Applications of magnetic nanoparticles for nucleic acid extraction and analysis in biological and biomedical fields.

Objective To establish a method of nucleic acid extraction and enrichment based on magnetic nanoparticle as medium for elevating the analytical sensitivity of domestic HBV real-time PCR kit and detection of the trace amount HBV DNA. Methods After receiving antiviral treatment, the serum samples of 50 hepatitis B patients with HBV DNA concentration ≤1×104 IU/ml were collected. The WHO HBV DNA calibrator was used as the standard material. Nanometer magnetic beads were used to adsorb and enrich the HBV nucleic acid and increase the concentration of the extracted HBV nucleic acid template. Compared with Roche HBV DNA detection reagent and four domestic reagent with conventional nucleic acid extraction and detection method, the improvement effect of this method on domestic nucleic acid detection reagent was evaluated. Results After application of nanometer magnetic extraction method to domestic regent, the analytical sensitivities of the domestic reagent reached 10 and 50 IU/ml, respectively,which was about the same detection level to 12 IU/ml of the imported Roche reagent. The positive rates of the detection of serum trace amount HBV DNA of hepatitis B patients with four kinds of domestic extraction reagent were 64% ( 32 ), 56% ( 28 ), 62% ( 31 ) and 58% (29), respectively. There were significant statistical differences between Roche reagent and four domestic extraction reagent kits(x2 = 7. 895, 12. 698,9. 013 and 11. 416 ,P ＜0. 05 ). With nanometer magnetic extraction method combined with domestic reagent kits, the detection rates were 88% (44), 88% (44), 88% (44) and 86% (43) ,respectively. There was no significant difference compared with the imported Roche reagent (x2 = 0. 000, 0. 000, 0. 000 and 0. 088,P ＞0. 05). Moreover, when the HBV nucleic acid concentration was 101-103 IU/ml, the logarithm value of viral nucleic acid concentration was in reverse correlation to Ct value, but the correlation decreased in the concentration range of 103-106 IU/ml. Conclusions The nucleic acid extraction method based on magnetic nanoparticle as medium can significantly improve the analytical sensitivity of domestic HBV DNA detection reagent, which can be used to monitor the trace amounts HBV DNA in the sera of the hepatitis B patients. Key words: Hepatitis B virus; Magnetic nanoparticle; Nucleic acid test; Analysis sensitivity

Natural products such as natural food are the richest bio-resource of bioorganic compounds for modern medicines, nutraceuticals, food supplements and pharmaceutical applications. The research and application on natural food started with the extraction techniques that play an important role to the extraction quantity (Yield), quality (extracted phytochemicals) and also to the subsequent analyses accomplished to evaluate the biological and chemicals activities. Various types of technologies with different principles of extraction of bioorganic compounds are available today. Based on the literature the conventional extraction methods show better recoveries of bioorganic substances of natural food. Also, conventional extraction methods facilitate the extraction of high concentration of bioorganic substances with the safe solvents system such as pure ethanol. Moreover, conventional extraction methods is still widely used due to its simplicity. However, the conventional extraction methods is not always suitable for industrial uses due to long extraction time and large consumption of harmful solvents systems such as methanol. Therefore, modern non-conventional extraction methods could be an alternative extraction method. Hence, in spite of good results achieved with the conventional extraction methods, modern non-conventional extraction methods was established to search for a faster and better extraction method consuming less solvent, especially those that are unattractive in food industry. This chapter is intended to provide insights on conventional and non-conventional extraction methods with their advantages and disadvantage or limitation.

