Browsing by Author "Sidhu, Jagpreet Singh"

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Acetylcholine structure-based small activatable fluorogenic probe for specific detection of acetylcholinesterase
(ACS, 2023-05) Sidhu, Jagpreet Singh
Early detection of Alzheimer’s disease (AD) is important for taking proper measures against AD pathogenesis. Acetylcholinesterase (AChE) is widely reported to be associated with the pathogenicity of AD. Here, employing the “acetylcholine-mimic” approach, we designed and synthesized a new class of naphthalimide (Naph)-based fluorogenic probes for specific detection of AChE and avoiding interference of butyrylcholinesterase (BuChE), the pseudocholinesterase. We investigated the action of the probes on Electrophorus electricus AChE, and the native human brain AChE that we expressed in Escherichia coli and purified in the active form for the first time. The probe Naph-3 exhibited a substantial fluorescence enhancement with AChE and majorly avoided BuChE. Naph-3 successfully crossed the cell membrane of the Neuro-2a cells and fluoresced upon reaction with endogenous AChE. We further established that the probe could be effectively used for screening AChE inhibitors. Our study provides a new avenue for the specific detection of AChE, which can be extended to the diagnosis of AChE-related complications.
Acetylcholine Structure-Based Small Activatable Fluorogenic Probe for Specific Detection of Acetylcholinesterase
(ACS, 2023-05) Sidhu, Jagpreet Singh
Early detection of Alzheimer’s disease (AD) is important for taking proper measures against AD pathogenesis. Acetylcholinesterase (AChE) is widely reported to be associated with the pathogenicity of AD. Here, employing the “acetylcholine-mimic” approach, we designed and synthesized a new class of naphthalimide (Naph)-based fluorogenic probes for specific detection of AChE and avoiding interference of butyrylcholinesterase (BuChE), the pseudocholinesterase. We investigated the action of the probes on Electrophorus electricus AChE, and the native human brain AChE that we expressed in Escherichia coli and purified in the active form for the first time. The probe Naph-3 exhibited a substantial fluorescence enhancement with AChE and majorly avoided BuChE. Naph-3 successfully crossed the cell membrane of the Neuro-2a cells and fluoresced upon reaction with endogenous AChE. We further established that the probe could be effectively used for screening AChE inhibitors. Our study provides a new avenue for the specific detection of AChE, which can be extended to the diagnosis of AChE-related complications.
Carbon Dot Based, Naphthalimide Coupled FRET Pair for Highly Selective Ratiometric Detection of Thioredoxin Reductase and Cancer Screening
(ACS, 2017-07) Sidhu, Jagpreet Singh
The fluorescence resonance energy transfer (FRET) mechanism has been established between carbon dots (CDs) and naphthalimide to monitor the activity of thioredoxin reductase (TrxR), which is often overexpressed in many cancer cells. The naphthalimide moiety was covalently attached to the surface of CDs through a disulfide linkage. In normal cell conditions (when devoid of high concentrations of TrxR), the CDs act as an energy donor and naphthalimide acts as an acceptor, which establishes the FRET pair as interpreted from the emission at λem = 565 nm, when excited at λex = 360 nm. However, contrary to this, the elevated levels of TrxR cause the breakage of disulfide bonds and consequently abolishes the FRET pair through the release of the naphthalimide moiety from the surface of CDs. This process was studied by monitoring of fluorescence intensity at λem = 565 and 440 nm, when excited at the same wavelength (λex = 360 nm). The TrxR based ratiometric quenching and enhancement of fluorescence intensity offers an interesting opportunity to monitor the enzyme activities and has many advantages over conventional monitoring of fluorescence intensity at a single wavelength to avoid interference of external factors. Fluorescence images of cancer cells in response to the nanosensor were visualized under a confocal microscope. Cytotoxicity study of nanosensor retards the growth of HeLa and MCF-7 cell lines in the presence of visible light. Therefore, the nanosensor also acts as a theranostic agent to diagnose as well as killing of cancer cells.
