Synthesis and characterization of a nano-formulation for long lasting sterilization effect.

The current COVID-19 pandemic has increased the use of alcohol based hand sanitisers globally. These available alcohol based sanitisers cannot provide an antibacterial effect for an extended period of time, after the evaporation of ethanol. Hence, the need for a sanitiser with an anti-microbial activity combined with a long lasting effect is the need of the hour. In this study, we report the synthesis of a long lasting sanitiser from ozonated omega 9 fatty acid esters in an ethanolic medium. The formed vesicles made of the fatty acids have been characterized by DLS, Zeta potential, and time resolved fluorescence anisotropy studies. Ethanol although, provides an antibacterial effect, the effect is more pronounced in our prepared formulation owing to its high peroxide value that generates additional oxidative stress. Finally, this additional antimicrobial effect will have relevance in the current COVID-19 scenario in providing a long lasting hand sanitiser.

A portable spectroscopic instrument for multiplexed monitoring of acute water toxicity: Design, testing, and evaluation.

The deteriorating water environment worldwide, mainly due to population explosion and uncontrolled direct disposal of harmful industrial and farming wastes, earnestly demands new approaches and accurate technologies to monitor water quality before consumption overcoming the shortcomings of the current methodologies. A spectroscopic water quality monitoring and early-warning instrument for evaluating acute water toxicity are the need of the hour. In this study, we have developed a prototype capable of the quantification of dissolved organic matter, dissolved chemicals, and suspended particulate matter in trace amounts dissolved in the water. The prototype estimates the water quality of the samples by measuring the absorbance, fluorescence, and scattering of the impurities simultaneously. The performance of the instrument was evaluated by detecting common water pollutants such as Benzopyrene, Crystal Violet, and Titanium di-oxide. The limit of detection values was found to be 0.50, 23.9, and 23.2 ppb (0.29 µM), respectively.

An evidence of pores in phospholipid membrane induced by an antimicrobial peptide NK-2.

NK-2, a peptide derived from a cationic core region of NK-lysin, has emerged as a promising candidate for new antibiotics. In contrast to classical antibiotics, antimicrobial peptides target bacterial membranes and disintegrate the membrane by forming the transmembrane pores. However, complete understanding of the precise mechanisms of cellular apoptosis and molecular basis of membrane selectivity is still in dispute. In the present study, we have shown that NK-2 forms trans-membrane pores on negatively charged phospholipid membranes using phase contrast microscopy. As bacteria mimicking membranes, we have chosen large unilamellar vesicles (LUV) and giant unilamellar vesicles (GUV) composed of negatively charged phospholipid, dioleoyl phosphatidyl glycerol (DOPG) and neutral phospholipid, dioleoyl phophatidylcholine (DOPC). Leakage of internal fluid of giant unilamellar vesicles (GUV), leading to decrease in intensity in the halo region of phase contrast micrographs, suggests the formation of transmembrane pores. No such reduction of intensity in the halo region of DOPC was observed, indicating, neutral vesicles does not exhibit pores. Rate constant reckoned from the decaying intensity in the halo region was found to be 0.007 s-1. Further, significant interaction of NK-2 with anionic membranes has been envisaged from zeta potential and dynamic light scattering. Binding free energy and other interaction parameters have been delineated using theoretical ansatz. A proliferation of average Size of anionic LUV on increasing NK-2 concentration indicates membrane-membrane interaction leading to peptide induced large aggregates of vesicles.

The metal cofactor zinc and interacting membranes modulate SOD1 conformation-aggregation landscape in an in vitro ALS model.

Aggregation of Cu-Zn superoxide dismutase (SOD1) is implicated in the motor neuron disease, amyotrophic lateral sclerosis (ALS). Although more than 140 disease mutations of SOD1 are available, their stability or aggregation behaviors in membrane environment are not correlated with disease pathophysiology. Here, we use multiple mutational variants of SOD1 to show that the absence of Zn, and not Cu, significantly impacts membrane attachment of SOD1 through two loop regions facilitating aggregation driven by lipid-induced conformational changes. These loop regions influence both the primary (through Cu intake) and the gain of function (through aggregation) of SOD1 presumably through a shared conformational landscape. Combining experimental and theoretical frameworks using representative ALS disease mutants, we develop a 'co-factor derived membrane association model' wherein mutational stress closer to the Zn (but not to the Cu) pocket is responsible for membrane association-mediated toxic aggregation and survival time scale after ALS diagnosis.

