Rapid, instrument-free colorimetric quantification of DNA using Nile Blue

The use of nucleic acid tests (NAT) for sensitive and rapid detection of pathogens relevant to human health has increased due to the ubiquity of nucleic acid amplification techniques such...

Polyhydroxyalkanoate Recovery From Newly Screened Bacillus Sp. LPPI-81 Using Various Methods of Extraction From Loktak Lake Sediment Sample

Nowadays the conventional plastic wastes are very challenging to environments and its production cost also creates an economic crisis due to petrochemical-based plastic. In order to solve this problem, the current studies were aimed at screening and characterizing these PHA producing isolates and evaluating the suitability of some carbon source for newly screened PHA producing isolates. Some carbon sources such as D-fructose, glucose, molasses, D-ribose and sucrose were evaluated for PHA production. Data were analyzed using SPSS version 20. The 16SrRNA gene sequence of these isolates was performed. This newly isolated taxa was related to Bacillus species. It was designed as Bacillus sp. LPPI-18 and affiliated Bacillus cereus ATCC 14577T (AE01687) (99.10%). Paenibacillus sp. 172 (AF273740.1) was used as an out-group. Bacillus sp. LPPI-18 is a gram-positive, rod-shaped, endospore former, and citrate test positive. This isolate showed positive for amylase, catalase, pectinase, and protease test. They produced intracellular PHA granules when this isolate was stained with Sudan Black B (SBB) and Nile Blue A (NBA) preliminary and specific staining dyes, respectively. Both Temperature and pH used to affect PHA productivity. Bacteria are able to reserve PHA in the form of granules during stress conditions. This isolate produces only when supplied with carbon sources. More PHA contents (PCs) were obtained from glucose, molasses, and D-fructose. In this regard, the maximum mean value of PC was obtained from glucose (40.55±0.7%) and the minimum was obtained from D-Ribose (12.4±1.4%). Great variations (p≤0.05) of PCs were observed among glucose & sucrose, molasses & sucrose and D-fructose & sucrose carbon sources for PHA productivity (PP) of Cell Dry Weight (CDW) g/L. After extraction, PHA film was produced for this typical isolate using glucose as a sole carbon source. Fourier transform infrared spectrum was performed for this isolate and showed the feature of polyester at 1719.64 to 1721.16 wavelength for these extracted samples. The peak of fingerprinting (band of carboxylic acid group) at this wave-length is a characteristic feature of PHB and corresponds to the ester functional group (C=O).

Novel Nile Blue Analogue Stains Yeast Vacuolar Membrane, Endoplasmic Reticulum and Lipid Droplets, Inducing Cell Death through Vacuole Membrane Permeabilization

Phenoxazine derivatives such as Nile Blue analogues are assumed to be increasingly relevant in cell biology due to their fluorescence staining capabilities and antifungal and anticancer activities. However, the mechanisms underlying their effects remain poorly elucidated. Using S. cerevisiae as a eukaryotic model, we found that BaP1, a novel 5- and 9-N-substituted benzo[a]phenoxazine synthesized in our laboratory, when used in low concentrations, accumulates and stains the vacuolar membrane and the endoplasmic reticulum. In contrast, at higher concentrations, BaP1 stains lipid droplets and induces a regulated cell death process mediated by vacuolar membrane permeabilization. BaP1 also induced mitochondrial fragmentation and depolarization but did not lead to ROS accumulation, changes in intracellular Ca2+, or loss of plasma membrane integrity. Additionally, our results show that the cell death process is dependent on the vacuolar protease Pep4p and that the vacuole permeabilization results in its translocation from the vacuole to the cytosol. In addition, although nucleic acids are commonly described as targets of benzo[a]phenoxazines, we did not find any alterations at the DNA level. Our observations highlight BaP1 as a promising molecule for pharmacological application, using vacuole membrane permeabilization as a targeted approach.

Fundamental Investigations into Single Molecule Surface Enhanced Raman Spectroscopy