Carbon dots as analytical tools for sensing of thioredoxin reductase and screening of cancer cells
(RSC, 2018-02) Sidhu, Jagpreet Singh
Thioredoxin Reductase (TrxR) is a redox regulating enzyme which is predestined for the maintenance of redox homeostasis of mammalian cells. However, the elevated level of TrxR is associated with the progress of various types of tumors and therefore, this is a significant target for the detection of cancer cells. Herein, an easily engineered ‘Turn ON’ fluorescent sensor probe has been synthesized for the detection of TrxR and cell imaging using carbon dots. The emission intensity of fCDs on complexation with Cu2+ ions was drastically quenched. Subsequently, the addition of TrxR to the solution of the fCDs-Cu2+ complex leads to the cleavage of the disulfide bond of the fCDs, which acclaim the release of 3-mercaptopropionic acid. 3-Mercaptopropionic acid, being a strong bi-dentate chelating agent for Cu2+ ions, extracted Cu2+ from the coordination sphere of fCDs and restored the original fluorescence intensity of fCDs. 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 application to monitor TrxR activities in the complex biological system. The fluorescence images of MCF-7 and HeLa cells after incubation with the fCDs-Cu2+ complex were recorded under a confocal laser scanning microscope (CLSM) as a function of time. Enhancement in the emission intensity of cancer cells after 2 h of treatment demonstrates the potential application of the sensor probe for the bioimaging of endogenous TrxR in living cells and screening of cancer cells. Such fluorescent probes will open the door for the development of promising clinical devices for the diagnosis of cancer cells.
A carbon quantum dot and rhodamine-based ratiometric fluorescent complex for the recognition of histidine in aqueous systems
(RSC, 2019-01) Sidhu, Jagpreet Singh
Histidine is an essential α-amino acid that plays a crucial role in tissue development and helps in the transmission of metallic ions during biological events. However, an abnormal level of histidine in the body is associated with various physiological conditions such as arthritis, liver cirrhosis, kidney diseases, and asthma. Herein, a unique ratiometric fluorescence sensing system has been developed for the recognition of histidine. The sensing system was developed using carbon quantum dots (CQDs) as an energy donor and a rhodamine 6G derivative (HS30) as an energy acceptor unit. Interestingly, upon the addition of Fe(III) into the mixture of CQDs and HS30, the phenomenon of fluorescence resonance energy transfer (FRET) was observed when excited at 350 nm. The emergence of a strong emission peak at 551 nm on the addition of Fe(III) suggested the formation of a ratiometric fluorescent complex “CQDs–Fe–HS30”. The ratiometric behavior of “CQDs–Fe–HS30” was studied by monitoring fluorescence emissions at 425 nm and 551 nm with an excitation wavelength of 350 nm. Furthermore, “CQDs–Fe–HS30” was employed for the recognition of histidine in an aqueous system. Due to the high affinity of histidine to Fe(III), the addition of histidine to an aqueous solution of “CQDs–Fe–HS30” resulted in the displacement of the Fe(III) cation from the complex, and the simultaneous quenching and enhancement of the emission peaks at 551 nm and 425 nm, respectively, was observed. The developed sensing system was successfully employed for a histidine recovery experiment in human urine samples with satisfactory results. Furthermore, the mixture of CQDs and HS30 was successfully utilized to implement an inhibit logic gate with Fe(III) and histidine as inputs and emission at 551 nm as output.
Cystamine-cobalt complex based fluorescent sensor for detection of NADH and cancer cell imaging
(Elsevier, 2019-08) Sidhu, Jagpreet Singh
The pronounced demand of sensing of biological analytes which play a significant role in progress of cancer cells encouraged us to develop a novel chemosensor having high selectivity and sensitivity for NADH. Therefore, imine linked dipodal receptor (L1) was synthesized and its organic nanoparticles (L1-ONP) were prepared using reprecipitation method to enhance their water solubility. These ONPs form a metal complex with Co(II) which was characterized by UV–vis absorption and Fluorescence Spectroscopy. The ONPs show highly sensitive affinity having a low detection limit (LOD) of 16.95 nM for the cobalt ions. The prepared ONPs metal complex exhibits high selectivity and sensitivity to sense the NADH with a very low LOD of 83.89 nM without any interference from other potentially interfering biological molecules. Fluorescence response from NADH was also not affected by pH and UV irradiation. The sensor was also employed for detection of NADH in MCF-7 cells.