Variants of Coconut cadang-cadang viroid (CCCVd) with over 90% sequence similarity to CCCVd in coconut have recently been associated with orange spotting disease in oil palm, especially in low concentrations in infected hosts. Thus, there is a need to extract high-quality nucleic acid for molecular detection of the viroid. Total nucleic acid (TNA) was extracted from oil palm leaf samples with orange spotting symptoms collected from different states in Malaysia using a modified and optimized version of the conventional natrium chloride EDTA Tris-HCL mercaptoethanol extraction method. The modifications involved additional ethanol and lithium chloride precipitation stages, thereby eliminating the need for further purification through non-denaturing polyacrylamide gel electrophoresis (PAGE). The modified procedure yielded a mean volume of 60 μg RNA per 10 g of fresh tissue which is approximately four times the volume produced by the conventional extraction method. Furthermore, the modified method resulted in higher purity than the conventional method according to the ratios of absorbance at wavelengths of 260/280 nm and 260/230 nm, which were 1.838 and 1.883 with the modified method and 1.085 and 0.765 with the conventional method. The modified method enabled extraction of high-quality RNA from all samples investigated. The extracted RNA was suitable for cDNA synthesis and PCR amplification and showed consistent detection of CCCVd-like RNA at approximately 250 amplicons. Using the conventional method, detection of CCCVd-like RNA was inconsistent and only feasible after the PAGE purification step.

Microwave-assisted extraction (MAE) is an effective green extraction method of value-added and bioactive compounds. The impact of different extraction times (2-7 min), microwave power (360-760 W), and solvent-to-sample ratio (20 : 1-40 : 1 ml/g) on the extraction yield of Ocimum basilicum var. album L. using MAE was investigated. Maximum extraction yield ( 17.00 ± 0.14 %) was obtained at optimal conditions for extraction using response surface methodology, including extraction time of 4.33 min, power of 570.32 W, and the solvent-to-sample ratio of 40 : 1 ml/g, which was very close to the model prediction (17.01%). The yield of the conventional extraction (CE) method (50°C, 100 rpm, 1 h extraction time, and solvent-to-sample ratio of 40 : 1 ml/g) was 14.53 ± 0.25 %. Comparisons were made between the functional and structural characteristics of the mucilage extracted under optimal conditions and the CE method. Fourier transforms infrared (FTIR) spectroscopy was utilized to study the changes in functional groups, and scanning electron microscopy (SEM) was used to determine the morphological characteristics. Emulsion stability against heat, water absorption capacity, foaming capacity, and foaming stability of the extracted mucilage under optimal conditions were 80.73 ± 0.08 %, 48.71 ± 0.32 g/g, 24.55 ± 0.42 %, and 91.6 ± 0.49 %, respectively, which were higher than the CE method. SEM results showed a more porous structure in mucilage obtained by the MAE method, while no changes were observed by FTIR analysis between the functional groups of extracted mucilage obtained from the utilized extraction methods. Therefore, the application of the MAE method was superior to CE in terms of yield, structural and functional characteristics, and significantly shorter extraction time. The findings show the great potential of microwave processing in commercial and laboratory extraction of mucilage without deteriorative effects on the structural and functional properties of the extracted material.

Gleevec (STI571) Influences Metabolic Enzyme Activities and Glucose Carbon Flow toward Nucleic Acid and Fatty Acid Synthesis in Myeloid Tumor Cells

