Simple Single-step Method Research Articles

A rapid One-Pot RNA isolation method for simplified clinical detection of SARS-COV-2 infection in India

BackgroundWith the rapid increase in COVID-19 cases and the discovery of new viral variants within India over multiple waves, the expensive reagents and time-consuming sample pretreatment required for qPCR analysis have led to slower detection of the disease. The vast Indian population demands an inexpensive and competent sample preparation strategy for rapid detection of the disease facilitating early and efficient containment of the disease.MethodsIn this study, we have surveyed the spread of COVID-19 infection over Faridabad, Haryana, India for 6 months. We also devised a simple single-step method for total RNA extraction using a single tube and compared its efficacy with the commercially available RNA isolation kits.FindingsOur findings suggest that determining Ct values for samples subjected to the One Pot RNA extraction method was as efficient as the commercially available kits but delivers a subtle advantage in a way, by minimizing the cost, labor and sample preparation time.ConclusionThis novel crude RNA extraction method is stable and capable of operating in developing countries like India for low resource settings, without the use of expensive reagents and instruments. Additionally, this method can be further adapted to pooling samples strategies owing to its high sensitivity.

Crack barriers for thick SiN using dicing.

Silicon nitride (SiN) waveguides need to be thick to show low dispersion which is desired for nonlinear applications. However, high quality thick SiN produced by chemical vapour deposition (CVD) contains high internal stress, causing it to crack. Crack-free wafers with thick SiN can be produced by adding crack barriers. We demonstrate the use of dicing trenches as a simple single-step method to produce high quality (loss<0.5 dB/cm) crack-free SiN. We show Kerr-comb generation in a ring resonator to highlight the high quality and low dispersion of the waveguides.

Non-innocent Intercalation of Diamines into Tetragonal FeS Superconductor

We report two solvothermal pathways toward intercalated iron sulfide, [Fe8S10]Fe(en)3·en0.5 (en = ethylenediamine), featuring [Fe8S10]2– layers stacked by [Fe(en)3]2+ cations and free ethylenediamine molecules. [Fe8S10]Fe(en)3·en0.5 is synthesized in a simple single-step method from Fe and S in ethylenediamine with addition of NH4Cl mineralizer as well as from solvothermal treatment of mackinawite, tetragonal FeS. In situ synchrotron powder X-ray diffraction experiments reveal a clear transformation of tetragonal FeS into [Fe8S10]Fe(en)3·en0.5 upon reaction with ethylenediamine. In-house control synthetic experiments confirmed the chemical process, whereby ethylenediamine leaches iron solely from the tetragonal Fe–S layers to form [Fe(en)3]2+ complexes and thereby oxidize the intralayer iron to Fe2.25+. Our report emphasizes that, in layered iron chalcogenides, diamines can intercalate as charged coordination complexes in tandem with neutral diamine molecules.

A rapid and simple single-step method for Mg separation from carbonates for isotopic analysis by MC-ICP-MS

A new simple and rapid method was developed for the purification of Mg from carbonate samples, especially for calcite-rich carbonates.

A rapid and simple single-step method for the purification of Toxoplasma gondii tachyzoites and bradyzoites.

This study describes a simple method for the large‐scale isolation of pure Toxoplasma gondii tachyzoites and bradyzoites. T. gondii tachyzoites were obtained from infected human foreskin fibroblasts (HFFs) and peritoneal exudates of mice, while tissue cysts containing bradyzoites were collected from chronically infected mice. Harvested cells and brain tissues were incubated in Hanks balanced salt solution (HBSS), containing 0.25% trypsin and 0.5% taurodeoxycholic acid (TDC) for 5 min. Subsequent washes in phosphate buffered saline (PBS) were conducted, and the cell viability of the preparations was good, as determined by flow cytometry and ability to reinfect HFF cells and propagate in mice. The purification procedure allowed for a rapid preparation of pure T. gondii tachyzoites and bradyzoites in sufficient quantity that can be used for downstream procedures. The advantage of the new method is that it is convenient and inexpensive.