FRET and PET paired dual mechanistic carbon dots approach for tyrosinase sensing
(RSC, 2018-05) Sidhu, Jagpreet Singh
A dual mechanistic FRET and PET paired ratiometric fluorescence sensor probe has been prepared using carbon dots and naphthalimide fluorophores. The carbon dots are covalently joined with a naphthalimide moiety to develop the FRET phenomenon, which emits at two different wavelengths (i.e., λmax = 440 and 540 nm). However, on catalytic reaction of tyrosinase, the fluorescence emission intensity of the acceptor unit at 540 nm is quenched gradually, owing to the switching on of the PET mechanism; while emission of the donor unit remains significantly unaffected. The probe exhibits high selectivity and specificity towards tyrosinase in complex biological medium with a detection limit of 1.2 U mL−1. Moreover, endogenous images of tyrosinase in B16 cells have been observed under a confocal laser-scanning microscope.
Gold conjugated carbon dots nano assembly: FRET paired fluorescence probe for cysteine recognition
(Elsevier, 2019-03) Sidhu, Jagpreet Singh
The detection and discrimination of Cys amino acid from numerous other related biomolecules has great importance in clinical field for diagnosis of various diseases. Herein, to detect the Cys, we embedded the carbon dots (CDs), gold, and naphthalimide (L1) into a single ratiometric fluorescence sensor assembly. Sensor assembly works on the principle of FRET mechanism between CDs and naphthalimide when CDs and L1 adhered on gold nanoparticles surface. 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 corresponded to naphthalimide emission was emerged that signifies the existence of a FRET between the CDs and naphthalimide fluorophores. With the addition of Cys, the FRET mechanism eliminated and the change in the fluorescence emission at two different wavelengths (450 nm and 568 nm) was recorded. The endogenous images of Cys was recorded by collecting the fluorescence images of HeLa cells under fluorescence microscope and also applied for the assay of Cys in blood serum. Cytotoxicity studies of CDs and sensor assembly were evaluated by performing the MTT assay.
A highly selective naphthalimide-based ratiometric fluorescent probe for the recognition of tyrosinase and cellular imaging
(RSC, 2018) Sidhu, Jagpreet Singh
Tyrosinase is polyphenolic oxidase enzyme associated with the progression of various diseases. Therefore, for the recognition of tyrosinase, naphthalimide-based ratiometric fluorescent sensor probe was designed and synthesized. 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. Furthermore, live cell images were recorded to assay the endogenous enzyme in A375 cells, which also show a dual color change in the presence of the L3 probe.
Indole Derivatives as Anticancer Agents for Breast Cancer Therapy: A Review
(Bentham Science, 2016) Sidhu, Jagpreet Singh
Breast cancer (BC) is the second most common cause of cancer-related deaths in women throughout the world. Multiple drugs have been approved by US-FDA for breast related malignancies. Frequent emergence of resistances creates the severe need of newer moieties that are free from such problems. Drugs targeting breast cancer have been observed to be based on the multiple mechanisms of action, and various indole based anticancer agents have also been explored. Moreover, indoles have promising anti-cancer potential; there has been the emphasis on the synthesis of indole derivatives to overcome problems faced by existing therapeutic agents. Taking into consideration the above-mentioned facts we have analyzed in detail the possible role of indole based anticancer agents typically for breast related malignancies. This is the first exhaustive review that jointly covers various synthetic anticancer indole derivatives and related signaling pathways by which these derivatives have shown promising anti-breast cancer potential.
