Probe for Recognition of Thioredoxin Reductase and Imaging of Cancer Cells This chapter is divided into two parts for the recognition of Thioredoxin reductase. Part A includes the synthesis of CDs as a Turn On probe for TrxR. The emission intensity of fCDs on complexation with Cu2+ ion was drastically quenched. Subsequently, the addition of TrxR to the solution of fCDs-Cu2+ complex leads to the cleavage of disulfide bond of the fCDs, that acclaim the release of 3-mercaptopropionic acid. The 3- mercaptopropionic acid, being a strong bi-dentate chelate for Cu2+ ion, extracted the Cu2+ from the coordination sphere of fCDs and the original fluorescence intensity of fCDs was restored. Thus, the probe is operating with a simple process of “ON-OFF” emission switching due to Cu2+ and “OFF-ON” switching with TrxR. The probe has been successfully used for real-time applications to monitor TrxR activities in the complex biological system. In advance to Part A, ratiometric fluorescence sensor using CDs and 1,8-naphthalimide fluorophore was developed and discussed in part B. The Fluorescence resonance energy transfer (FRET) mechanism was established between the carbon dot and naphthalimide. The naphthalimide moiety was covalently attached to the surface of carbon dot through a disulphide linker. In the normal cell conditions (when devoid of high concentrations of TrxR); the carbon dot act as an energy donor and naphthalimide act as acceptor; hence establish the FRET pair as interpreted from the emission at λem =565 nm, when exited at λex =360 nm. However, contrary to this the elevated levels of TrxR leads to the breakage of disulfide bonds and consequently break the FRET pair through the release of naphthalimide moiety from the surface of carbon dots. The event is monitored through the quenching at λem = 565 nm and enhancement at λem = 440 nm, when excited at the same wavelength (λex =360 nm). The TrxR based ratiometric quenching and enhancement fluorescence intensity; offered an interesting opportunity to monitor the enzyme activities ratiomatrically; which has advantages over the conventional monitoring the fluorescence intensity at single wavelength. Eventually, the applications of nanosensor were tested for the real application in cancer cells and results are supported with confocal microscopy. UV-vis UV-vis Cu2+ TrxR Ex Em 0 100 200 300 400 Intensity (a.u.) 400 450 500Wavelen5g5h0t (nm) 600 650 700 0 200 400 600 800 1000 B A 400 450 500 550 600 650 700 0 200 400 600 800 1000 B A Wavelength (nm) 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 I450/I565 Time (min) 0 4 8 12 16 20 30 40 50 6 Chapter 3: Fluorescence and Colorimetric Detection of Cysteine Interlinked to Reduced Cellular Environment and Cellular Imaging This chapter is divided into two parts on the basis of fluorescence and colorimetric detection of Cysteine. Part A briefly discussed the detection of Cysteine using naphthalimide and CDs based fluorogenic probe. Herein, to detect the Cys, we embedded the carbon dots (CDs), gold, and naphthalimide (L1) into a single ratiometric fluorescence sensor assembly. Sensor assembly worked on the principle of FRET mechanism between CDs and naphthalimide when CDs and L1 adhered on gold nanoparticles surface. The gold metal was turned into solid support by in situ reduction of HAuCl4 in the presence of CDs and L1. When the assembly was excited at 360 nm, emission maxima at 568 nm that corresponded to naphthalimide emission has emerged that signified the existence of a FRET between the CDs and naphthalimide fluorophores. With the addition of Cys to sensor solution, the FRET mechanism eliminated and change in the fluorescence emission at two different wavelengths (450 nm and 568 nm) was recorded.Part B dealt with the colorimetric detection of cysteine. we have synthesized azophenol based chromogenic sensor for recognition of Cys in aqueous medium. Azophenol dye fabricated with 2-chloroacetyl chloride group which reduces the electron conjugation effect of 4-hydroxy group of dye. Upon addition of Cys, this conjugated system was retrieved and sharp change in the color was observed by a naked eye. The reaction mechanism was established with the help of LC-MS chromatogram. The solid state assembly of sensor probe was developed by adsorbing the dye over silica and change in its color upon addition of Cys was extracted into red, green and blue components. The detection limit in aqueous system was found to be 0.5 μM. Dip based sensor was developed by coating the probe solution