Biosynthesis of Gold Nanoparticles Using Litsea cubeba Fruit Extract for Catalytic Reduction of 4-Nitrophenol

This work describes a simple single-step method for green synthesis of colloidal gold nanoparticles (AuNPs) using Litsea cubeba (LC) fruit extract as a reducing as well as stabilizing agent simultaneously. Major parameters affecting the formation of LC-AuNPs, including gold ion concentration, reaction time, and reaction temperature were optimized using ultraviolet-visible (UV-Vis) measurements at a characteristic maximum absorbance of 535 nm. The functional groups responsible for reducing gold ions and capping AuNPs were examined by Fourier-transform infrared (FTIR) spectroscopy. Powder X-ray diffraction (XRD) analysis revealed the crystalline nature of AuNPs. Transmission electron microscopy (TEM) measurements showed that the biosynthesized LC-AuNPs were mostly spherical with an average size of 8-18 nm. The nanoparticles also demonstrated excellent ultrarapid catalytic activity for the complete reduction of 4-nitrophenol to p-aminophenol in the presence of NaBH4 within 10 min with a reaction rate constant of 0.348 min-1.

Protein extraction and cytotoxicity abilities of colloidal gold-coated silica hybrid nanoparticles

Monodisperse non-hybrid silica and hybrid colloidal silica of ≤ 200 nm decorated with small Au nanoparticles (NPs) were synthesized in a simple single-step method. Non-hybrid silica NPs were synthesized in the absence and presence of different twin tail cationic surfactants, while tiny Au NPs were grown under in situ reaction conditions on non-hybrid silica synthesized previously by using cationic dextran. Bio-applicability and cytotoxicity of both hybrid as well as non-hybrid silica NPs were tested by using them for the extraction of protein fractions from complex aqueous protein solutions and treating them with blood cells, respectively. Both non-hybrid and hybrid silica NPs demonstrate excellent ability to extract proteins fractions predominantly of relatively low molecular masses, i.e., ~ 80 kDa. Extraction preferences between both kinds of silica became prominent when predominantly hydrophobic proteins such zein and rice proteins were used rather than mainly polar protein like BSA. Applicability for more complex biological fluid like serum indicated the competitive extractions among strongly versus weakly bound proteins. With significant bearing in in vivo conditions, hybrid silica was potentially toxic towards the blood cells in comparison to non-hybrid silica. It stems from the collective interactions of silica as well as nanometallic surfaces of Au NPs to interact with the blood cells causing hemolysis and hence may not be the suitable vehicles for drug release in systemic circulation.

Preparation of Ultraviolet Curing Type Silicone Rubbers Containing Mesoporous Silica Fillers.

Here we have been focusing on mesoporous silica (MPS) as inorganic filler material to improve the mechanical strength of silicone rubbers. The MPS particles are more effective in reducing the coefficient of thermal expansion (CTE) and hardening silicone rubber composites when compared to commercially available nonporous silica particles. In this study, we utilize ultraviolet curing type silicone rubbers and prepare MPS composites according to a simple single-step method. From an industrial viewpoint, simplifying the fabrication processes is critical. The thermal stability and mechanical strength are examined in detail in order to showcase the effectiveness of MPS particles as filler materials.