Amyotrophic lateral sclerosis, or ALS, is an incurable neurodegenerative disease in which a person slowly loses specialized nerve cells that control voluntary movement. It is not fully understood what causes this fatal disease. However, it is suspected that clumps, or aggregates, of a protein called SOD1 in nerve cells may play a crucial role. More than 140 mutations in the gene for SOD1 have been linked to ALS, with varying degrees of severity. But it is still unclear how these mutations cause SOD1 aggregation or how different mutations influence the survival rate of the disease. The protein SOD1 contains a copper ion and a zinc ion, and it is possible that mutations that affect how these two ions bind to SOD1 influences the severity of the disease. To investigate this, Sannigrahi, Chowdhury, Das et al. genetically engineered mutants of the SOD1 protein which each contain only one metal ion. Experiments on these mutated proteins showed that the copper ion is responsible for the protein's role in neutralizing harmful reactive molecules, while the zinc ion stabilizes the protein against aggregation. Sannigrahi et al. found that when the zinc ion was removed, the SOD1 protein attached to a structure inside the cell called the mitochondria and formed toxic aggregates. Sannigrahi et al. then used these observations to build a computational model that incorporated different mutations that have been previously associated with ALS. The model suggests that mutations close to the site where zinc binds to the SOD1 protein increase disease severity and shorten survival time after diagnosis. This model was then experimentally validated using two disease variants of ALS that have mutations close to the sites where zinc or copper binds. These findings still need to be tested in animals and humans to see if these mechanisms hold true in a multicellular organism. This discovery could help design new ALS treatments that target the zinc binding site on SOD1 or disrupt the protein's interactions with the mitochondria.

An Energy-Resolved Optical Non-invasive Device Detects Essential Electrolyte Balance in Humans at Point-of-Care.

Regular monitoring of electrolyte balance is essential for patients suffering from chronic kidney disease (CKD), particularly those undergoing dialysis. In the context of the recent COVID-19 pandemic, more severe forms of infection are observed in elderly individuals and patients having co-morbidities like CKD. The repeated blood tests for the monitoring of electrolyte balance predispose them not only to COVID-19 but also other to hospital-acquired infections (HAI). Therefore, a non-invasive method for easy detection of essential electrolyte (K+ and Na+) levels is urgently needed. In this study, we developed an optical emission spectroscopy-based non-invasive device for simultaneous monitoring of salivary Na+ and K+ levels in a fast and reliable way. The device consisted of a closed spark chamber, micro-spectrometer, high voltage spark generator, electronic circuits, optical fiber, and an indigenously developed software based on the LabVIEW platform. The optical emission originating from the biological sample (i.e., saliva) due to recombination of ions energized by impingement of electrons returning from high voltage spark provides necessary information about the concentration of electrolytes. A small-scale clinical trial on 30 healthy human subjects shows the potential of the indigenously developed device in determining salivary Na + and K+ concentration. The low-cost, portable, point-of-care device requires only 2 mL of sample, and can simultaneously measure 1.0-190.0 mM Na+, and 1.0-270.9 mM K+ . To our understanding, the present work will find its relevance in combating COVID-19 morbidities, along with regular CKD patient-care.

Spectroscopic Studies on the Biomolecular Recognition of Toluidine Blue: Key Information Towards Development of a Non-Contact, Non-Invasive Device for Oral Cancer Detection.

Molecular interaction of aromatic dyes with biological macromolecules are important for the development of minimally invasive disease diagnostic biotechnologies. In the present work, we have used Toluidine Blue (TB) as a model dye, which is a well-known staining agent for the diagnosis of oral cancer and have studied the interaction of various biological macromolecules (protein and DNA) with the dye at different pH. Our spectroscopic studies confirm that TB interacts with Human Serum Albumin (HSA), a model protein at very high pH conditions which is very hard to achieve physiologically. On the other hand, TB significantly interacts with the DNA at physiological pH value (7.4). Our molecular studies strengthen the understanding of the Toluidine Blue staining of cancer cells, where the relative ratio of the nucleic acids is higher than the normal intracellular content. We have also developed a non-invasive, non-contact spectroscopic technique to explore the possibility of quantitatively detecting oral cancer by exploiting the interaction of TB with DNA. We have also reported development of a prototype named "Oral-O-Scope" for the detection of Oral cancer and have carried out human studies using the prototype.