<p>After the first claim of single molecule (SM) detection by surface enhanced Raman spectroscopy (SERS) was published in 1997 and years of debate and maturing, SM-SERS can now be considered as an established subfield of SERS. Besides the obvious promising advances in analytical spectroscopy that SM-SERS enables, some more fundamental studies are now also accessible. The main focus of this thesis is to understand certain aspects and tackle some outstanding issues in SM-SERS, both in methods and applications. In the first part of this thesis, we focus on the application of SM-SERS to the study of the homogeneous broadening of molecular vibrations. We show that the homogeneous linewidth of the dye Nile blue as measured on single molecule SERS spectra is much smaller than the inhomogeneous broadening obtained from the average signal. Individual molecules having the central Raman frequency occurring at slightly different positions is therefore the main cause of the inhomogeneous broadening in this system. Furthermore, we show that the homogeneous broadening of the mode of single molecules exhibits a strong temperature dependence from 80K to 300 K. This is suggestive of the vibrational energy exchange model which explicitly relates the temperature dependence of the linewidth of a vibrational mode to its interaction with other modes of the molecule or its environment. The average signal does not show this temperature dependence, this property is indeed washed out by ensemble averaging and its unravelling is made possible by SM-SERS. This study is the first example of direct measurement and study of the homogeneous broadening of a Raman peak. In the second part of this work, we focus on a particular method to prove single molecule sensitivity and demonstrate the single molecule detection of the iconic C₆₀ by SM-SERS using its peculiar spectral properties regarding isotopic substitution. A change in one unit mass in one of the carbon atoms is readily observed as a detectable frequency shift in the Ag(2) mode on the Raman spectrum of one C₆₀. This remarkable result is a direct consequence of the high symmetry of the molecule and is only accessible experimentally by measuring individual molecules. We perform SM-SERS detection of a isotopically enriched C₆₀ and show how the distribution of frequencies for the Ag(2) mode reflects the isotopic spread of the sample. Density Functional Theory (DFT) calculations support the experimental results. This provides the first ever evidence of single molecule detection of C₆₀ via SERS. Finally, we focus on the photostability of dyes excited resonantly in SERS conditions. Photobleaching of the molecule is an issue when doing SERS (and SM-SERS) at resonance. Nile blue is deposited on a highly ordered gold nanolithographic substrate and the time dependence of the SERS signal is monitored. Using a simple two-level system model to describe the mechanisms of photobleaching and express the photobleaching rate, we analyse the SERS intensity decay at different powers. This study is the first to be dedicated to the photobleaching decay rates of molecules on metallic surfaces and to highlight that the decay dynamics contains rates spanning four orders of magnitude. This work can potentially reveal information on the distribution of SERS enhancement factors on the surface.</p>

Fundamental Investigations into Single Molecule Surface Enhanced Raman Spectroscopy

<p>After the first claim of single molecule (SM) detection by surface enhanced Raman spectroscopy (SERS) was published in 1997 and years of debate and maturing, SM-SERS can now be considered as an established subfield of SERS. Besides the obvious promising advances in analytical spectroscopy that SM-SERS enables, some more fundamental studies are now also accessible. The main focus of this thesis is to understand certain aspects and tackle some outstanding issues in SM-SERS, both in methods and applications. In the first part of this thesis, we focus on the application of SM-SERS to the study of the homogeneous broadening of molecular vibrations. We show that the homogeneous linewidth of the dye Nile blue as measured on single molecule SERS spectra is much smaller than the inhomogeneous broadening obtained from the average signal. Individual molecules having the central Raman frequency occurring at slightly different positions is therefore the main cause of the inhomogeneous broadening in this system. Furthermore, we show that the homogeneous broadening of the mode of single molecules exhibits a strong temperature dependence from 80K to 300 K. This is suggestive of the vibrational energy exchange model which explicitly relates the temperature dependence of the linewidth of a vibrational mode to its interaction with other modes of the molecule or its environment. The average signal does not show this temperature dependence, this property is indeed washed out by ensemble averaging and its unravelling is made possible by SM-SERS. This study is the first example of direct measurement and study of the homogeneous broadening of a Raman peak. In the second part of this work, we focus on a particular method to prove single molecule sensitivity and demonstrate the single molecule detection of the iconic C₆₀ by SM-SERS using its peculiar spectral properties regarding isotopic substitution. A change in one unit mass in one of the carbon atoms is readily observed as a detectable frequency shift in the Ag(2) mode on the Raman spectrum of one C₆₀. This remarkable result is a direct consequence of the high symmetry of the molecule and is only accessible experimentally by measuring individual molecules. We perform SM-SERS detection of a isotopically enriched C₆₀ and show how the distribution of frequencies for the Ag(2) mode reflects the isotopic spread of the sample. Density Functional Theory (DFT) calculations support the experimental results. This provides the first ever evidence of single molecule detection of C₆₀ via SERS. Finally, we focus on the photostability of dyes excited resonantly in SERS conditions. Photobleaching of the molecule is an issue when doing SERS (and SM-SERS) at resonance. Nile blue is deposited on a highly ordered gold nanolithographic substrate and the time dependence of the SERS signal is monitored. Using a simple two-level system model to describe the mechanisms of photobleaching and express the photobleaching rate, we analyse the SERS intensity decay at different powers. This study is the first to be dedicated to the photobleaching decay rates of molecules on metallic surfaces and to highlight that the decay dynamics contains rates spanning four orders of magnitude. This work can potentially reveal information on the distribution of SERS enhancement factors on the surface.</p>

Two-Step Targeted Drug Delivery Via Proteinaceous Barnase-Barstar Interface and PLGA-Based Nano-Carrier

