Subodh Kumar

Serum albumins, such as bovine serum albumin (BSA) and human serum albumin (HSA), play a crucial role in various biological processes. Discrimination between BSA and HSA is challenging due to their similar structures and reactivity. Here, we report mitochondria-targeted red fluorescent tripodal nanoprobe DMAS-TP, forming spherical nano-aggregates (∼90 nm) in water for discrimination of BSA over HSA and detection and imaging of viscosity in HeLa cells. DMAS-TP exhibits an ∼56-fold fluorescence intensity increase in 95% glycerol compared to water, which indicates restricted movement in high-viscosity solvents. Furthermore, the addition of 5 equiv. BSA and HSA to DMAS-TP solution displays ∼50-fold and ∼10-fold fluorescence intensity increases at 630 nm (λ_ex 490 nm), respectively, and can detect as low as 20 nM BSA and 140 nM HSA. The fluorescence anisotropy plot shows that the DMAS-TP anisotropy values decrease from 0.234 to 0.185 with increasing [BSA/HSA], suggesting a conformational change from Y- to ω-like structure. The fluorescence lifetime of DMAS-TP increases gradually with BSA/HSA, suggesting dynamic complexation. DMAS-TP aggregates diminish to 6 nm particles when encapsulated in BSA confirmed by DLS, SEM, and TEM, with reduced fluorescence intensity in the presence of bilirubin, indicating that DMAS-TP binds within the BSA cavity near site IB. DMAS-TP is highly permeable to HeLa cells and shows a strong affinity for mitochondria, making it suitable for imaging viscosity and BSA via strong fluorescence in the red channel.

Exploitation of billirubin drug binding cavity of HSA for the endogeneous and exogeneous detection of HSO₃⁻ in HeLa cell mitochondria and detection of HSO₃⁻ in root using CTAB.

Nitroreductases are the enzymes that can convert nitroaromatic substances including explosives like TNT to their corresponding nitrite, hydroxylamine or amino derivatives with reduced NADH or NADPH as an electron donor. A wide range of nitroreductases are known to present in bacterial genomes that are explored for their potential in the reduction of nitroaromatic compounds and they are used in converting prodrugs to toxic hydroxylamine derivatives causing tumor cell killing. Likewise, in cancer cells, the low oxygen condition called hypoxia is a characteristic of solid tumors and during hypoxia nitroreductase gene expression occurs. Moreover, due to the ability of nitroreductases to reduce nitroaromatic compounds, phytoremediation potential of plants was inspected by expressing bacterial nitroreductase genes in plants through genetic engineering. Due to its diversity and widespread applications in different biosystems, its detection has gained momentum among the scientific community. Designing fluorescent probes for detecting nitroreductases has emerged as a beneficial tool compared to conventional monitoring approaches due to their high sensitivity, selectivity, and real‐time monitoring ability. This review aims to provide an understanding of the activity and the status of nitroreductases in bacteria, plants and animal cells as well as the significance of fluorescent imaging‐related studies by employing recently designed probes.

Spermine is a vital biomarker for clinical diagnosis of cancer and estimating food spoilage. Here, supramolecular assemblies of two donor–π–acceptor dipods R-SPM (λ_em 640 nm) and NIR-SPM (λ_em 720 nm) with SDS have been discovered for the detection of spermine and spermidine under physiological conditions at nanomolar levels. The addition of SDS to R-SPM and NIR-SPM results in the formation of self-assemblies (DLS, zeta-potential and UV-vis studies) with no significant change in their fluorescence but further addition of spermine/spermidine to the R-SPM∩SDS and NIR-SPM∩SDS assemblies results in a 30–80 fold increase in fluorescence intensity, respectively at 640 nm and 720 nm. The LOD for spermine and spermidine detection is 22 nM (4.4 ppb) and 67 nM (9.7 ppb). The ensembles show nominal interference from other biogenic amines, amino acids, metal ions, and anions. Both R-SPM∩SDS and NIR-SPM∩SDS ensembles can be stored in the dark for >3 months without affecting their performance. The potential of these ensembles for real world applications like analysis of spermine in urine, human serum and food spoilage in the case of cheese, mushrooms, chicken and mutton has been demonstrated. The smartphone relied RGB analysis facilitates the on-site determination of spermine in food samples.

A stable radical anion with t_1/2 = 96 minutes in 50% water is characterized using EPR, CV, I–V, optical, SEM and AFM techniques, and developed as an assay for the rapid detection of 26.6 pM H₂O₂.

The displacement of bilirubin in human serum with THYQ provides fluorescence “turn on” detection for bilirubin, which is in good agreement with the clinical results.