Large scale validation of a new non-invasive and non-contact bilirubinometer in neonates with risk factors.

The study was aimed to evaluate the performance of a newly developed non-invasive and non-contact bilirubin measurement device (AJO-Neo) as an alternative to the conventional invasive biochemical method of total serum bilirubin (TSB) estimation in preterm and term neonates suffering from hyperbilirubinemia associated with risk factors, and/or undergoing phototherapy. The safety and efficacy of the device were assessed in 1968 neonates with gestational ages ranging from 28 to 41 weeks and suffering from incidences of hyperbilirubinemia. Linear regression analysis showed a good correlation between AJO-Neo and the conventional method of TSB (Pearson's coefficient, r = 0.79). The small bias (0.27 mg/dL) and limits of agreements (- 3.44 to 3.99 mg/dL) were within the range of clinical acceptance. The device was also precise in the measurement of bilirubin levels in all subgroups of the study. The receiver operator curve (ROC), that takes account of both sensitivity and specificity of a device showed high efficacy of the device (area under the curve, AUC = 0.83) in the detection of bilirubin. While monitoring the bilirubin level during phototherapy, the device indicated promising results showing good agreement with TSB. Specificities and sensitivities of the device indicated a much higher accuracy in neonates with associated risk factors for hyperbilirubinemia. Hence, the newly developed device (AJO-Neo) is reliable in measuring bilirubin level in preterm, and term neonates irrespective of gestational or postnatal age, sex, risk factors, feeding behavior or skin color.

Kinetoplastid Membrane Protein-11 Induces Pores in Anionic Phospholipid Membranes: Effect of Cholesterol.

Kinetoplastid membrane protein-11 (KMP-11), expressed in all stages of leishmanial life cycle, is considered a potential candidate for leishmaniasis vaccine. KMP-11 is found on the membrane surface of the parasite. Although the biological function of KMP-11 is unknown, we hypothesize from its sequence analysis that it may interact with the macrophage membrane and may influence the entry process of the parasite into the host cell. To validate this hypothesis, we have investigated the interaction of KMP-11 with unilamellar anionic phospholipid vesicles and explored its pore-forming activity. The decrease in negative ζ-potential of the vesicles and reduction in the fluorescence intensity of membrane-bound dye DiI C-18 suggest a strong association of KMP-11 with the membrane. The fluorescence leakage experiment as well as phase contrast microscopy shows direct evidence of KMP-11-induced pore formation in an anionic membrane. Incorporation of cholesterol into the membrane has been found to inhibit pore formation induced by KMP-11, suggesting an important role of cholesterol in leishmaniasis. Interestingly, vesicles containing only neutral phospholipid do not exhibit any tendency toward pore formation.

Nanoparticle-based 'turn-on' scattering and post-sample fluorescence for ultrasensitive detection of water pollution in wider window.

Ultrasensitive detection of heavy metal ions in available water around us is a great challenge for scientists since long time. We developed an optical technique that combines Rayleigh scattering of UV light (365 nm) and post-sample fluorescence detection from colloidal silver (Ag) nanoparticles (NPs) having a surface plasmon resonance (SPR) band at 420 nm. The efficacy of the technique is tested by the detection of several model toxic ions, including mercury, lead, and methylmercury in aqueous media. The light scattering from the Hg-included/inflated Ag NPs at 395 nm was observed to saturate the light sensor even with ppm-order concentrations of Hg ions in the water sample. However, the pollutant is not detected at lower concentrations at this wavelength. Instead, the fluorescence of a high-pass filter (cut-off at 400 nm) at 520 nm is applied to detect pollutant concentrations of up to several hundreds of ppm in the water sample. We also detected lead and methylmercury as model pollutants in aqueous media and validated the efficacy of our strategy. Finally, we report the development of a working prototype based on the strategy developed for efficient detection of pollutants in drinking/agricultural water.

Wide bandgap semiconductor-based novel nanohybrid for potential antibacterial activity: ultrafast spectroscopy and computational studies.