A naphthalimide-based novel “Turn-On” fluorescence approach for the determination of uric acid and monitoring of xanthine oxidase activity
(RSC, 2019) Sidhu, Jagpreet Singh
Uric acid is an important inflammatory component, which is produced by the 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, a naphthalimide-based turn-on fluorescent probe was developed to monitor the uric acid and xanthine oxidase concentration in an aqueous system. The probe exhibited high selectivity towards uric acid and the xanthine oxidase-catalyzed reaction of xanthine. The fluorescence emission of the probe was enhanced upon the binding of uric acid, generated by the XO-assisted oxidation reaction. The detection limit for xanthine oxidase was calculated to be 0.7 U mL−1. The specificity of the probe for xanthine oxidase was confirmed by treating the enzyme with allopurinol. The endogenous images of uric acid in HeLa cells were obtained using a fluorescence microscope.
Oxidative decarboxylation of fatty acids to terminal alkenes by a membrane-bound metalloenzyme, UndB
(CUP, 2023-04) Sidhu, Jagpreet Singh
Biosynthetically produced alkenes are high-value molecules that can serve as ‘drop-in’ replacements for fossil fuels. Alkenes are also heavily used in the polymer, lubricant, and detergent industries. UndB is the only known membrane-bound fatty acid decarboxylase that catalyzes the conversion of fatty acids to terminal alkenes at the highest reported in vivo titers. However, the enzyme remains poorly understood and enigmatic. Here, we demonstrate the first-time purification of UndB and establish that it is an oxygen-dependent, non-heme diiron enzyme that engages conserved histidine residues at the active site. We also identify redox partners that support the activity of UndB and determine the enzyme's substrate specificity and kinetic properties. We detect CO2 as the co-product of the UndB-catalyzed reaction and provide the first evidence in favor of the hydrogen atom transfer (HAT) mechanism of the enzyme. Our findings decipher the biochemistry of an enigmatic metalloenzyme that catalyzes 1-alkene biosynthesis at the membrane interface with the highest known efficiency.
Phenoxy-1,2-dioxetane-based activatable chemiluminescent probes: tuning of photophysical properties for tracing enzymatic activities in living cells
(RSC, 2024-11) Sidhu, Jagpreet Singh; Taliyan, Rajeev
The use of chemiluminophores for tracing enzymatic activities in live-cell imaging has gained significant attention, making them valuable tools for diagnostic applications. Among various chemiluminophores, the phenoxy-1,2-dioxetane scaffold exhibits significant structural versatility and its activation is governed by the chemically initiated electron exchange luminescence (CIEEL) mechanism. This mechanism can be initiated by enzymatic activity, changes in pH, or other chemical stimuli. The photophysical properties of phenoxy-1,2-dioxetanes can be fine-tuned through the incorporation of different substituents on the phenolic ring and by anchoring them with specific triggers. This review discusses the variations in physicochemical properties, including emission maxima, quantum yield, aqueous solubility, and pKa, as influenced by structural modifications, thereby establishing a comprehensive structure–activity relationship. Furthermore, it categorises the probes based on different enzyme classes, such as hydrolase-sensitive probes, oxidoreductase-responsive probes, and transferase-activatable phenoxy-1,2-dioxetanes, offering a promising platform technology for the early diagnosis of diseases and disorders. The summary section highlights key opportunities and limitations associated with applying phenoxy-1,2-dioxetanes in achieving precise and effective enzyme assays.
The Photochemical Degradation of Bacterial Cell Wall Using Penicillin-Based Carbon Dots: Weapons Against Multi-Drug Resistant (MDR) Strains
(Wiley, 2017-10) Sidhu, Jagpreet Singh
Inability of antibiotics repertoire to effectively control the progress of multi-drug resistant (MDR) bacteria has prompted the substantial curiosity among the scientists to seek new tactics to combat the bacterial growth. Therefore, to eradicate the pathogenic bacteria with least cytotoxicity, we employed carbon dots as a broad spectrum of antibacterial weapons in the presence of visible light. Instead of using citric acid, we engaged the penicillin G as a carbon source for the synthesis of penicillin carbon dots (PCDs), which made the carbon dots more aggressive towards pathogenic microbes. Penicillin was also covalently attached to –NH2 containing citric acid based CDs (CDs-Penicillin) via an amide bond to evaluate whether penicillin in the form of PCD has retained its activity or in its conjugated form (CDs-Penicillin). Synthesized dots were assessed for their antibacterial activity against Staphylococcus aureus, Escherichia coli (DH5α), MDR Escherichia coli and Methicillin-resistant Staphylococcus aureus in the presence as well as the absence of visible light. The mechanism of bacteria-killing through cell wall rupturing was investigated using scanning electron microscopy. Antibacterial assay demonstrates that penicillin in the form of PCDs retained its activity and possess great prospects in the development of new bactericidal therapies to invade the MDR bacteria. Cytotoxicity of both PCDs and CDs-Penicillin has been evaluated by measuring the viability of human HeLa cells. Fluorescence images of bacteria collected using different excitation wavelength.