The limitations of prevailing probes for the detection of human serum albumin (HSA) and HSO₃ ^- make it challenging to apprehend the cooperative effect of both HSA and HSO₃ ^- in biological systems. Herein, we present a multi-responsive fluorescent probe MGTP, which distinguishes HSA from bovine serum albumin (BSA) through an ∼104-fold fluorescence enhancement at an emission maximum of 595 nm with HSA and only an ∼10-fold increase at an emission maximum of 615 nm with a shoulder at 680 nm with BSA. The absorbance spectrum of MGTP also discriminates HSA and BSA with the respective absorption maxima at 543 nm and at 580 nm. MGTP in the confined space of HSA or BSA undergoes instantaneous conjugate addition of HSO₃ ^- and results in a ratiometric change in fluorescence intensity with diminishing of red fluorescence (600 nm) and emergence of green fluorescence (515 nm). MGTP in the absence of SAs does not react with HSO₃ ^- in phosphate-buffered saline buffer and reacts sluggishly in the dimethyl sulfoxide-water 1:1 mixture. The limit of detection values for the detection of HSA and HSO₃ ^- are 4 and 6.88 nM, respectively. The drug binding studies reveal that MGTP preferably confines itself at the bilirubin site of HSA. In MCF-7 cancer cells, MGTP is localized into mitochondria and reveals both exogenous and endogenous visualization of HSO₃ ^- through a change in fluorescence from the red to green channel.

The present study provides an insight into the difference in binding of monopodal (MPMB) and dipodal (DPDM) fluorescent probes in the cavity of serum albumins (SAs). The aggregates of DPDM show nearly 4-fold preference towards BSA (66-fold enhancement) over HSA (17-fold enhancement) in fluorescence intensity but aggregates of MPMB exhibit a similar response towards BSA, HSA and RNA. DPDM and MPMB do not show fluorescence responses to other proteins and biomolecules. The mechanism of interaction of these probes with SAs has been investigated through drug binding, fluorescence lifetime, fluorescence anisotropy, FT-IR, DLS, circular dichroism and docking studies. The docking and drug binding studies in consonance with each other show that MPMB preferably binds at the bilirubin site (subdomain IB) of BSA but DPDM undergoes a Z- to U-like conformation change during binding in the cavity of BSA and binds near to both the bilirubin and ibuprofen sites. The increase in lifetime of both DPDM and MPMB in the presence of BSA/HSA points to a dynamic mechanism of interaction. Probe DPDM detects as low as 14 nM BSA and 141 nM HSA but in the case of MPMB, the detection limits are 90 nM BSA and 30 nM HSA. The decrease in size of aggregates of DPDM from 34 nm to 6 nm in the presence of BSA (DLS) confirms the complexation of DPDM in its cavity.

Onosma bracteata Wall. is an important medicinal and immunity-enhancing herbs. This plant is commonly used in the preparation of traditional Ayurvedic drugs to treat numerous diseases. Inspired by the medicinal properties of this plant, the present study aimed to investigate the antiproliferative potential and the primary molecular mechanisms of the apoptotic induction against human osteosarcoma (MG-63) cells. Among all the fractions isolated from O. bracteata, ethyl acetate fraction (Obea) showed good antioxidant activity in superoxide radical scavenging assay and lipid peroxidation assay with an EC₅₀ value of 95.12 and 80.67 µg/mL, respectively. Silica gel column chromatography of ethyl acetate (Obea) fraction of O. bracteata yielded a pure compound, which was characterized by NMR, FTIR, and HR-MS analysis and was identified as 1,2-benzene dicarboxylic acid, bis (2-methyl propyl) ester (BDCe fraction). BDCe fraction was evaluated for the antiproliferative potential against human osteosarcoma MG-63, human neuroblastoma IMR-32, and human lung carcinoma A549 cell lines by MTT assay and exhibited GI₅₀ values of 37.53 μM, 56.05 μM, and 47.12 μM, respectively. In MG-63 cells, the BDCe fraction increased the level of ROS and simultaneously decreased the mitochondria membrane potential (MMP) potential by arresting cells at the G₀/G₁ phase, suggesting the initiation of apoptosis. Western blotting analysis revealed the upregulation of p53, caspase3, and caspase9 while the expressions of p-NF-κB, p-Akt and Bcl-xl were decreased. RT-qPCR studies also showed upregulation in the expression of p53 and caspase3 and downregulation in the expression of CDK2, Bcl-2 and Cyclin E genes. Molecular docking analysis displayed the interaction between BDCe fraction with p53 (−151.13 kcal/mol) and CDK1 (−133.96 kcal/mol). The results of the present work suggest that the BDCe fraction has chemopreventive properties against osteosarcoma (MG-63) cells through the induction of cell cycle arrest and apoptosis via Akt/NF-κB/p53 pathways. This study contributes to the understanding of the utilization of BDCe fraction in osteosarcoma treatment.