The conventional methods of treating cancer with chemo- and radiotherapy present plenty of serious problems, such as low therapeutic index and high systemic toxicity. The advanced cancer treatment strategies utilize nanoformulations of drugs that can enter a tumor due to the enhanced permeability and retention (EPR) effect. However, EPR fails in the treatment of several human diseases. Mainstream biomedical studies are focused on creating the drugs that would enter the tumor with higher effectiveness and require smaller doses for administration. A two-stage drug delivery system is an encouraging alternative solution. At first, the primary, non-toxic targeting module is delivered to the tumor cells, followed by injection of the second complementary targeting module at a considerably lower dose, thus decreasing systemic toxicity. To meet the challenge, we have developed a two-stage drug delivery system (DDS), mediated by the high-affinity binding of the Barnase*Barstar protein pair. Barnase and Barstar act as lego bricks linking the first and the second modules on the surface of the cancer cell. Barnase (12 kDa) is a natural ribonuclease from Bacillus amyloliquefaciens, while Barstar (10 kDa) is its natural inhibitor. The Barnase*Barstar is one of the strongest known protein*protein complexes with Kaff = 1014 M−1 exhibiting extraordinarily stability in severe conditions. Artificial scaffold polypeptide DARPin9_29 genetically fused with Barstar served is a first module of the developed two-step DDS. DARPin9_29 (14 kDa) specifically recognizes the tumor marker HER2 overexpressed on human breast cancer cells. As a second module, a therapeutic nano-cargo was developed based on fluorescent polymer PLGA nanoparticles loaded with diagnostic Nile Blue dye and the chemotherapeutic drug doxorubicin. This nano-PLGA structure was covalently coupled to Barnase. We showed two-stage efficient labeling of HER2-overexpressing cancer cells using the first non-toxic module DARPin9_29-Barstar and the second toxic nano-module PLGA-Barnase. We demonstrated the doxorubicin-induced cytotoxicity of this two-step DDS based on polymer nanoparticles and proteinaceous Barnase-Barstar interface and showed more than 10-fold therapeutic dose reduction versus free doxorubicin. We believe that the developed two-step DDS based on PLGA nano-cargo and protein interface will promote the creation of new-generation cancer treatment strategies.

Understanding of Förster Resonance Energy Transfer (FRET) in Ionic Materials

Herein, an ionic material (IM) with Förster Resonance Energy Transfer (FRET) characteristics is reported for the first time. The IM is designed by pairing a Nile Blue A cation (NBA+) with an anionic near-infrared (NIR) dye, IR820−, using a facile ion exchange reaction. These two dyes absorb at different wavelength regions. In addition, NBA+ fluorescence emission spectrum overlaps with IR820− absorption spectrum, which is one requirement for the occurrence of the FRET phenomenon. Therefore, the photophysical properties of the IM were studied in detail to investigate the FRET mechanism in IM for potential dye sensitized solar cell (DSSCs) application. Detailed examination of photophysical properties of parent compounds, a mixture of the parent compounds, and the IM revealed that the IM exhibits FRET characteristics, but not the mixture of two dyes. The presence of spectator counterion in the mixture hindered the FRET mechanism while in the IM, both dyes are in close proximity as an ion pair, thus exhibiting FRET. All FRET parameters such as spectral overlap integral, Förster distance, and FRET energy confirm the FRET characteristics of the IM. This article presents a simple synthesis of a compound with FRET properties which can be further used for a variety of applications.

Polyhydroxyalkanoate production from rice straw hydrolysate obtained by alkaline pretreatment and enzymatic hydrolysis using Bacillus strains isolated from decomposing straw

Rice straw is an important low-cost feedstock for bio-based economy. This report presents a study in which rice straw was used both as a source for isolation of bacteria producing the biodegradable polyester polyhydroxyalkanoate (PHA), as well as the carbon source for the production of the polymer by the isolated bacteria. Of the 100 bacterial isolates, seven were found to be positive for PHA production by Nile blue staining and were identified as Bacillus species by 16S rRNA gene sequence analysis. Three isolates showed 100% sequence identity to B. cereus, one to B. paranthracis, two with 99 and 100% identity to B. anthracis, while one was closely similar to B. thuringiensis. For use in PHA production, rice straw was subjected to mild alkaline pretreatment followed by enzymatic hydrolysis. Comparison of pretreatment by 2% sodium hydroxide, 2% calcium hydroxide and 20% aqueous ammonia, respectively, at different temperatures showed maximum weight loss with NaOH at 80 °C for 5 h, but ammonia for 15 h at 80 °C led to highest lignin removal of 63%. The ammonia-pretreated rice straw also led to highest release of total reducing sugar up to 92% on hydrolysis by a cocktail of cellulases and hemicellulases at 50 °C. Cultivation of the Bacillus isolates on the pretreated rice straw revealed highest PHA content of 59.3 and 46.4%, and PHA concentration of 2.96 and 2.51 g/L by Bacillus cereus VK92 and VK98, respectively.