Recent advancements in parallel and tandem reaction-based fluorogenic probes for tracing enzymatic activities
(Elsevier, 2025-11) Sidhu, Jagpreet Singh
Optical imaging is a non-invasive and indispensable technique for tracing enzymatic activities at a molecular level. However, unintended activation or off-target accumulation of imaging probes often leads to false-positive signals. Moreover, the use of multiple fluorophores to detect distinct biomarkers is limited by variability in cellular uptake and subcellular localization within complex biological environments. To address these deficiencies, multi-locked fluorogenic probes have been developed to target multiple biomarkers simultaneously. These probes typically employ parallel or tandem-locked configuration, wherein enzyme-specific substrates quench the fluorescence of reporter motifs until activation occurs. Recent advancements in multi-locked probes have revealed many opportunities for advancing non-invasive diagnostic chemical tools. This review systematically compiles recent progress in the development of enzyme-responsive dually locked probes for bio-imaging applications. It provides a comprehensive analysis of their underlying mechanisms and design strategies. The review concludes with a summary of current achievements and future perspectives in this rapidly evolving field.
Structural Diversity of D-Alanine: D-Alanine Ligase and Its Exploration in Development of Antibacterial Agents Against the Multi-Variant Bacterial Infections
(Wiley, 2022-04) Sidhu, Jagpreet Singh
D-alanine: D-alanine ligase (Rv2981c or Ddl) (EC 6.3.2.4) is a bacterial protein that performs critical functions for the proper growth and development of bacterial cells. Understanding the activity profile of Ddl within the various strains of the bacteria seems vital in broad-spectrum antimicrobial drug discovery. Therefore, to understand this heterologous nature, we focused on understanding the functional impact of the structural differences in the Ddl protein from Legionella pneumophila and E. coli bacteria. The structural features and dynamic behavior of Ddl, the interaction pattern, and the docking score of the Ddl-ATP/ADP are also found significantly different from each other. In-depth analysis viz molecular dynamics simulation and residue interaction network (RIN) studies provided us the detailed insight into the differences in the Ddl proteins from both the bacteria. In conclusion, understanding the inter-specific differences in the antibiotic targets Ddl in the case of diverse bacterial strains is vital for rationalizing the treatment of these infectious diseases. Therefore, the current work attempts to foresee the development of more efficacious antibacterial agents devoid of emerging resistance to bacterium strains.
Study of Mechanical properties of Epoxy composites filled with Graphite & TiO2
(International Journal of Engineering Research and Applications, 2021-02) Sidhu, Jagpreet Singh
Epoxy Based Resin filled with Graphite particle & TiO2 particle were made by hand layup technique. Tensile strength, Flexural strength, Impact, hardness & density were determined as per ASTMD 256, ASTMD 792, and ASTMD 2240. Composite containing 3 wt % of graphite particle exhibits the optimum mechanical & wears performances. A further increase in the graphite content increases the specific wear rate & deteriorates the mechanical performance. The performance of composites can further be improved by adding TiO2 filler in polymer. The present work includes the processing & mechanical characterization of the composite. The experimental results showed that as TiO2 increases (upto 4 to 5 wt %) density, hardness, Tensile strength & Flexural strength also increases & Impact strength decreases after increase in TiO2. The systematic experimentation leads to determination of significant process parameters & material variables that predominantly influence the mechanical properties.