Differential Adhesive and Bioactive Properties of the Polymeric Surface Coated with Graphene Oxide Thin Film

Surface engineering of implantable devices involving polymeric biomaterials has become an essential aspect for medical implants. A surface enhancement technique can provide an array of unique surface properties that improve its biocompatibility and functionality as an implant. Polyurethane-based implants that have found extensively acclaimed usage as an implant in biomedical applications, especially in the area of cardiovascular devices, still lack any mechanism to ward off bacterial or platelet adhesion. To bring out such a defense mechanism we are proposing a surface modification technique. Graphene oxide (GO) in very thin film form was wrapped onto the electrospun fibroporous polycarbonate urethane (PCU) membrane (GOPCU) by a simple method of electrospraying. In the present study, we have developed a simple single-step method for coating a polymeric substrate with a thin GO film and evaluated the novel antiadhesive activity of these films. SEM micrographs after coating showed the presence of very thin GO films over the PCU membrane. On the GOPCU surface, the contact angle was shifted by ∼30°, making the hydrophobic PCU surface slightly hydrophilic, while Raman spectral characterization and mapping showed the presence and distribution of GO over 75% of the membrane. A reduced platelet adhesion on the GOPCU surface was observed; meanwhile, bacterial adhesion also got reduced by 85% for Staphylococcus aureus (Gram positive, cocci) and 64% for Pseudomonas aeruginosa (Gram negative, bacilli). A cell adhesion study conducted using mammalian fibroblast cells projected its proliferation percentage in a MTT assay, with 82% cell survival on PCU and 86% on GOPCU after 24 h culture, while a study for an extended period of 72 h showed 87% of survival on PCU and 88% on GOPCU. This plethora of functionalities by a simple modification technique makes thin GO films a self-sufficient surface engineering material for future biomedical applications.

A simple approach for the preparation of simazine molecularly imprinted nanofibers via self-polycondensation for selective solid-phase microextraction

A novel molecularly imprinted sol-gel material based on polysiloxane nanofiber was introduced as a solid-phase microextraction coating on a stainless steel wire for the extraction of simazine. The nanostructured molecularly imprinted fiber was prepared by a simple single step method at room temperature, using methyltriethoxysilane as the sol-gel precursor and simazine as the template molecule. The fiber was applied for the extraction of simazine in different water samples followed by gas chromatography and mass spectrometry detection. The extraction capacity of the molecularly imprinted fiber was 8 ng, and it had better extraction efficiency than the non-imprinted fiber and commercial fibers (PDMS and PA). The fiber had also a good selectivity for simazine and its analogous compounds. Important parameters affecting the extraction and desorption efficiency, such as salt concentration, stirring rate, pH of sample solution, extraction time and temperature, temperature and time of desorption, were investigated. The intra- and inter-day relative standard deviations were in the range of 4.3–7.6%. The fiber-to-fiber reproducibility was 7.7–8.5%. The method showed a good linearity (r2> 0.9980) in the range of 0.02–20 μg L−1 with the detection limit of 0.005 μg L−1. The relative recoveries were also in the range of 94–97% for different water samples.

Fast method for skeletal tissue gene expression analysis.

Several chronic diseases have been associated with bone alteration in the last few years. Despite the wealth of information provided by the analysis of the transcriptome in affected tissues, only a limited number of studies evaluated gene expression in bone tissue due to the difficulty to obtain high quality RNA. Therefore, skeletal pathologies have been often associated to a defective maturation process that occurs during recruitment of progenitor stem cells. In order to explore the possibility of analysing the gene expression during osteogenic differentiation in skeletal tissue, a single-step method to extract well-preserved RNA from bone specimens was performed. A comparison between this technique and a traditional method was made by analysing the quality and yield of RNA obtained. In addition, RNAs were assayed by reverse transcription-quantitative polymerase chain reaction to analyse the expression levels of the bone genes associated with the differentiation process in a mouse model. The present data showed that good quality RNA can be obtained from bone tissue by a simple single-step method allowing the expression analysis of the genes encoded by skeletal tissue. In conclusion, the present study allows the possibility to easily obtain good quality RNA from bone tissue that is suitable for gene expression studies of bone diseases.