of cotton plug.Chapter 4: Synthesis of Naphthalimide and Carbon Dot Based Fluorescence Sensor for Tyrosinase and Cellular Imaging A dual mechanistic FRET and PET paired ratiometric fluorescence sensor probe has been prepared using carbon dots and naphthalimide fluorophores. To develop the FRET phenomenon, carbon dots were covalently joined with naphthalimide moiety, which emits at two different 440 and 540 nm wavelength. However, on catalytic reaction of tyrosinase, the fluorescence emission intensity of acceptor unit at 540 nm started to decrease gradually, owing to switch on of PET mechanism while emission of donor unit remained significantly unaffected. The probe exhibited high selectivity and specificity towards tyrosinase in complex biological medium with detection limit 1.2 U/mL. Moreover, endogenous images of tyrosinase in B16 cells observed under the confocal laser-scanning microscope. In part B, only organic fluorophore 1,8-naphthalimide was chosen to recognize the tyrosinase enzyme. 3-Hydroxyphenyl, as the substrate unit for the enzyme, is an important feature of this design, which avoids the interference of other bio-analytes for the recognition of tyrosinase. When the sensor probe was excited at 425 nm, an intense blue emission band emerged at 467 nm. However, upon the addition of tyrosinase to the probe solution, the monophenolic unit oxidized to o-dihydroxy and consequently released the 4-aminonaphthalimide unit. As the oxidation reaction proceeded, the fluorescence emission at 535 nm started to increase gradually with an increase in the concentration of enzyme. Therefore, the sensor probe gives the ratiometric changes via fluorescence spectroscopy. The probe affords high selectivity and sensitivity to tyrosinase with a detection limit of 0.2 U mL−1.Chapter 5: Synthesis of Naphthalimide Based Receptor for Detection of Uric acid and Monitoring of Xanthine Oxidase Activity Uric acid is an important inflammatory component which is produced from xanthine oxidase (XO) catalyzed reaction of xanthine. The presence of uric acid in blood serum above the normal value leads to the formation of urate crystals and causes the growth of gout. Therefore, herein naphthalimide based Turn-On fluorescence probe has been developed to monitor the uric acid and xanthine oxidase concentration in an aqueous system. The probe exhibited high selectivity towards uric acid and xanthine oxidase catalyzed reaction of XO. The fluorescence emission of the probe was enhanced upon binding of uric acid, generated by XO assisted oxidation reaction. The detection limit for xanthine oxidase was calculated to be 0.7 U/mL. The specificity of probe for xanthine oxidase was confirmed by treating the enzyme with Allopurinol. The endogenous images of uric acid were collected in HeLa cells using fluorescence microscope.Chapter 6: Synthesis of Naphthalimide Based Receptor and Their Metal Complexes for Inhibition Assay of Acetylcholineterase via Organophosphate Hydrolysis Inhibition of Acetylcholinesterase leads to a higher level of acetylcholine in blood plasma that causes the hyper activation of the parasympathetic system and results into the death of living being. Chlorpyriphos and parathion methyl are foremost used highly toxic pesticide which blocks the acetylcholinesterase activity in living beings. Therefore, to degrade the highly toxic pesticide into less harmful components and measuring the acetylcholine esterase activity, we synthesized four nickel complexes of naphthalimide based organic ligands. The metal complexes ([Ni(L1)2] -[Ni(L4)2]) were synthesized by the electrochemical method and characterized using single-crystal X-ray crystallography and mass spectrometry. Analytical techniques revealed that complexes are mononuclear and possess octahedral geometry. The photophysical properties in the solution phase of ligands and metal complexes were evaluated using UV-vis absorption spectroscopy. The rate of degradation of chlorpyriphos and parathion methyl was evaluated using 31P NMR and LC-MS chromatogram. The time-dependent change in LC-MS chromatogram was recorded and the byproduct of chlorpyriphos upon catalytic degradation with complex was confirmed from mass spectrometry. Moreover, the inhibition assay of Acetylcholinesterase was performed for pesticides in the presence of metal complex. Chapter 7: Conclusion This chapter summarized the overall work discussed in all chapters.
URI: http://localhost:8080/xmlui/handle/123456789/1713
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