Synthesis of a 3,4-Disubstituted 1,8-Naphthalimide-Based DNA Intercalator for Direct Imaging of Legionella pneumophila
(ACS, 2019-03) Sidhu, Jagpreet Singh
The development of organic molecules to target nucleic acid is an active area of research at the interface of chemistry and biochemistry, which involves DNA binding, nuclear imaging, and antitumor studies. These molecules bind with DNA through covalent interactions, electrostatic interactions, or intercalation. However, they are less permeable to membrane, and they have a significant cytotoxicity, which limits their application under in vivo conditions. In the present work, various mono- and disubstituted 1,8-naphthalimides-based derivatives (S-12, S-13, S-15, and S-21) have been synthesized and characterized through various spectroscopic techniques. Among these, 3-amino-4-bromo-1,8-naphthalimide (S-15) was found to have an attractive water solubility and act as a nuclear imaging agent. The spectroscopic absorption and emission data showed that S-15 has a strong affinity for salmon sperm DNA with a binding constant of 6.61 × 104 M–1, and the ratiometric fluorescence intensity (I489/I552) of S-15 has a linear relationship in the 0–50 μM range of DNA concentrations. It intercalates with DNA through the hydrophobic planar naphthalimide core as confirmed through cyclic voltammetry, circular dichroism, 1H NMR titration, and thermal denaturation studies. Positively charged amine groups also participate in H-bonding with the bases and backbone of DNA. The S-15 intercalator showed a large Stokes shift and photostability, which made it attractive for direct imaging of Legionella pneumophila, without the need for a prior membrane permeabilization.
Synthesis of Nickel(II) Complexes of Novel Naphthalimide Based Heterodipodal Schiff Base Ligands, Structure, Characterization and Application for Degradation of Pesticides
(Wiley, 2020-06) Sidhu, Jagpreet Singh
To degrade the highly toxic pesticide into less harmful components, we have synthesized four nickel complexes of naphthalimide based organic ligands. These complexes catalyze the hydrolysis of phosphorothioate bonds of organophosphates in an aqueous medium. 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 rate of degradation of chlorpyriphos and parathion methyl was evaluated using 31P NMR and LC-MS chromatogram. The by-product of chlorpyriphos upon catalytic degradation with complex was confirmed from mass spectrometry. It was found that chlorpyriphos degrade into 3,5,6-trichloropyridin-2-ol after 50 minutes of incubation with catalyst. However, parathion methyl took only 20 minutes to hydrolyze into its by-product. Moreover, the inhibition assay of acetylcholinesterase was performed for pesticides in the presence of metal complex and the interesting outcome was recorded.
Trends in small organic fluorescent scaffolds for detection of oxidoreductase
(Elsevier, 2021-11) Sidhu, Jagpreet Singh
Oxidoreductases are diverse class of enzymes engaged in modulating the redox homeostasis and cellular signaling cascades. Abnormal expression of oxidoreductases including thioredoxin reductase, azoreductase, cytochrome oxidoreductase, tyrosinase and monoamine oxidase leads to the initiation of numerous disorders. Thus, enzymes are the promising biomarkers of the diseased cells and their accurate detection has utmost significance for clinical diagnosis. The detection method must be extremely selective, sensitive easy to use, long self-life, mass manufacturable and disposable. Fluorescence assay approach has been developed potential substitute to conventional techniques used in enzyme's quantification. The fluorescent probes possess excellent stability, high spatiotemporal ratio and reproducibility represent applications in real sample analysis. Therefore, the enzymatic transformations have been monitored by small activatable organic fluorescent probes. These probes are generally integrated with enzyme's substrate/inhibitors to improve their binding affinity toward the enzyme's catalytic site. As the recognition unit bio catalyzed, the signaling unit produces the readout signals and provides novel insights to understand the biochemical reactions for diagnosis and development of point of care devices. Several structural modifications are required in fluorogenic scaffolds to tune the selectivity for a particular enzyme. Hence, the fluorescent probes with their structural features and enzymatic reaction mechanism of oxidoreductase are the key points discussed in this review. The basic strategies to detect each enzyme are discussed. The selectivity, sensitivity and real-time applications are critically compared. The kinetic parameters and futuristic opportunities are present, which would be enormous benefits for chemists and biologists to understand the facts to design and develop unique fluorophore molecules for clinical applications.