Microfabrication of Fresnel zone plates by laser induced solid ablation

A novel and simple single-step method of inscribing optical elements on metal-coated transparent substrates is demonstrated. Laser induced solid ablation (LISA) demands very low laser energies (nJ), as can be amply provided by a femtosecond laser oscillator. Here, LISA is used to write Fresnel zone plates on indium and tungsten coated glass. With up to 100 zones, remarkable agreement is obtained between measured and expected values of the focal length. LISA has enabled attainment of focal spot sizes that are 38% smaller than what would be obtained using conventional lenses of the same numerical aperture. The simplicity with which a high degree of automation can readily be achieved using LISA makes this cost-effective method amenable to a wide variety of applications related to microfabrication of optical elements.

Spatially graded TiO₂-SiO₂ Bragg reflector with rainbow-colored photonic band gap.

A simple single-step method to fabricate spatially graded TiO2-SiO2 Bragg stack with rainbow colored photonic band gap is presented. The gradation in thickness of the Bragg stack was accomplished with a modified glancing angle deposition (GLAD) technique with dynamic shadow enabled by a block attached to one edge of the rotating substrate. A linear gradation in thickness over a distance of about 17 mm resulted in a brilliant colorful rainbow pattern. Interestingly, the photonic band gap position can be changed across the whole visible wavelength range by linearly translating the graded Bragg stack over a large area substrate. The spatially graded Bragg stack may find potential applications in the tunable optical devices, such as optical filters, reflection gratings, and lasers.

Synthesis, photophysics and pyrolytic ceramization of a platinum(II)-containing poly(germylacetylene) polymer

A synthetic route to a new polymeric platinum(II)-containing germylacetylene has been developed. Soluble and thermally stable platinum(II) polyyne polymer trans-[–Pt(PBu3)2CCGePh2CC–]n (P1) was prepared in good yield by CuI-catalyzed polymerization of trans-[Pt(PBu3)2Cl2] and Ph2Ge(CCH)2. We report the optical absorption and photoluminescence spectra of P1 and the results are compared with platinum(II) polyynes with purely acetylenic and other (hetero)aromatic conjugated units. Harvesting of organic triplet emission harnessed through the strong heavy-atom effect of Pt and Ge was examined. Polymer P1 was found to have a high optical bandgap (ca. 3.84 eV). The influence of metal and sp3-hybridized germyl-based conjugation interrupter on the intersystem crossing rate and the spatial extent of the lowest singlet and triplet excitons were fully elucidated. Our investigations indicate that a high-bandgap polymer P intrinsically gives rise to a very efficient phosphorescence with fast radiative decay. Remarkably, a simple single-step method was also established for the synthesis of platinum–germanium (PtGe) alloy nanoparticles using P1 as the polymer precursor and the resulting ceramic metal alloys have been characterized by powder X-ray diffraction, energy dispersive X-ray analysis and transmission electron microscopy.

Preparation of Mesoporous/Macroporous Materials in Highly Concentrated Emulsions Based on Cubic Phases by a Single-Step Method

A novel and simple single-step method for the preparation of meso/macroporous silica materials is described, which consists in templating in highly concentrated emulsions with a cubic liquid crystal in the continuous phase. Tetraethyl orthosilicate (TEOS) was solubilized in the aqueous continuous phase of highly concentrated emulsions stabilized by C(12)(EO)(8) and a PEO-PPO-PEO block copolymer nonionic surfactant, with a cubic liquid crystalline phase of the Fd3m type. The resulting silica materials were characterized by small-angle X-ray scattering, nitrogen sorption and transmission electron microscopy. The results showed that a dual pore size distribution was obtained, consisting of mesopores in the nanometer range and macropores between 1 and 5 μm. These dual meso/macroporous silicas with bimodal pore size distribution can possess specific surface areas higher than 400 m(2)/g.

