BITS Faculty Publications
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Item Recent advancements in parallel and tandem reaction-based fluorogenic probes for tracing enzymatic activities(Elsevier, 2025-11) Sidhu, Jagpreet SinghOptical 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.Item Acetylcholine structure-based small activatable fluorogenic probe for specific detection of acetylcholinesterase(ACS, 2023-05) Sidhu, Jagpreet SinghEarly 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.Item Unraveling the Conversion of Fatty Acids into Terminal Alkenes by an Integral Membrane Enzyme, UndB(ACS, 2023-11) Sidhu, Jagpreet SinghBiosynthetically produced 1-alkenes hold immense value as green alternatives to fossil fuels and find widespread applications in the polymer, lubricant, and detergent industries. UndB is the only known membrane enzyme capable of converting fatty acids into 1-alkenes with unprecedented in vivo titers. However, despite diverse applications, UndB has remained poorly understood since its discovery nearly a decade ago. We present here insights into the molecular basis of UndB catalysis and the mechanism of the UndB reaction at the membrane interface. We unravel UndB as a diiron-enzyme that utilizes a conserved histidine cluster at the active site. We decipher the dependency of UndB activity on molecular oxygen and electrons and identify the most efficient redox partners of UndB. We elucidate the catalytic intricacies of UndB and establish it as the most efficient decarboxylase in producing industrially valuable medium-chain 1-alkenes. We further identify CO2 as the C1-derived coproduct of the UndB-catalyzed reaction and provide compelling evidence supporting the hydrogen atom transfer mechanism of UndB. These results now place our understanding of UndB on a molecular level, paving the way for its application for sustainable 1-alkene production.Item 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, RajeevThe 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.Item Study of Mechanical properties of Epoxy composites filled with Graphite & TiO2(International Journal of Engineering Research and Applications, 2021-02) Sidhu, Jagpreet SinghEpoxy 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.Item Acetylcholine Structure-Based Small Activatable Fluorogenic Probe for Specific Detection of Acetylcholinesterase(ACS, 2023-05) Sidhu, Jagpreet SinghEarly 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.Item Indole Derivatives as Anticancer Agents for Breast Cancer Therapy: A Review(Bentham Science, 2016) Sidhu, Jagpreet SinghBreast 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.Item Carbon Dot Based, Naphthalimide Coupled FRET Pair for Highly Selective Ratiometric Detection of Thioredoxin Reductase and Cancer Screening(ACS, 2017-07) Sidhu, Jagpreet SinghThe 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.Item Carbon dots as analytical tools for sensing of thioredoxin reductase and screening of cancer cells(RSC, 2018-02) Sidhu, Jagpreet SinghThioredoxin 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.Item FRET and PET paired dual mechanistic carbon dots approach for tyrosinase sensing(RSC, 2018-05) Sidhu, Jagpreet SinghA 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.
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