The properties of nanomaterials generated by external stimuli are considered an innovative and promising replacement for the annihilation of bacterial infectious diseases. The present study demonstrates the possibility of getting the antibiotic-like drug action from our newly synthesized nanohybrid (NH), which consists of norfloxacin (NF) as the photosensitive material covalently attached to the ZnO nanoparticle (NP). The synthesized NH has been characterized using various microscopic and spectroscopic techniques. Steady state fluorescence and time-correlated single photon counting (TCSPC)-based spectroscopic studies demonstrate the efficient electron transfer from NF to ZnO. This enhances the reactive oxygen species (ROS) production capability of the system. First principles density functional theory has been calculated to gain insight into the charge separation mechanism. To explore the electron densities of the occupied and unoccupied levels of NH, we have verified the nature of the electronic structure. It is observed that there is a very high possibility of electron transfer from NF to ZnO in the NH system, which validates the experimental findings. Finally, the efficacy of NH compared to NF and ZnO has been estimated on the in vitro culture of E. coli bacteria. We have obtained a significant reduction in the bacterial viability by NH with respect to control in the presence of light. These results suggest that the synthesized NH could be a potential candidate in the new generation alternative antibacterial drugs. Overall, the study depicts a detailed physical insight for nanohybrid systems that can be beneficial for manifold application purposes.

Spectroscopy of an intrinsic fluorophore in animal and plant milk for potential identification of their quality.

Riboflavin (RF), commonly known as vitamin B2, is an essential ingredient in any milk variety of animal origin. The photophysics of the molecule RF, including its interaction with biological macromolecules, are well studied. Here, we have investigated the possibility of the molecule as a potential biomarker of milk quality. We also found omnipresence of this molecule in milk of plant origin (soy milk). Spectroscopic studies on various animal and plant milks of different commercial origins confirmed the potential of RF for use in identifying the quality of the milk varieties. Our developed strategy involved identification or spectroscopic signature of RF by measuring optical density at 365 nm (quality factor 1) and fluorescence intensity around 520 nm (excitation at 365 nm; quality factor 2) on a very small amount of whole milk (10 µL). We also developed a prototype device called Mil-Q-Way to be used in the real field. The required interfacing software in the LabView platform was also developed. A 2-parameter plot (quality factor 1 on the x-axis and quality factor 2 on the y-axis) called the Mil-Q-Way plot clearly differentiates the quality of milks of different commercial origins. The low-cost device based on simple spectroscopy was shown to screen for the presence of harmful adulterants in drinkable milk.

Conformational Switch Driven Membrane Pore Formation by Mycobacterium Secretory Protein MPT63 Induces Macrophage Cell Death.

Virulent Mycobacterium tuberculosis (MTB) strains cause cell death of macrophages (Mϕ) inside TB granuloma using a mechanism which is not well understood. Many bacterial systems utilize toxins to induce host cell damage, which occurs along with immune evasion. These toxins often use chameleon sequences to generate an environment-sensitive conformational switch, facilitating the process of infection. The presence of toxins is not yet known for MTB. Here, we show that MTB-secreted immunogenic MPT63 protein undergoes a switch from ß-sheet to helix in response to mutational and environmental stresses. MPT63 in its helical form creates pores in both synthetic and Mϕ membranes, while the native ß-sheet protein remains inert toward membrane interactions. Using fluorescence correlation spectroscopy and atomic force microscopy, we show further that the helical form undergoes self-association to produce toxic oligomers of different morphology. Trypan blue and flow cytometry analyses reveal that the helical state can be utilized by MTB for killing Mϕ cells. Collectively, our study emphasizes for the first time a toxin-like behavior of MPT63 induced by an environment-dependent conformational switch, resulting in membrane pore formation by toxic oligomers and Mϕ cell death.

A Novel Whole Spectrum-Based Non-Invasive Screening Device for Neonatal Hyperbilirubinemia.

OBJECTIVE: Careful screening of bilirubin level in newborns is mandatory as per American Academy of Pediatrics (2004), to reduce incidents of kernicterus and acute bilirubin encephalopathy. Although, invasive capillary collection of blood and subsequent biochemical test is considered a gold standard for jaundice detection in neonates, transcutaneous bilirubin measurement using various non-invasive instruments is also used sporadically across the globe. The major aim of this study was to develop a non-invasive spectrometry-based technique for measurement of neonatal bilirubin level as an alternative of total serum bilirubin (TSB) test without limitations of other available bilirubinometers. METHODS: The instrument comprises of a light source and a spectroscopic detector. A light beam from source incident on the neonatal nail plate through optical fibers. The retro reflected light is acquired using the detector. An indigenously developed software is used to acquire and analyze the optical signal and to calculate the bilirubin value. The instrument was calibrated and validated in reference to TSB on 1033 subjects. MAJOR RESULTS: The result (r = 0.95 and P < 0.001) indicates a strong correlation between the bilirubin value obtained from our method and TSB. Time variant analysis of the subjects undergoing phototherapy provided a good correlation (r = 0.98). The repeatability test result shows the mean coefficient of variation is less than 5.0%. CONCLUSIONS: The indigenously developed non-invasive technique successfully detects the bilirubin level in newborns under various physiological conditions with high accuracy and precision.