Production of aqueous spherical gold nanoparticles using conventional ultrasonic bath

A conventional ultrasonic bath was used to examine the feasibility of forming aqueous spherical gold nanoparticles (GNPs) under atmospheric conditions. The effects of ultrasonic energy on the size and morphology of GNPs were also investigated. Highly monodispersed spherical GNPs were successfully synthesised by sodium citrate reduction in a conventional ultrasonic bath, without an additional heater or magnetic stirrer, as evidenced by ultraviolet–visible spectra and transmission electron microscopy. Ultrasonic energy was shown to be a key parameter for producing spherical GNPs of tunable sizes (20 to 50 nm). A proposed scheme for understanding the role of ultrasonic energy in the formation and growth of GNPs was discussed. The simple single-step method using just a conventional ultrasonic bath as demonstrated in this study offers new opportunities in the production of aqueous suspensions of monodispersed spherical GNPs.

Sonochemical synthesis and characterization of Cl-doped TiO2 and its application in the photodegradation of phthalate ester under visible light irradiation

Chlorine-doped anatase titanium dioxide (TiO2) nanocrystallites were prepared via a simple single-step method by sonicating a solution of tetraisopropyl titanate and sodium chloride in water/ethanol at 70°C. The as-prepared sample was characterized using X-ray diffraction, transmission electron microscopy, X-ray photoelectron spectroscopy, and ultraviolet–visible spectroscopy. The presence of the chloride dopant is confirmed by the Cl 2p XPS peak with a binding energy of 198.2eV, which is assigned to the anionic Cl incorporated into TiO2. Cl-doping of TiO2 shifts the absorption edge toward a higher wavelength. The as-prepared samples were successfully used in the photodegradation of aqueous butyl benzyl phthalate (BBP) under visible light irradiation. The photodegradation rates of BBP reached 92% under visible light irradiation for 240min. BBP mineralization was also realized by further increasing the irradiation time.

Bacterial expression and purification of recombinant bovine Fab fragments.

We have previously described a recombinant phagemid expression vector, pComBov, designed for the production of native sequence bovine monoclonal antibodies (mAb) generated by antibody phage display. Bovine mAb Fab fragments isolated from libraries constructed using pComBov in Escherichia coli strain XL1-Blue, which is routinely used for antibodies expressed on the surface of phage, were expressed at very low yields. Therefore, a study was undertaken to determine optimal growth conditions for maximal expression of bovine Fab fragments in E. coli. By varying the E. coli strain, and the temperature and length of the culture growth, we were able to substantially increase the yield of soluble Fab fragments. A high yield of Fab fragments was found in the culture growth medium, which enabled us to devise a rapid and simple single-step method for the purification of native (nondenatured) Fabs based on immobilized metal affinity chromatography against a six-histidine amino acid carboxyl-terminal extension of the heavy-chain constant region. Using these methods we were able to express and purify antigen-specific bovine Fab fragments from E. coli.

A simple single-step method for the synthesis of recombinant non-homologous competitor cDNA and its implications for quantitative PCR in biological sciences and molecular diagnostics.

In recent years there has been great interest in quantitative polymerase chain reaction. Consequently, a large number of assays have been developed, of which the one using non-homologous competitors is arguably the most precise. Despite widespread applications, currently there is no simple method to synthesize such competitors. Here a facile and cost-effective, single-step method for synthesis of recombinant, non-homologous, competitor complementary DNA is described. The method can be adapted to generate competitors of any size and sequence. The entire procedure is quick, straightforward and does not require any specialized equipment except a standard thermocycler.

Development of a pH titration method for the simultaneous determination of uranium, nitrate and free-acid in the feed solution of the sol-gel process of nuclear fuel fabrication

pH titration curves generated by slow addition of alkali to solutions containing varying concentrations of uranyl nitrate and nitric acid were studied using an autotitrator linked to a personal computer. A procedure with multiple choice of equations has been developed for the estimation of free acid, nitrate and uranium in pure uranyl nitrate solution by a single titration. The technique provides a simple single-step method with required accuracy and precision for the simultaneous estimation of the three quantities in the uranyl nitrate feed solution of the sol-gel process for making UO3 microspheres. The relative standard deviations in the determination of uranium and nitrate were ±0.82% and ±1.52%, respectively, in 15 determinations.