BackgroundOil from oleaginous yeasts has the potential to replace non-sustainable vegetable oil as raw material to produce food, feed, biofuels, or biochemicals. Co-produced compounds like carotenoids may be helpful to obtain economically viable bioprocesses. Identifying appropriate extraction methods is a bottleneck both for establishing oleaginous yeasts as cell factories for both oil and carotenoids production and for analysis of intracellular compounds like lipids and carotenoids. We conducted extractions using supercritical carbon dioxide (SC-CO2) and conventional solvent methods to extract and analyze lipids and carotenoids from R. toruloides CBS 14 cells grown on wheat straw hydrolysate. The lipid extracts were analyzed using gas chromatography (GC), and the carotenoids were identified and quantified using ultra-high-performance liquid chromatography (UHPLC).ResultsFour main carotenoids in the extracts from both extraction methods were identified including β-carotene, γ-carotene, torularhodin, and torulene. Interestingly, torularhodin was the major carotenoid extracted using SC-CO2 extraction, followed by torulene. This was different from the conventional acetone extraction method, where β-carotene was the main carotenoid. After the conventional extraction, torularhodin and torulene underwent degradation due to the saponification step, which was necessary to remove lipids before UHPLC analysis. The total carotenoid concentration obtained from SC-CO2 extraction was 332.09 ± 27.32 μg/g dry weight compared to 19.9 ± 2.74 μg/g dry weight in acetone extraction. A small amount of carotenoids was observed to be lost into the lipid extract, but this loss was not as substantial as that seen with acetone extraction. Additionally, the total lipid content in samples extracted using SC-CO2 was significantly lower than that obtained using the conventional Folch method. GC analysis revealed that oleic acid was the major fatty acid in both lipid extracts, followed by palmitic acid and linoleic acid. Notably, the proportion of unsaturated fatty acids was higher in the extracts from the SC-CO2 method compared to the conventional method.ConclusionThese findings indicate that the SC-CO2 extraction method outperformed conventional methods by preserving the integrity of unsaturated lipids and retaining an abundance of carotenoids, resulting in high-quality extracts.

A microfluidic system for rapid nucleic acid analysis based on real-time convective PCR is developed. To perform ‘sample-in, answer-out’ nucleic acid analysis, a microfluidic chip is developed to efficiently extract nucleic acid, and meanwhile convective PCR (CPCR) is applied for rapid nucleic acid amplification. With an integrated microfluidic chip consisting of reagent pre-storage chambers, a lysis & wash chamber, an elution chamber and a waste chamber, nucleic acid extraction based on magnetic beads can be automatically performed for a large size of test sample within a limited time. Based on an easy-to-operate strategy, different pre-stored reagents can be conveniently released for consecutive reaction at different steps. To achieve efficient mixing, a portable companion device is developed to introduce properly controlled 3-D actuation to magnetic beads in nucleic acid extraction. In CPCR amplification, PCR reagent can be spontaneously and repeatedly circulated between hot and cool zones of the reactor for space-domain thermal cycling based on pseudo-isothermal heating. A handheld real-time CPCR device is developed to perform nucleic acid amplification and in-situ detection. To extend the detection throughput, multiple handheld real-time CPCR devices can be grouped together by a common control system. It is demonstrated that influenza A (H1N1) viruses with the reasonable concentration down to 1.0 TCID50/ml can be successfully detected with the microfluidic system.

Although numerous approaches were proposed for the nucleic acid (NA)-based SARS-CoV-2 detection, the nonideal NA desorption efficiency of conventional magnetic beads (MBs) limits their widespread application. In this study, we developed solvent-responsive MBs (called responsive MBs), which, in the presence of buffers, modulated the absorption and desorption capacities of NA by flipping the surface -COO-. Relative to other commercial MBs, responsive MBs exhibited similar absorption profiles and markedly enhanced desorption profiles. When applied for NA detection of complex samples, responsive MBs exhibited better performance of RNA detection than DNA, with obvious advantages in sensitivity. Specifically, the RNA and DNA desorption rates of commercial MBs were ∼85 and 82.5%, while those of responsive MBs were nearly 94 and 93.5%, respectively. Furthermore, responsive MBs exhibited remarkable extraction ability in a wide range of tissues and better performance of RNA extraction than DNA. When applied for SARS-CoV-2 detection, the responsive MBs along with the simulated digital RT-LAMP (a previously established apparatus) further improved detection efficiency, yielding a precise quantitative detection as low as 25 copies and an ultimate sensibility detection of 5 copies/mL. It was also successfully employed in numerous NA-based technologies such as polymerase chain reaction (PCR), sequencing, and so on.

Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease

Nucleic acid amplification-based diagnostics is known as one of the molecular diagnostic systems that allows higher sensitive detection of pathogens than test methods such as immunoassay. However, it has not been widely used because it is complicated to use and takes a long time to generate results. On the other hand, development of fully automated molecular diagnostic systems has been growing around the world as demand for such systems from physicians and laboratory technicians has increased. To meet this demand, we have developed the “Simprova” fully automated molecular diagnostic system, which takes advantage of LAMP (Loop-mediated Isothermal Amplification), a method Eiken Chemical Co., Ltd. invented. Simprova comprises a master unit that controls the entire system and a test unit that extracts and purifies nucleic acid from samples (pretreatment), and uses the LAMP method to detect and amplify nucleic acid. Users can obtain test results automatically by simply installing a pretreatment cartridge, a multi-well testing chip and the sample in the test unit. The multi-well testing chip has 25 reaction wells connected by channels and enables simultaneous testing of multiple targets with one sample. Turnaround time for one test is approximately 30 minutes. Since a conventional extraction and purification method using magnetic-bead separation is used for the pretreatment, nucleic acid can be extracted from serum, plasma, whole blood, urine, and sputum, for example. In addition, the system can perform random-access testing by connecting four test units to the master unit to realize near-the-patient testing. Simprova is therefore a robust and useful system for a wide variety of applications.

Magnetic engineering nanoparticles: Versatile tools revolutionizing biomedical applications

In cotyledons ofChenopodium rubrum L. polydisperse RNA is synthesized in the region of the low molecular weight RNAs during photoperiodic induction. After short-time labelling the rate of 4s RNA synthesis was always higher in induced plants than in plants having obtained a light-break in the middle of the dark period. When glucose was added to the nutrient medium during the dark period of a single photoperiodic cycle the rate of nucleic acid (NA) synthesis was higher in non-induced plants than in induced ones at the termination of the dark period. In plants induced by two cycles in the absence of glucose the rate of NA synthesis at the termination of the second dark period was higher in induced than in non-induced plants. This difference is due to the differential kinetics of NA synthesis during darkness. In plants induced in the presence of glucose the peak of the rhythm in NA synthesis was advanced by 4 h relative to that found in plants induced in the absence of sugar. Thus, the termination of the dark period coincided with the negative slope of the oscillation in plants induced in the presence of glucose, while in plants having obtained a light-break NA synthesis decreased only slightly after having attained its peak. In plants induced in the absence of glucose the termination of the dark period coincided with the peak in the rhythm in NA synthesis. The rhythm in NA synthesis of the cotyledons during the dark period of an inductive cycle is out of phase with the rhythm in flower initiation.

High-efficient nucleic acid separation from animal tissue samples via surface modified magnetic nanoparticles

Brown seaweeds are rich in fucoidan, a sulfated polysaccharide with antimicrobial, immunomodulatory, antioxidant, and anticancer properties. The brown algae Sargassum sp. has not been thoroughly investigated for fucoidan extraction using various techniques and evaluations of their effects on extraction yield and its structural properties. The purpose of this study was to compare the structural characteristics and extraction yield of fucoidan from Sargassum sp. using conventional, microwave, and ultrasonic-assisted extraction methods. The results showed that a slightly higher yield was obtained by using the ultrasonic-assisted extraction method (2.772%) followed by the microwave-assisted extraction method (2.494%) and conventional extraction method (2.399%). However, the IC50 values for antioxidants were found to be lower (less value is preferable) for crude fucoidan obtained by microwave-assisted extraction method (175 ?g/ml) than for conventional (195 ?g/ml) and ultrasound-assisted extraction methods (230 ?g/ml). The crude fucoidan obtained from the three different extraction methods showed moderate antioxidant strength Contribution to Sustainable Development Goals (SDGs)SDG 3: Good Health and Well-beingSDG 9: Industry, Innovation, and InfrastructureSDG 12: Responsible Consumption and ProductionDG 14: Life Below Water

Utilization of electrocoagulation for the isolation of alkaloids from the aerial parts of Stemona aphylla and their mosquitocidal activities against Aedes aegypti