Lipid chain saturation and the cholesterol in the phospholipid membrane affect the spectroscopic properties of lipophilic dye nile red.

We have studied the effect of composition and the phase state of phospholipid membranes on the emission spectrum, anisotropy and lifetime of a lipophilic fluorescence probe nile red. Fluorescence spectrum of nile red in membranes containing cholesterol has also been investigated in order to get insights into the influence of cholesterol on the phospholipid membranes. Maximum emission wavelength (λem) of nile red in the fluid phase of saturated and unsaturated phospholipids was found to differ by ~10nm. The λem was also found to be independent of chain length and charge of the membrane. However, the λem is strongly dependent on the temperature in the gel phase. The λem and rotational diffusion rate decrease, whereas the anisotropy and lifetime increase markedly with increasing cholesterol concentration for saturated phosoholipids, such as, dimyristoyl phosphatidylcholine (DMPC) in the liquid ordered phase. However, these spectroscopic properties do not alter significantly in case of unsaturated phospholipids, such as, dioleoyl phosphatidylcholine (DOPC) in liquid disordered phase. Interestingly, red edge excitation shift (REES) in the presence of lipid-cholesterol membranes is the direct consequences of change in rotational diffusion due to motional restriction of lipids in the presence of cholesterol. This study provides correlations between the membrane compositions and fluorescence spectral features which can be utilized in a wide range of biophysical fields as well the cell biology.

Development and validation of a noncontact spectroscopic device for hemoglobin estimation at point-of-care.

Anemia severely and adversely affects human health and socioeconomic development. Measuring hemoglobin with the minimal involvement of human and financial resources has always been challenging. We describe a translational spectroscopic technique for noncontact hemoglobin measurement at low-resource point-of-care settings in human subjects, independent of their skin color, age, and sex, by measuring the optical spectrum of the blood flowing in the vascular bed of the bulbar conjunctiva. We developed software on the LabVIEW platform for automatic data acquisition and interpretation by nonexperts. The device is calibrated by comparing the differential absorbance of light of wavelength 576 and 600 nm with the clinical hemoglobin level of the subject. Our proposed method is consistent with the results obtained using the current gold standard, the automated hematology analyzer. The proposed noncontact optical device for hemoglobin estimation is highly efficient, inexpensive, feasible, and extremely useful in low-resource point-of-care settings. The device output correlates with the different degrees of anemia with absolute and trending accuracy similar to those of widely used invasive methods. Moreover, the device can instantaneously transmit the generated report to a medical expert through e-mail, text messaging, or mobile apps.

Charge-Driven Interaction of Antimicrobial Peptide NK-2 with Phospholipid Membranes.

NK-2, derived from a cationic core region of NK-lysin, displays antimicrobial activity toward negatively charged bacterial membranes. We have studied the interaction of NK-2 with various phospholipid membranes, using a variety of experimental techniques, such as, isothermal titration calorimetry (ITC), ζ potential, and dynamic light scattering. As bacteria mimicking membranes, we have chosen large unilamellar vesicles (LUVs) composed of negatively charged phospholipid and neutral phospholipids. ITC and ζ potential results show the stronger binding affinity of NK-2 to negatively charged membranes than to neutral membranes. Saturation of the isotherm, obtained from ITC, at a given lipid to NK-2 ratio, was found to be consistent with the charge compensation, determined from ζ potential. A surface partition model with electrostatic contribution was used to estimate the intrinsic binding constant and other thermodynamical parameters of binding kinetics of NK-2. The size distribution of negatively charged LUV in the presence of NK-2 was found to increase drastically, indicating the presence of large aggregates. Such a large aggregate has not been observed in neutral membranes, which supports the ITC and ζ potential results.